Discerning the Role of Krüppel-Like Factor 4 in Breast Cancer

DISCERNING THE ROLE OF KRÜPPEL-LIKE FACTOR 4

IN BREAST CANCER

JENNIFER L. YORI

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Dissertation Advisor: Dr. Ruth A. Keri

Department of Pharmacology

CASE WESTERN RESERVE UNIVERSITY

May, 2011 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

______

candidate for the ______degree *.

(signed)______(chair of the committee)

______

(date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein. DEDICATION

This work is dedicatied to Beverly Ann McMillan for all that she taught me

and Fiona Campbell Yori for all that she teaches me everyday.

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TABLE OF CONTENTS

List of Tables vii

List of Figures viii

Acknowledgements x

Abbreviations xii

Abstract 1

Chapter I INTRODUCTION, REVIEW OF THE LITERTURE, AND 3

STATEMENT OF PURPOSE

1.1 Molecular/Histopathological subtypes and cellular origin of 3

breast cancer

1.1.1 Luminal breast cancer 5

1.1.2 HER2/ERRB2 breast cancer 6

1.1.3 Basal-like breast cancer 8

1.1.4 Comparison of mouse models and human tumors generate a 10

new molecular subtype

1.2 Metastasis, epithelial-to mesenchymal transition (EMT) and 12

the cancer stem cell theory

1.2.1 The epithelial-to-mesenchymal transition 13

1.2.1.1 Transcriptional regulation of EMT 15

1.2.2 The cancer stem cell theory 19

1.2.2.1 Cancer stem cells and EMT 21

1.2.2.2 iPS cells 23

1.3 Krüppel-like factor 4 (KLF4) 24

1.3.1 The Sp1/Krüppel-like factor family 24

1.3.2 Krüppel-like factor 4 (KLF4) 26

1.3.2.1 Structure and regulation 26

1.3.2.2 Mechanisms of activation and repression 28

1.3.3 Function of KLF4 in normal biological processes 30

1.3.3.1 KLF4 as a regulator of development and differentiation 30

1.3.3.2 KLF4 regulation of proliferation and apoptosis 31

1.3.4 The role of KLF4 during carcinogenesis 34

1.3.4.1 KLF4 as tumor suppressor 34

1.3.4.2 KLF4 as an oncogene 35

1.3.4.3 The role of KLF4 in breast cancer and metastasis 35

1.3.4.4 Krüppel-like factors, EMT and metastasis 37

1.4 Statement of Purpose 39

Chapter 2 KRÜPPEL-LIKE FACTOR 4 (KLF4) INHIBITS 52

EPITHELIAL-TO-MESENCHYMAL TRANSITION

THROUGH REGULATION OF E-CADHERIN GENE

EXPRESSION

2.1 Introduction 53

2.2 Materials and Methods 56

2.3 Results 61

2.4 Discussion 67

2.5 Acknowledgements 74

Chapter 3 KRÜPPEL-LIKE FACTOR 4 INHIBITS TUMORIGENIC 88

PROGRESSION AND METASTASIS IN A MOUSE MODEL

OF BREAST CANCER

3.1 Introduction 89

3.2 Materials and Methods 91

3.3 Results 95

3.4 Discussion 101

3.5 Acknowledgements 105

Chapter 4 SUMMARY AND FUTURE DIRECTIONS 120

4.1 Summary 120

4.2 Future directions 121

4.2.1 Pharmacological targeting of KLF4 121

4.2.2 Does TGF-β signaling regulate KLF4 expression during EMT? 130

4.2.3 Can forced expression of KLF4 block TGF-β-induced EMT? 131

4.2.4 Does the TGF-β “switch” during tumorigenesis alter its effect 132

on the regulation of KLF4?

4.2.5 What are the mechanisms by which KLF4 suppresses Snail? 133

4.2.6 Does HER2 signaling regulate KLF4 expression? 134

4.2.7 What is the role of KLF4 in the maintenance and self-renewal 135

of MaSCs and CSCs?

References 139

LIST OF TABLES

Table 1.1 Regulators and Targets of KLF4 48

Table 3.1 Incidence of lung and liver micrometastases in AdGFP-4T1 and 106

AdKLF4-4T1 tumor bearing mice at 21 days post injection

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LIST OF FIGURES

Figure 1.1 The link between normal mammary epithelial hierarchy, 41

molecular subtypes, gene expression signatures and clinical

markers of breast cancer

Figure 1.2 Organization of the human KLF4 gene and protein 43

Figure 1.3 KLF4 is a central player in the regulatory networks modulating 45

EMT and MET during generation of iPSCs and CSCs

Figure 2.1 KLF4 is required for the maintenance of mammary epithelial 75

cell morphology

Figure 2.2 KLF4 silencing results in loss of acinus formation and 77

decreased proliferation of mammary epithelial cells

Figure 2.3 KLF4 is required to sustain E-cadherin expression in non- 79

transformed mammary epithelial cells

Figure 2.4 KLF4 binds and activates the E-cadherin promoter 81

Figure 2.5 KLF4 induces expression of E-cadherin protein and a 83

transition to epithelial morphology in the mesenchymal-like

MDA-MB-231 breast cancer cells

Figure 2.6 KLF4 transcriptional activation of E-cadherin results in 85

decreased migration and invasion of MDA-MB-231 cells

Figure 3.1 Loss of KLF4 expression in Her2/Neu-induced mouse 107

mammary tumors

Figure 3.2 KLF4 expression inversely correlates with metastatic 110

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progression

Figure 3.3 KLF4 inhibits growth of 4T1 cells 112

Figure 3.4 KLF4 inhibits primary tumor growth of 4T1 cells 114

Figure 3.5 KLF4 overexpression decreases lung and liver micrometastases 116

Figure 3.6 KLF4 inhibits Snail expression in mammary epithelial cells and 118

tumor cells

ACKNOWLEDGEMENTS

It is very rare that one gets the opportunity to spend their time pursuing a career in something they are genuinely passionate about. I am ever grateful to the numerous people in my life who have made this possible. To Dr. John Nilson, who first brought me into the Pharmacology Department, imparting upon me his great words of wisdom, “It doesn‟t matter if you‟re not as smart as everyone else, it just means you have to work harder than everyone else”. His support and advice through my first year of graduate school made me realize this truly was an attainable goal. To Helai Mohammad, who taught me everything I needed to know in order to realize how much I really didn‟t know,

I thank you for your persistence and attention to detail, as well as a solid understanding of cloning!

I would especially like to thank Dr. Ruth Keri, who upon John‟s departure accepted me into her laboratory, allowing me to work in the area of breast cancer research. As a mentor, she has provided me with the opportunity to develop a research project from the ground up and call it my own. She has challenged me to critically evaluate my own experimental designs and data as well as the scientific literature. In addition, unlike most mentors, she has allowed her students to be an integral part of the grant writing process, contributing at all levels, from suggested experiments to critical review. She is truly a role model for young women in science. I‟m also grateful to my committee members,

Dr. Amy Wilson-Delfosse, Dr. Mukesh Jain, and Dr. Noa Noy for their input and guidance.

During my time in the lab, many new members have come as old ones have moved on.

Throughout the duration there has been comfort in consistency. To Darcie Seachrist and

Kristen Lozada, who have been with me since the beginning and are still there today, I don‟t think I could have endured this journey without your friendships and support. Kris, thank you for teaching me all the ways to manipulate a mouse and for your many hours in the mouse room, especially when I was on maternity leaves. Darcie, your willingness to listen and offer advice, both scientific and personal, is so appreciated. I cannot thank you enough for all the hard work you put in towards the end to help me finish up. I‟d like to also thank several past lab member, including Jonathan Mosley, Erin Milliken, and

Melissa Landis, for keeping me excited about science and showing me how it was done.

To Monty Johnson, our first post-doc, I owe many thanks. His willingness to share his experience, knowledge and time was really a turning point for me in my graduate career, and I am forever grateful.

Finally, I would like to thank my family and friends who have provided me with all the encouragement, love and support I could ever need. Rick and Louise Yori, the best in- laws anyone could ask for, thank you for your generosity and willingness to help, always!

Dad, even though you‟ve teased me about being in school since I was five, you‟ve always found a way to let me know how proud you were of me, and that has meant so much.. To my husband Rich, who has helped me raise two wonderful children during these busy years, words alone could never convey my thanks and gratitude for all your sacrifice.

Most of all, I would like to thank my mother, who provided me with a true appreciation for education, an understanding of true commitment and strength, and the realization that life is what you alone make of it – Thank you.

ABBREVIATIONS

AI Aromatase inhibitor

AKT Protein kinase B

APC Adenomatous polyposis coli bHLH Basic helix-loop-helixβ

β-TrCP Beta-transducin repeat containing

BRCA1 Breast cancer 1, early onset

CBP CREB binding protein

Cdk Cyclin-dependent kinase

CK5/6 Cytokeratin 5/6

CRB3 Crumbs-3

CSC Cancer stem cell

CYP1A1 Cytochrome P450 family 1, member A1

EGFR Epidermal growth factor receptor (HER1)

EMT Epithelial-to-mesencymal transition

ER Estrogen receptor

ES Embryonic stem

FOXC2 Forkhead box C2

GSK3β Glycogen synthase kinase 3 beta

H3K4 Histone H3 lysine 4

HDAC1/2 Histone deacetylase 1/2

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HDC Histidine decarboxylase

HER2 Human epidermal growth factor receptor 2 (ERRB2, NEU)

HER3 Human epidermal growth factor receptor 3 hNMSC Human normal mammary stem cell

IHC Immunohistochemistry iPSC Induced pluripotent stem cell

KLF4/GKLF/EZF Krüppel-like factor 4 / Gut-enriched krüppel-like factor 4 /

Epithelial zinc finger

LBX1 Ladybird homeobox 1

LGL2 Lethal giant larvae 2

LSD1 Lysine-specific demethylase 1

MAPK Mitogen-activated protein kinase

MDCK Madin-Darby canine kidney cells

MET Mesenchymal-to-epithelial transition miR Micro-RNA

MYC Myelocytomatosis viral oncogene

NF-ĸB Nuclear factor kappa B

Oct-4 Octamer-binding transcription factor 4

PAK1 p21-activated kinase 1

PIAS1 Protein inhibitor of activated STAT-1

PI3K Phosphatidylinositol 3-kinase

PKD1 Protein kinase D1

PR Progesterone receptor

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RAS Rat sarcoma viral oncogene

RTK Receptor tyrosine kinase

RXR Retinoid X receptor

SC Stem cell

SERD Selective estrogen receptor down-regulator

SERM Selective estrogen receptor modulator

SMA Smooth muscle actin

Sox2 (Sex determining region Y)-box 2

Sp-1 Specificity protein 1

TGF-β Transforming growth factor beta

WNT Wingless-type MMTVintegration site family member

ZEB1/2 Zinc finger E-box binding homeobox 1/2

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Discerning The Role Of Krüppel-Like Factor 4 In Breast Cancer

Abstract

JENNIFER L. YORI

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor which plays an integral role in a myriad of cellular processes, ranging from regulation of the cell cycle in modulating proliferation and apoptosis, to exquisite control of differentiation and cell morphology during both development and normal tissue homeostasis, as well as disease progression. The advent of induced pluripotent stem (iPS) cell generation has expanded

KLF4‟s regulatory repertoire to include a functional role in the maintenance and renewal of the stem cell state. Initially discovered in a genetic screen to identify transcription factors involved in growth regulation, KLF4 was first described as gut-enriched Krüppel- like factor (GKLF) and epithelial zinc finger (EZF), as expression was found to be enriched in epithelial versus mesenchymal cells, and those of the gastrointestinal tract.

Subsequent studies have identified diverse regulatory roles for KLF4 in a multitude of cell types and tissues, from the cornea to the cardiovascular system.

The role of KLF4 during diseased states is as diverse as its function during normal development and tissue homeostasis. Predominantly recognized as an anti-proliferative factor, the first implications of its role as a tumor suppressor came from studies demonstrating loss of KLF4 in gastric and colorectal cancers. However, evidence of increased KLF4 expression in squamous cell carcinoma and ductal carcinoma of the

1 breast suggested an additional and opposing oncogenic function for this transcription factor.

To determine the functional role of KLF4 in breast cancer, we first sought to better understand its role in normal mammary epithelial cell biology. Using the non- transformed MCF-10A mammary epithelial cell line, we found that loss of KLF4 resulted in the conversion of these cells from a cuboidal, epithelial morphology to an elongated, fibroblastic phenotype. Additionally, molecular alterations, such as loss of E-cadherin, coupled with increased migration were suggestive of an epithelial-to-mesenchymal transition (EMT). Conversely, overexpression in a highly metastatic tumor line was able to reduce their migratory and invasive capacity, suggesting an inhibitory role for KLF4 in the regulating the metastatic potential of breast cancer cells.

Using two independent mouse models of mammary gland tumorigenesis, we were able to demonstrate that KLF4 expression is lost in HER2/Neu-induced mammary tumors, while overexpression of KLF4 in an orthotopic 4T1 tumor model reduced primary tumor growth as well as inhibited distant metastases. These data lead us to conclude that KLF4 functions as tumor and metastasis suppressor in the breast.

CHAPTER 1

INTRODUCTION, REVIEW OF THE LITERATURE, AND STATEMENT OF

PURPOSE

1.1 Molecular/Histopathological subtypes and cellular origin of breast cancer

Breast cancer is the second leading cause of death in women, and aside from non- melanoma skin cancer, the most common form of cancer among women

(http://www.cdc.gov/cancer/breast/statistics/). It is estimated that in 2010, there will be over 207,000 newly diagnosed cases and 40,000 breast cancer related deaths

(http://www.cancer.gov/cancertopics/types/breast). While early detection and targeted therapies have greatly reduced the incidence of mortality from this disease, the prognosis for women with locally advanced and metastatic disease remains poor.

Breast cancer is a heterogeneous disease as evidenced by distinct biological features and clinical behaviors that cannot be understood based solely on traditional histopathological tumor assessment (Weigelt and Reis-Filho, 2009). In the past decade, seminal work by several groups has led to the molecular classification of breast cancer subtypes (Perou et al., 2000; Sorlie et al., 2001). Perou and colleagues used microarray analysis of a small number of invasive breast cancers to define a list of close to 500 “intrinsic genes” whose expression varied most among tumors from different patients (Perou et al., 2000).

Hierarchical clustering of these genes revealed the existence of four molecular subtypes including basal, human epidermal growth factor receptor (HER2) positive, normal breast- like and luminal. Subsequent work further divided the luminal group into luminal A and

3 luminal B based on expression differences in proliferation-related genes (Sorlie et al.,

2001). More recently, additional subtypes have been described to identify tumors enriched for stem-like cells or cancer stem cells (Herschkowitz et al., 2007; Prat et al.,

2010). Of clinical importance, these different molecular subtypes have prognostic significance. This has led to the development of multiple prognostic gene signature tests being used to predict the likelihood of relapse, including the 70-gene prognostic signature

(MammaPrint), the Oncotype DX, and the Genomic Grade Index (GGI) (Ma et al., 2004;

Paik et al., 2004; Sotiriou et al., 2006; van 't Veer et al., 2002; Wang et al., 2005).

However, widespread use of gene-expression profiling in the clinical setting remains limited. Traditionally, diagnosis and treatment involves identification of pathological parameters including tumor size, lymph node status, endocrine receptor status and HER2 status, which are used as prognostic predictors and adjuvant therapy determinants. In an effort to correlate these clinical parameters with gene-expression driven subtypes, five immunohistochemical (IHC) markers including estrogen receptor (ER), progesterone receptor (PR), HER2, cytokeratin 5/6 (CK5/6) and epidermal growth factor receptor

(EGFR) have been used as surrogates to define the different subtypes (Blows et al.,

2010). Furthermore, it has been demonstrated that the IHC marker defined phenotypes are associated with clinical characteristics similar to those seen in studies using expression array defined subtypes, and are able to identify distinct biological characteristics that are associated with differences in short and long-term outcomes

(Carey et al., 2006; Tamimi et al., 2008; Yang et al., 2007) (Figure 1.1). The comparison of genomic assays and use of old and new molecular markers in patient treatment and/or selection for clinical trials remains challenging, however consensus recommendations

4 have recently been made regarding the incorporation of these parameters in breast cancer management (Kaufmann and Pusztai, 2010).

1.1.1 Luminal breast cancer

The luminal subtype(s) account for roughly 70% of all breast cancers (Bhargava et al.,

2009). These cancers are estrogen receptor (ER) or progesterone receptor (PR) positive, and often have a higher expression level of genes that are generally co-expressed with

ER. Luminal A cancers have the lowest proliferation indices of all subtypes and are usually low grade, resulting in the best prognosis (Sorlie et al., 2001). Luminal B tumors tend to be of a higher grade, and more proliferative, while several studies have suggested, based on IHC definitions of molecular subtypes, that these tumors, in addition to ER or

PR, are often HER2 positive (Carey et al., 2006; Cheang et al., 2009; Sihto et al., 2008;

Tamimi et al., 2008; Yang et al., 2007). Luminal type breast cancers are most commonly treated with estrogen receptor targeted therapies including selective estrogen receptor modulators (SERMs), selective estrogen receptor down-regulators (SERDs) and aromatase inhibitors (AIs). Tamoxifen is a SERM that has proven effective in treating most luminal subtype breast cancers. Results from several studies suggest that SERMs, such as tamoxifen and raloxifene may be beneficial as chemopreventative treatments in women with certain breast cancer risk factors (Vogel et al., 2010). In addition, AIs are currently being used as adjuvant hormone therapy to help prevent breast cancer recurrence, and studies looking at aromatase inhibitors such as anastrozole and letrozole are being conducted to determine if they can reduce the risk of developing breast cancer

5 in post-menopausal women (Dunn and Ryan, 2009; Howell, 2008). Although SERMs and AIs lower the risk of recurrence for several years post-treatment, more than half of all recurrences occur 6 to 15 years after diagnosis ((EBCTCG), 2005), thus late recurrence remains the major clinical challenge in hormone positive breast cancers.

1.1.2 HER2/ERRB2 breast cancer

HER2, also known as erythroblastic leukemia viral oncogene homolog 2 (ERBB2), is a member of the EGFR family of tyrosine kinases. Amplification of the HER2 gene or overexpression of its protein product in breast tumors accounts for approximately 25-

30% of all breast cancer cases (Slamon et al., 1989). HER2 tumors are associated with poor prognostic factors, including larger tumor size, higher grade and nodal involvement

(Carey et al., 2006; Tamimi et al., 2008). Correlations between HER2 positivity, morphological features such as apocrine differentiation and expression of basal phenotypic markers, including CK5/6 and EGFR have been shown (Bhargava et al.,

2010). HER2 tumors have been described as hormone receptor negative, however, as noted above, a subset of HER2 positive tumors with ER positivity are considered to be of the luminal B subtype. Trastuzumab (Herceptin, Genentech), a monoclonal antibody that targets the HER2 extracellular domain, has led to improved outcomes, including overall survival, as an adjuvant treatment of metastatic breast cancer (Murphy and Fornier,

2010). However, greater than 60% of patients with HER2+ cancers do not respond to trastuzumab monotherapy, and most initial responders develop resistance within 1 year

(Esteva et al., 2002). Several mechanisms of Herceptin resistance include: (a) the

6 inability of Herceptin to bind to a constitutively active truncated form of the HER2 receptor that is found in up to 60% of HER2+ tumors (Scaltriti et al., 2007) as well as epitope masking by Mucin 4 or CD44/hyaluronan complex (Carraway et al., 2001); (b) alternate signaling through the insulin-like growth factor-1 receptor (Lu et al., 2001),

EGFR/HER3 heterodimers (Narayan et al., 2009), or upregulation of other receptors including c-Met (Shattuck et al., 2008) and β-integrins (Yang et al., 2010); (c) upregulation of downstream signaling pathways (Nagata et al., 2004); and (d) failure to trigger immune-mediated responses (Scaltriti et al., 2009).

Since the introduction of Herceptin as adjuvant treatment for HER2 positive breast cancer, the prognostic stratification of breast tumor types has changed, from luminal A having the best prognosis, to luminal B now having a decreased risk of distant recurrence when compared with luminal A tumors (Dawood et al., 2010). These data suggest that

HER2+/ER+ tumors have a better response to HER2 inhibition than do HER2+/ER- tumors. The implicit contribution of ER positivity to these findings was recently examined by Loi and colleagues. Preliminary data presented at the 2010 IMPAKT Breast

Cancer Conference suggests HER2+/ER+ tumors preferentially activate a different set of biologic pathways (PI3K/AKT) compared with HER2+/ER- (EGFR/RAS/MAPK) breast cancers (Loi, 2010). These findings have several clinical implications. First, antiestrogen therapy often overlaps Herceptin treatment in the postoperative adjuvant setting, which may be counter-indicated for women with double positive tumors.

Second, while Herceptin blocks the PI3K/AKT pathway, combinatorial therapy with an

EGFR/RAS/MAPK inhibitor such as lapatinib (Tykerb, GlaxoSmithKline) may be

7 indicated for HER2+/ER- tumors. Recently it has been shown in vitro that activated

PI3K/AKT signaling in HER2-amplified cell lines confers resistance to Herceptin but not

Tykerb (O'Brien et al., 2010). Interestingly, all the HER2-amplified resistant lines were also ER negative. While Herceptin has been the most widely used and successful therapy in treating HER2 positive breast cancers, an improved understanding of the mechanisms of resistance to this drug have led to the discovery of multiple valid therapeutic targets that may be used in combination with or in place of Herceptin as adjuvant therapy for this disease subset.

1.1.3 Basal-like breast cancer

Basal-like cancers, which account for up to 15% of all breast cancers, are a group of ER- negative tumors named for the consistent expression of genes found in normal basal/myoepithelial cells of the breast. While the majority of basal-like breast cancers have a triple negative phenotype (ER-negative, PR-negative, HER2-negative), the two are not synonymous. However characteristic similarities include the fact that they both more frequently affect younger women and are more prevalent in African-American women (Dent et al., 2007; Morris et al., 2007). Basal-like breast cancers have been defined through microarray-based expression profiling and surrogate markers, such as the triple negative phenotype and expression of high molecular weight cytokeratins (CKs) including CK5/6, 14 and 17, however, as a group, basal-like tumors are very heterogeneous, having distinctive clinical presentations, histological features, responses to chemotherapy and outcomes (Carey et al., 2007; Carey et al., 2006; Livasy et al., 2006;

Sorlie et al., 2001). The aggressive clinical behavior of these tumors is evidenced by their high propensity for metastatic spread, however, unlike non-basal-like cancers which frequently disseminate to axillary nodes and bones, basal-like cancers favor haematogenous spread, with a proclivity for metastasis to the brain and lungs (Fulford et al., 2007; Tsuda et al., 2000).

While breast cancer is commonly a result of dysregulated or aberrant endocrine signaling, or gene amplification, as is the case with HER2 positive tumors, genetic factors can also influence tumor type. Tumors arising in carriers of germ-line mutation in the breast cancer susceptibility gene BRCA1 are commonly diagnosed with basal breast tumors which are ER, PR and HER2 negative (Sorlie et al., 2003). In fact, the majority of basal- like breast cancers show a dysfunctional BRCA1 pathway (Turner and Reis-Filho, 2006).

However, sporadic cases differ by the lack of BRCA1 somatic mutations, suggesting epigenetic mechanisms of downregulation (Esteller et al., 2000; Turner et al., 2007).

Sporadic cases are also more likely to be luminal in nature (Turner et al., 2004).

Recently it has been proposed that BRCA1 acts as a stem-cell regulator and that basal- type cancers originate from stem cells that have lost their ability to differentiate (Dontu et al., 2004; Foulkes, 2004). Support for this notion comes from Wicha and colleagues, who demonstrate that loss of BRCA1 may limit the differentiation potential of mammary stem cells and prevent the formation of ER-positive luminal cells (Liu et al., 2008b). The role of stem/cancer stem cells in breast cancer will be discussed further in section 1.2.2.

1.1.4 Comparison of mouse models and human tumors generate a new molecular subtype

Currently there are numerous murine models of breast carcinoma that recapitulate many properties of human cancers (Van Dyke and Jacks, 2002), however it is not always clear which features of a human cancer are most relevant for disease comparison in the mouse.

Furthermore, is unknown as to the extent to which these models faithfully represent clinically significant human phenotypes of breast cancer. To address this issue, a recent study was undertaken in which gene expression analysis was used to classify a large set of human breast tumors and mouse mammary tumors, providing some biologically and clinically significant shared features between the two species. Of significance, these studies resulted in the recent subclassification of a new molecular subtype know as the claudin-low subtype (Herschkowitz et al., 2007). Clinical presentation of these tumors is most similar to the triple-negative breast cancers, while molecular characterization reveals that these tumors are enriched in epithelial-to-mesenchymal transition (EMT) features and stem like features, have low expression of luminal and proliferation- associated genes and display a breast cancer differentiation hierarchy that resembles the normal epithelial mammary developmental cascade (Prat et al., 2010). Interestingly, claudins have been implicated in the induction of KLF4 during formation of the epidermal permeability barrier (Turksen and Troy, 2002). It is therefore tempting to speculate, in light of the EMT-characteristics of claudin-low tumors, in conjunction with our findings supporting an inhibitory role for KFL4 during EMT (Yori et al., 2010a), that loss of KLF4 may be associated with the claudin-low subtype.

It can be argued that the ability to identify cancer subgroups based on gene expression profiling has not provided significant advances over current clinico-pathological practice

(Sotiriou and Piccart, 2007). Indeed, it has been shown that ER signaling, ERBB2 amplification and proliferation are the three key biological drivers in at least nine different prognostic signatures derived from expression data (Wirapati et al., 2008).

Furthermore, when histological data are compared with molecular data various discrepancies are evident. For example, as mentioned above, basal-like tumors can be defined based on expression of certain cytokeratins, however it has been shown that these cytokeratins are present in both luminal and myoepithelial cells of the breast (Gusterson,

2009). This begs the question, are luminal tumors derived from luminal cells and basal- like tumors from basal/myoepithelial cells as molecular profiling nomenclature would suggest? In fact, the cellular origins of breast cancer are currently quite unclear, though it has been established that most breast cancers arise in the terminal duct lobular units of the mammary gland, rather than the luminal and myopeithelal cells that line the ducts

(Wellings and Jensen, 1973). It has been difficult to reconcile the simple cytoarchitecture of the mammary gland, comprised of primarily luminal epithelial and myoepithelial cells, with the highly heterogeneous and diverse breast cancer phenotypes, including differences in histopathologies and clinical behavior, leading to much debate regarding the cellular origin of the different breast cancer subtypes (Pece et al., 2010; Stingl and

Caldas, 2007), (Figure 1.1). In general, the phenotypic diversity of breast tumors has been attributed to the subtype-specific genetic and epigenetic alteration, however, it has also been suggested that this heterogeneity is a result of tumors arising from a variety of distinct normal epithelial cell types (Bocker et al., 2002; Dontu et al., 2003b; Welm et al.,

2003). Whether tumor heterogeneity reflects differences in the target cell population, differences in oncogene activation and loss of tumor suppressor function in these cells or perhaps dysfunction in a common mammary stem cell remains to be determined.

1.2 Metastasis, epithelial-to mesenchymal transition (EMT) and the cancer stem cell theory

The ability of cells to first form a tumor and subsequently metastasize to distant organs is a complex multistep process (Nguyen et al., 2009). Tumor initiation is often triggered by a genetic mutation(s), coupled with the accumulation of subsequent transforming events that lead to dysregulated and aberrant cell growth. Once established, tumor recruitment of blood vessels and evasion of immune surveillance permits a rich microenvironment in which these cells can survive. These processes also provide a portal which facilitates the dissemination of tumor cells throughout the body. First a tumor cell must acquire the ability to migrate away from the tumor and extravasate into the local vasculature or lymph system. The next step requires cells to exit the circulation through intravasation into a distant tissue that the tumor cell has somehow deemed amenable for survival.

Once there, tumor cells may remain dormant for years before an autonomous signals from the cell, or paracrine communication from the distant organ elicits a “regrowth” program, and the development of a metastasis (see review (Nguyen et al., 2009). It has been proposed that this reinitiation step requires the tumor cell to either be a stem-like or progenitor cell, or have acquired self-renewal properties (Gupta et al., 2007), and recently

12 it has been suggested that the acquisition of this stem-like phenotype is a result of an epithelial-to-mesenchymal transition (Mani et al., 2008).

1.2.1 The epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT) describes a series of molecular and morphologic changes that occur in epithelial cells, resulting in decreased cell adhesion and altered polarity [due to loss of cell junctional proteins (epithelial cadherins/adherens junction proteins), tight junction and desmosomal proteins, lateral integrins and cytoskeletal components (cytokeratins)], increased motility (reflected in altered actin organization from cortical bundles into stress fibrers), and resistance to anoikis

(Stockinger et al., 2001; Valdes et al., 2002). First described and defined in the developmental context of heart morphogenesis (Bolender and Markwald, 1979), and later with the first three-dimensional model study in culture (Trelstad et al., 1982), EMT has long been recognized as a crucial process in development, as early as gastrulation

(Trelstad et al., 1967). EMT is a transient and reversible process. While it is important, for example, in formation of the mesoderm and derivation of neural crest cells (Gammill and Bronner-Fraser, 2003), the mesenchymal cells of the mesoderm eventually give rise to epithelial organs by undergoing mesenchymal-to-epithelial transition (MET) (Davies,

1996), the reverse of EMT.

More recently, the relevance of EMT and tumor progression became apparent as many of the genes which were discovered to be activated (or suppressed) during tumorigenesis

13 and metastasis were also know to be modulated during development (Berx et al., 2007;

Thiery and Sleeman, 2006). While an EMT-like phenotype has often been observed in carcinoma, the idea that EMT occurs in vivo has met with resistance, particularly by pathologists, citing absence of direct clinical evidence. A limitation of clinical studies in determining the contribution of EMT to cancer progression is the difficulty in ascertaining whether an undifferentiated phenotype is a result of an active EMT, or reflects a lack of differentiation. Some examples of breast cancers believed to have some component of EMT include rare carcinosarcomas and metaplastic carcinomas that have both an epithelial compartment and a mesenchymal compartment. Cytogenetic studies indicate that these two compartments originate from a common precursor cell population that undergoes EMT, giving rise to the mesenchymal component (Zhuang et al., 1997).

Infiltrating lobular carcinomas account for 10-15% of invasive ductal carcinomas and are characterized by the lack of E-cadherin expression (Berx et al., 1996; Moll et al., 1993), a hallmark of EMT. They also express high levels of a classic EMT master regulator,

Twist (Yang et al., 2004). Interestingly, these tumors maintain expression of epithelial cytokeratins, suggesting a case of partial EMT. Basal-like carcinomas often have increased activation of several pathways that are also activated during EMT, including the oncogenic cMyc pathway, which has been shown to activate the Snail/GSK induction of EMT (Cho et al., 2010). Furthermore, a number of studies have linked the overexpression of Snail family members in breast carcinoma to tumor aggressiveness

(Storci et al., 2008). Snail has also been shown to mediate tumor recurrence in a mouse model of HER2/ERBB2/NEU-induced mammary tumorigenesis (Moody et al., 2005), supporting a role for EMT-mediated resistance of HER2 breast cancers to Herceptin

(Ostler et al., 2008; Wang et al., 2008b). While the majority of these finding are correlative, only recently has direct in vivo evidence for EMT in breast cancer emerged

(Trimboli et al., 2008). In order to directly visualize EMT during cancer progression,

Trimboli et al. used genetically marked tumor epithelial and stromal cells in three different mouse models of breast cancer to determine their fate during tumor progression.

Their findings revealed that stromal fibroblasts associated with Myc-induced tumors were cells of epithelial origin that had undergone EMT.

1.2.1.1 Transcriptional regulation of EMT

Both in vitro and in vivo model systems have led to the characterization of five major

“EMT pathways” that have been found to trigger EMT-associated processes including receptor tyrosine kinases (RTKs), integrins, WNT, NF-ĸB, and TGF-β pathways

(Klymkowsky and Savagner, 2009). These EMT-inducing signals exert their action through repression of epithelial genes, while activating transcriptional cascades that specify motility and invasion (Peinado et al., 2007; Thiery and Sleeman, 2006). Most of the present knowledge regarding the regulation of EMT comes from mechanistic studies examining the loss of E-cadherin, one of the earliest steps in the EMT cascade. Many transcription factors that drive the process of EMT in both embryonic and cancer cells directly repress the adherens junction protein E-cadherin, resulting in decreased cell-cell adhesion. These transcriptional repressors include the Snail superfamily including

SNAI1 (Snail), SNAI2 (Slug), the bHLH family (E47/TCF3 and Twist), two ZEB factors,

ZEB1 (δEF1/TCF8) and ZEB2 (SIP1) (Peinado et al., 2007), and FOXC2 (Mani et al.,

2007). These factors share a similar mechanism of repression by binding to conserved E- box sequences in the proximal promoter regions of E-cadherin and other epithelial genes

(Peinado et al., 2007; Peinado et al., 2004). Snail and ZEB factors can downregulate several tight junction proteins including occludin and members of the claudin family

(Ikenouchi et al., 2003; Vandewalle et al., 2005), while MDCK cells induced to undergo

EMT have reduced expression of claudin-4 (De Craene et al., 2005). ZEB1 silencing in undifferentiated breast carcinoma cells results in upregulation of several core polarity components, including CRB3 (Crumbs3) and PATJ (Aigner et al., 2007). Snail also targets several polarity genes, including CRB3 and LGL2, however, in contrast to ZEB1, which targets the proximal E-box elements of the CRB3 promoter, Snail preferentially acts through distal E-box sequences (Aigner et al., 2007; Spaderna et al., 2008;

Whiteman et al., 2008).

In addition to direct transcriptional regulation of E-cadherin, a majority of E-cadherin- negative tumors result from silencing due to promoter hypermethylation, with increased

E-cadherin expression at metastatic sites compared to the primary tumor, suggesting a mesenchymal-to-epithelial transition (MET) (Chao et al., 2010; Kowalski et al., 2003;

Yates et al., 2007). Furthermore, many tumor suppressor genes are frequently methylated and thus silenced in breast cancer lymph node metastases (Feng et al., 2010). Given the transient nature of EMT during development and the phenotypic plasticity of cancer cells during metastasis, an epigenetic component to the regulation of this differentiation program has been suggested (Feinberg, 2007). Recently it has been shown that Snail mediates the demethylation of H3K4 and deacetylation of histone H3 and H4 through

LSD1 and the sin3A-HDAC1/2 complex respectively, to control gene expression of E- cadherin by altering chromatin structure (Lin et al., 2010). The manner in which these

Snail-modulated histone modifications contribute to E-cadherin promoter methylation remains to be determined.

A plethora of signaling pathways have been shown to converge on Snail/Slug both during development and in tumor cells, including the five “EMT pathways” referenced earlier in this section (Moustakas and Heldin, 2007; Thiery, 2003; Thiery and Sleeman, 2006).

Recently, a positive role for Myc in TGF-β-induced Snail transcription and EMT has been demonstrated (Smith et al., 2009), providing mechanistic insight into the in vivo

EMT seen in Myc-induced mammary tumors (Trimboli et al., 2008). LBX1, a developmentally regulated inducer of EMT during myogenesis and neurogenesis, has been shown to be upregulated in triple-negative breast cancers and overexpression of

LBX1 in mammary epithelial cells in vitro leads to morphologic transformation and enhanced migration through upregulation of ZEB1, ZEB2, Snail and TGFB2 (Yu et al.,

2009).

In addition to transcriptional regulation, post-translational mechanisms affecting protein stability and nuclear/cytoplasmic location have emerged as crucial regulators of various

EMT-inducers. One such example is the GSK3β-regulated phosphorylation of Snail

(Dominguez et al., 2003; Zhou et al., 2004). In the un-phosphorylated state, Snail resides in the nucleus, where it is transcriptionally active. Phosphorylation at one of two consensus motifs results in translocation to the cytoplasm, where a second

17 phosphorylation event targets it for β-TrCP-mediated ubiquitination and degradation.

Interestingly, GSK3β inhibition of NFĸB, a transcriptional activator of Snail, is another mechanism whereby this ubiquitously expressed kinase can suppress Snail expression and EMT in epithelial cells (Bachelder et al., 2005). Recently it has been shown that

Snail phosphorylation at Ser11 by another serine/threonine kinase, protein kinase D1

(PKD1, originally described as PKCμ), which is downregulated in advanced prostate, breast, and gastric cancers, is a potential mechanism by which PKD1 inhibits tumor growth and metastasis in a tumor xenograft model of prostate cancer (Du et al., 2010a).

Conversely, p21-activated kinase 1 (PAK1) phosphorylation of Snail at Ser246 increases its repression activity by sequestering it in the nucleus (Yang et al., 2005b).

The emergence of regulatory feedback loops between microRNAs (miRs) and EMT factors represents a novel post-transcriptional mechanism controlling EMT and the acquisition of a stem-like phenotype. MicroRNAs are short non-coding RNAs that control gene expression by pairing to complementary sequences in the 3‟-UTR region of target mRNAs (He and Hannon, 2004), leading to degradation and/or translational inhibition (Filipowicz et al., 2008). Modulation of the E-cadherin repressors ZEB1/ZEB2 by five members of the miR-200 family and miR-205 RNA have been recently reported to prevent the induction of EMT (Christoffersen et al., 2007; Gregory et al., 2008;

Hurteau et al., 2009; Korpal et al., 2008; Park et al., 2008). In agreement with a proposed function in regulating EMT, these miRs are downregulated in response to EMT stimuli, including transcriptional repression by ZEB1 and Snail (Burk et al., 2008). Furthermore,

ZEB1 has been shown to promote tumorigenicity by repressing stemness-inhibiting miRs

18 that target the stem-like factors Sox2 and KLF4 (Wellner et al., 2009), supporting a role for EMT in the generation of cells with stem-like characteristics. The relationship between EMT and stem cells will be further discussed in section 1.2.2.2. Interestingly, expression of the miR-200 family of RNAs is lost in invasive breast cancer cells and metaplastic breast carcinomas (Gregory et al., 2008). While these are just a sampling of recent, significant advances that have contributed to a mechanistic understanding of

EMT, many questions remain regarding the role of EMT in the establishment of metastasis, tumor recurrence, and the cancer stem cell phenotype.

1.2.2 The cancer stem cell theory

The resemblance between stem cells (SCs) and cancer was first noted in 1875 by

Cohnheim who hypothesized that tumors were formed from „misplaced‟ stem cells during embryonic development (Cohnheim, 1875). The first evidence supporting this hypothesis came nearly 100 years later with the discovery that a single leukemic tumor cell could generate a completely new tumor (Bruce and Van Der Gaag, 1963; Kleinsmith and Pierce, 1964; Makino, 1956; Park et al., 1971). Today, the „stem cell origin of cancer‟ hypothesis maintains that stem cells or cells having acquired the ability to self renew are the cells that give rise to tumors, through the accumulation of genetic changes and escape from environmental controls (Shipitsin and Polyak, 2008). The cancer stem cell (CSC) hypothesis postulates that within a differentiated tumor resides a population of cells with stem-like features that gave rise to the tumor (Dalerba et al., 2007; Shipitsin and Polyak, 2008; Wicha et al., 2006). Currently, it is well established that cancer cells

19 with stem-like features exist in the majority of tumor types, and cancer is frequently characterized by alterations in pathways that control the homeostasis of normal stem cells

(Visvader and Lindeman, 2008). Recent evidence suggests that both hereditary (BRCA1) and sporadic (HER2 amplification) breast cancers may result from dysregulation of pathways involved in stem cell self-renewal (Korkaya et al., 2008; Liu et al., 2008b).

Furthermore, the hypothesis that CSCs or tumor initiating cells are intrinsically resistant to therapy is supported by preliminary data from a neoadjuvant lapatinib study on HER2- positive tumors (Li et al., 2008). Tumors from the group receiving chemotherapy treatment had an increase in the percentage of TICs, while the lapatinib treated group did not, suggesting that TICs in these cancer may be targeted by anti-HER2 therapy.

Addition support comes from another study indicating that stem cells are enriched in tumors treated with anti-estrogens and conventional chemotherapy(Creighton et al.,

2009). The isolation and transcriptional characterization of human normal mammary stem cells (hNMSCs) (Pece et al., 2010), has provided an hNMSC gene signature which was used to identify poorly differentiated, aggressive breast tumors as having a higher content of CSCs. More importantly, their findings suggest that the biological and molecular heterogeneity of breast cancers (i.e., different sub-types) can be correlated with their CSC content. However, the question as to the origin of tumor initiating cells remains. While one possibility is that CSCs are derived from malignant transformation of a terminally differentiated cell, resulting in dedifferentiation to a primitive stem cell- like state, it is equally likely that CSCs arise from existing stem cells, or progenitor cells that have reacquired self-renewal capabilities, as such is the case with induced pluripotent stem (iPS) cells (Meissner et al., 2007; Takahashi and Yamanaka, 2006).

1.2.2.1 Cancer stem cells and EMT

There is growing body of evidence suggesting an association among EMT induction, the emergence of CSCs and drug resistance that has gained great momentum over the past few years (Singh and Settleman, 2010). Prior to the 2008 landmark study by Weinberg and colleagues, empirically connecting EMT to the emergence of stem cells (Mani et al.,

2008), several observations had indicated such a relationship existed. For example, during tumor metastasis, a process often enabled by EMT (Thiery et al., 2009), disseminated tumor cells would seem to require self-renewal capability once they have reached the metastatic site. Indeed metastasis in and of itself is similar to those processes which occur during tissue repair and regeneration, where stem cells must exit the bone marrow and travel to secondary sites to participate in tissue reconstruction (Kondo et al.,

2003). Furthermore, a number of pleiotropically acting transcription factors which are involved in the process of EMT during embryogenesis have also been found to confer malignant traits on neoplastic cells (Briegel, 2006). These observations led to the discovery that immortalized human mammary epithelial cells, when induced to undergo

EMT, not only acquired mesenchymal traits previously associated with EMT, but also the expression of stem-cell markers (Mani et al., 2008). Of clinical significance, it has been shown that after a tumor initiated immune response, recruited CD8+ T-cells induce dedifferentiation of breast cancer cells, resulting in the formation of CD44high, CD24low stem-like cells (Santisteban et al., 2009).

A major driving force in many types of physiological and pathophysiological EMT is the

TGF-β signaling pathway (Thiery et al., 2009). TGF-β signals through its cognate, dual- specificity receptors, triggering a cascade of signaling events that activates a number of downstream effector proteins, most notably the Smad family of transcription factors. A potent growth suppressor and mediator of apoptosis during early stages of tumor progression, TGF-β switches to a pro-proliferative, pro-metastatic factor during late stages of tumorigenesis. It has been suggested that the upregulation of antiapoptotic signaling pathways in human cancers, notably those involving PI3K/AKT, provides a permissive context for the metastasis-inducing properties of TGF-β without the accompanying cell death (Conery et al., 2004; Gal et al., 2008). Association of the TGF-

β-mediating Smads with Zeb proteins during suppression of E-cadherin, together with an essential role for Zeb2 (SIP1) in human embryonic stem cell maintenance implicates

TGF-β-signaling in EMT-induced „stemness‟, and is further supported by the strong expression of a TGF-β signature in metastatic breast CSCs (Shipitsin et al., 2007).

Additionally, classical EMT-activators such as ZEB1 have been shown to promote tumorigenicity through repression of stemness-inhibiting miRs (Wellner et al., 2009).

This seems to contradict the recent findings that mesenchymal-to-epithelial transition

(MET), the reverse of EMT, is a critical initial step towards pluripotency (Li et al.,

2010b; Samavarchi-Tehrani et al., 2010) in the generation of iPS cells. However, CSCs, which are defined by their ability to seed new tumors and self-renew have not been shown to be pluripotent as is the case with SCs and iPS cells (Gupta et al., 2009; Polyak and Weinberg, 2009).

1.2.2.2 iPS cells

The inner cell mass of a blastocyst stage embryo, also known as embryonic stem (ES) cells, are capable of differentiating into any cell type, making them a most promising resource for the treatment of disease, and regenerative medicine. However, inherent bioethical issues, coupled with the risk of immune rejection prompted scientists to find alternative means of generating pluripotent cells from differentiated adult cells. In 2006, the Japanese group led by Yamanaka, were the first to successfully reprogram adult fibroblasts into induced pluripotent stem (iPS) cells (Takahashi and Yamanaka, 2006), through the forced expression of four transcription factors including Sox2, Klf4, Oct4 and

Myc. Known today as the “Yamanaka factors” or SKOM, subsequent generation of iPS cells from multiple groups and multiple cell types have demonstrated the pluripotency of these cells through the regeneration of various tissues and whole animals. In addition to the original four factors, other factors such as Nanog and Lin-28 (Yu et al., 2007) have been used to drive iPS cell selection. Furthermore, it has been shown that a core

Krüppel-like factor circuitry, involving KLF2, KLF4 and KLF5 regulates self-renewal of embryonic stem cells (Jiang et al., 2008a), and that the KLFs and the Oct4/Sox2/Nanog network are strongly interconnected (Bourillot and Savatier, 2010). Currently, the therapeutic utility for which iPS cells were initially created is impeded by the fact that all of the reprogramming factors are also oncogenes in certain contexts. Furthermore, it has been suggested that tissue specific stem/progenitor cells present in the somatic tissue are the source of iPS cells, as hard evidence proving the induction or generation of iPS cells from a truly differentiated adult cell is lacking (Liu, 2008). While several studies have

23 attempted to address the question as to the origin of iPS cells, currently no apparent consensus has been reached.

iPS cells offer a unique experimental system in which key questions regarding cell fate determination, epigenetic regulation and the mechanism of self-renewal can be investigated, yet the inefficient, stochastic process in which these cells are generated is not clearly understood. Earlier this year, two studies from different groups revealed that each exogenous reprogramming factor (SKOM) plays a critical role in activating an epithelial program by shutting down key mesenchymal genes to overcome the EMT epigenetic barrier (Li et al., 2010b; Samavarchi-Tehrani et al., 2010). The findings that a mesenchymal-to-epithelial transition (MET) is required to initiate nuclear reprogramming and self-renewal, in contrast to the apparent EMT-induced “stemness” of tumor cells will certainly provide a platform for much discussion regarding the relationship between these two processes among the cancer and stem cell communities.

1.3 Krüppel-like factor 4 (KLF4)

1.3.1 The Sp1/Krüppel-like factor family

Gene transcription is a highly complex and intricately orchestrated process, regulated in large part through transcription factors which bind regulatory sequences in the promoters of genes to tightly regulate expression and control nearly every cellular process. A common recurring set of motifs in both promoters and more distal regulatory elements

24 are known as GC and GT boxes. Identified in the early 1980‟s, Specificity Protein 1

(Sp1), one of the first mammalian transcription factors to be cloned, was shown to recognize and bind to GC-rich sites within promoters via three C-terminal Cys2His2 zinc finger motifs. The identification of a similar binding motif in the Drosophila segmentation gene product Krüppel (Kadonaga et al., 1987) led to the discovery of an extended Sp1/Krüppel-like factor (KLF) family consisting of twenty-five evolutionarily conserved zinc finger transcription factors (Simmen et al., 2010). Overall, the amino- acid similarity among the highly conserved C-terminal DNA binding domain of the

Sp1/KLF family members is greater than 65% (Kaczynski et al., 2003). Not surprisingly, many of these factors have similar DNA-binding affinities for different GC-rich sites, in addition to competition for the same binding sites (Cook et al., 1998; Hagen et al., 1992;

Kaczynski et al., 2001; Sogawa et al., 1993; Turner and Crossley, 1999; Zhang et al.,

1998). While most KLFs are ubiquitously expressed, it appears the highly variable amino terminus accounts for functional specificity in gene regulation and interactions with other transcription factors (Bieker, 2001; Suske et al., 2005). One of the first KLF factors to be described was erythroid KLF (EKLF) (Miller and Bieker, 1993), with subsequent factors being designated as xKLF, where “x” denotes the tissue in which the gene was first identified. However, a single nomenclature system in which KLF‟s are named in order of discovery (KLF1, KLF2, etc.) has more recently been adopted.

1.3.2 Krüppel-like factor 4 (KLF4)

1.3.2.1 Structure and regulation

Krüppel-like factor 4 (KLF4) was independently cloned from the mouse by two separate groups and named as gut-enriched Krüppel-like factor (GKLF) (Shields et al., 1996) and epithelial zinc finger (EZF) (Garrett-Sinha et al., 1996), due to its high expression in the intestine and skin epithelium, respectively. Since then, its expression has been detected in a variety of tissues including the lung (Shields et al., 1996), thymus (Panigada et al.,

1999), cornea (Chiambaretta et al., 2004), testis (Behr and Kaestner, 2002), and many others. The human KLF4 gene, which is located at chromosome 9q31.2, consists of 5 exons resulting in a transcript size of approximately 3.5 kilobases, and an open reading frame which encodes a 470-amino acid protein of approximately 55 kDa (Yet et al.,

1998), (Figure 1.2). However, it appears that at least 7 transcripts, consisting of 5 protein encoding splice variants currently exist (ENSEMBL and ACEVIEW databases).

Support of these predicted variants comes from the identification of 5 characterized isoforms in pancreatic cancer cells (Wei et al., 2010). The identification of 4 functional polyadenylation sites further increases transcript diversity (Godmann et al., 2005).

At the protein level, the appearance of two in-frame ATG codons located in the second exon allow for the possibility of a 479-amino acid product. It is currently not clear if or how these two different translational products, which differ by 9 amino acids, are regulated. A longer variant has been identified as the canonical sequence in the

UniProKB database, with the inclusion of an additional 34 amino acids located near the zinc finger region. The existence of both the 479 and 513-amino acid products is based on several large scale cDNA nucleotide sequence analysis studies (Gerhard et al., 2004;

Ota et al., 2004). The structure of the full length KLF4 protein conforms to the characteristic three domains common to all of the KLF family members, including a highly variable N-terminal activation domain, a C-terminal region containing the three highly conserved zing fingers, and a nuclear localization signal region adjacent to and included in the zinc finger DNA-binding domain (Shields and Yang, 1997). Other features include a transcriptional activation domain and a PEST sequence which likely contributes to its short (~2 hour) half life (Chen et al., 2005). In addition to post- translational ubiquitin-modification which regulates its degradation, several groups have identified other sites of modification, resulting in sumoylation-regulated transcriptional activity and expression (Du et al., 2010b; Kawai-Kowase et al., 2009) and phosphorylation-mediated transcriptional complex formation (Li et al., 2010a).

Acetylation of KLF4 can alter its transactivation potential both directly, by altering binding activity (Meng et al., 2009) and indirectly, through its ability to modify histone acetylation and chromatin structure via interactions with histone acetyltransferases

(Evans et al., 2007). The majority of known factors and conditions that modulate KLF4 expression including its own autoregulation (Mahatan et al., 1999) are listed in Table 1.1 and many of them will be discussed in subsequent sections.

1.3.2.2 Mechanisms of activation and repression

KLF4 is a pleiotropic transcription factor, acting as both a transcriptional activator and repressor. The N-terminus of KLF4 contains a strong transactivation domain defined by several clusters of acidic residues (Geiman et al., 2000), however the existence of a central repressive domain (Yet et al., 1998), and the ability of KLF4 to compete with other transcription factors for DNA-binding suggests mechanisms of both active and passive repression for this classically defined transcriptional activator. In some instances,

KLF4 and Sp1 synergistically activate gene transcription (Blanchon et al., 2006;

Brembeck and Rustgi, 2000; Higaki et al., 2002), however several genes containing overlapping Sp1 and KLF4 recognition sequences, including CYP1A1, HDC, and Sp1, are repressed as a direct result of KLF4 displacement of Sp1 from the target gene‟s promoter (Ai et al., 2004; Kanai et al., 2006; Zhang et al., 1998).

The transactivation potential of KLF4 appears to be dependent in large part on its interactions with other transcriptional coactivators, including those that act on chromatin.

One such example is the N-terminal interaction of KLF4with the transcriptional coactivators p300/CBP which is required for the transactivation function of KLF4 (Evans and Liu, 2006; Geiman et al., 2000). These histone acetyltransferases are permissive for gene transcription as they increase histone acetylation, converting chromatin from a state in which it is relatively inaccessible to the core basal transcription machinery to an open state that is more accessible. Furthermore, the interaction between KLF4 and other transcriptional cofactors such as Tip60 (Ai et al., 2007) and the p65/RelA subunit of NF-

ĸB (Feinberg et al., 2005) likely contribute to its functional capabilities as a transcriptional activator versus repressor. While Tip60 activates gene transcription through its intrinsic HAT activity, it can also acts as a corepressor for STAT3 by the recruitment of HDAC7 (Xiao et al., 2003). In addition, Tip60 has been implicated in transcriptional repression through interactions with CREB or the transcriptional repressor

ZEB (Hlubek et al., 2001). It is likely these interactions may affect the ability of KLF4 to act as a transactivator in the presence of Tip60.

In contrast to the ability of KLF4 to recruit p300/CBP and activate transcription, KLF4 can indirectly repress transcription through its ability to inhibit p300/CBP recruitment by other transcriptional activators such as β-catenin (Evans et al., 2010) and Smad3

(Feinberg et al., 2005). Furthermore, KLF4 has been shown to interact with and recruit various histone deacetylases (HDACs) to mediate transcriptional repression (Evans et al.,

2007; Noti et al., 2005; Wei et al., 2007). HDACs are present as subunits of larger corepressor complexes. Recruitment to specific genetic loci by other transcription factors results in the removal of histone acetyl groups and the remodeling of chromatin, causing the targeted gene to be silenced. Recently an interaction between KLF4 and the small ubiquitin modifier protein SUMO-1 has been described (Du et al., 2010b). While SUMO modification of proteins has been increasingly linked to transcriptional repression, the precise mechanism of repression is still unknown (Yang et al., 2003). However, this interaction between SUMO and KLF4 provides another potential mechanism whereby

KLF4 may exert a suppressive effect on gene transcription.

1.3.3 Function of KLF4 in normal biological processes

1.3.3.1 KLF4 as a regulator of development and differentiation

KLF4 is distributed throughout a wide variety of embryonic and adult tissues. Its developmentally regulated expression is highest during the later embryonic stages, but appears as early as embryonic day 4.5 in the mouse. Starting at embryonic day 10, KLF4 expression becomes highly dynamic throughout the developing embryo and is found in the eye, limb buds, thymus, testis, kidney, respiratory tract, skeleton, tongue and heart

(Behr and Kaestner, 2002; Chiambaretta et al., 2004; Ehlermann et al., 2003; Garrett-

Sinha et al., 1996; Ohnishi et al., 2000; Panigada et al., 1999; Yoshida et al., 2010).

Maximal expression is seen in the GI tract around embryonic day 15.5, and at day 16.5 within the developing layers of the skin (Conkright et al., 1999; Garrett-Sinha et al.,

1996; Segre et al., 1999). In adult tissue, such as the GI tract, skin, and thymus, KLF4 expression is found primarily in post-mitotic terminally differentiated cells (Garrett-Sinha et al., 1996; Jenkins et al., 1998; Shields et al., 1996). Upregulation of KLF4 in endothelial cells by shear stress (McCormick et al., 2001) and smooth muscle cells in response to vascular injury (Liu et al., 2005a) suggests a role for KLF4 in homeostasis of the vascular system.

Homologous gene targeting in mice has been a valuable tool to better understand the role of KLF4 during development. Klf4-knockout mice die within 15 hours after birth as a result of defects in skin development. Failure of normal basement membrane formation

30 and disruption in the skin‟s fat layer causes loss of skin barrier function and rapid loss of body fluids (Segre et al., 1999). These findings led to the in vitro identification of KLF4 as a transcriptional regulator of adipogenesis (Birsoy et al., 2008). Klf4-knockout mice also have a 90% decrease in goblet cells of the colon, and abnormal morphology in those that exist (Katz et al., 2002). Due to the early postnatal lethality of the conventional Klf4- knockout mice, tissue specific ablation or conditional knockout models have been useful in studying the role of KLF4 during postnatal development. Ablation of Klf4 in gastric epithelia results in the perturbation of differentiation into mature cell lineages as well as a marked hypertrophy (Katz et al., 2005). In the eye, lack of goblet cells in the conjunctiva, and abnormal corneal epithelium are a result of conditional Klf4 deletion

(Swamynathan et al., 2007). Smooth and cardiac muscle-selective knockout of Klf4 results in growth retardation and postnatal deaths due to reduced cardiac output (Yoshida et al., 2010). Taken together, the studies described above demonstrate an important function for KLF4 in controlling the in vivo differentiation and proliferation of many tissues.

1.3.3.2 KLF4 regulation of proliferation and apoptosis

KLF4 was first identified in a genetic screen for transcription factors involved in growth regulation (Shields et al., 1996). Since then, KLF4 has been shown to play important roles in many other cellular processes, including differentiation and apoptosis, during development and normal tissue homeostasis. The high expression of KLF4 in terminally differentiated, postmitotic intestinal epithelial cells suggested a link to growth arrest and

31 led to the identification of KLF4 as a critical regulator of cell cycle progression in vitro.

Expression profiling confirms that KLF4 activates numerous genes encoding negative cell cycle regulators while suppressing expression of genes that promote cell cycle progression (Chen et al., 2003). In addition, a global inhibitory function for KLF4 in regulating genes involved in macromolecular synthesis supports a role for KLF4 as an inhibitor of proliferation and growth-arrest associated gene (Whitney et al., 2006).

Progression through the cell cycle is driven by cyclins and their cognate cyclin-dependent kinases (Cdks), while inhibition of cell cycle progression is regulated by Cdk inhibitors including p16ink4a, p21Cip1/Waf1, p27Kip1 and p57Kip2. Two of the first cell-cycle genes identified as being regulated by KLF4 were CCND1 (cyclin D1) and p21Cip1/Waf1 (Shie et al., 2000a; Zhang et al., 2000). Cyclin D1 is required for entry into the cell cycle, and

KLF4 binds to an Sp1 motif in the CCND1 promoter and inhibits its expression. p21Cip1/Waf1 inhibits the activity of several cyclin-Cdk complexes, including D1/Cdk4,

E1/Cdk2 and A/Cdk2, resulting in cell cycle arrest at the G1-S transition (Xiong et al.,

1993). KLF4 binds the p21Cip1/Waf1 proximal promoter at a specific Sp1-like cis element and recruits p53 to activate expression of this Cdk inhibitor, preventing progression through the G1 phase of the cell cycle. Both p53 and p21 are also required for sustained

G2 arrest following γ-irradiation, and this is mediated, in part, through transcriptional activation by KLF4 (Yoon and Yang, 2004). Transactivation of the cyclin-Cdk inhibitor p27Kip1 (Wei et al., 2008) and repression of cell cycle promoting CCNB1 (Cyclin B1) gene (Yoon and Yang, 2004) are several other mechanisms whereby KLF4 exerts a coordinate control over cell cycle progression to inhibit proliferation.

Many of the studies delineating the mechanism whereby KLF4 contributes to cell cycle arrest have been in the context of DNA damage-induced growth arrest. In addition to regulation of various cyclins and Cdk inhibitors, KLF4 transcriptionally regulates p53 gene expression (Rowland et al., 2005; Wassmann et al., 2007). Whether KFL4 activates or suppresses p53 is variable, and may be cell type or context dependent. The tumor suppressor p53 is an integral player in cell cycle regulation of both the G1/S and G2/M transitions, in response to DNA damage (Vogelstein et al., 2000). DNA damage leads to the stabilization of p53 and the activation of pathways resulting in either cell cycle arrest or induction of apoptosis (Aylon and Oren, 2007). One mechanism whereby p53 induces apoptosis is through activation of the Bax promoter, which is inhibited by KFL4

(Ghaleb et al., 2007a). In response to cytostatic reparable DNA damage, p53 transcriptionally activates KLF4, which in turn is required for p53-mediated induction of p21, and cell cycle arrest. However, irreparable DNA damage leads to destabilization of

KLF4 mRNA, allowing p53 to activate the pro-apoptotic Bax gene (Zhou et al., 2009).

Thus, KLF4 appears to play a role in controlling the switch in p53 response to DNA damage. Another role of p53 during the cell cycle is the regulation of centrosome duplication. Numerous studies have implicated centrosome amplification and subsequent chromosomal instability in the development and pathogenesis of human cancers

(Fukasawa, 2005), often resulting from loss or mutation of p53. Klf4 is also sufficient to prevent radiation-induced centrosome amplification in part through suppression of Cyclin

E, a key regulator of centrosome duplication during G1(Yoon et al., 2005).

1.3.4 The role of KLF4 during carcinogenesis

1.3.4.1 KLF4 as tumor suppressor

KLF4 was initially identified as a growth arrest associated gene in the intestinal epithelium (Shields et al., 1996), and has been found to have altered expression in various models of intestinal tumorigenesis. Colon cancer cells, which have lost expression of

KLF4, undergo G1/S cell cycle arrest in the presence of exogenously expressed KLF4

(Chen et al., 2001), and restoration of KFL4 to these cells inhibits their tumorigenicity in vivo (Dang et al., 2003). KFL4 mRNA is significantly reduced in human gastric adenocarcinomas and colorectal cancer specimens, including those from patients with familial adenomatous polyposis, carrying germline APC (adenomatous polyposis coli) mutations (Dang et al., 2000; Shie et al., 2000b; Zhao et al., 2004). APC is the most frequently mutated tumor suppressor gene in colon cancer, and has been found to negatively regulate KLF4 (Stone et al., 2002). Several studies examining KLF4 expression in human gastric cancers and lymph node metastases have revealed that decreased KLF4 expression is correlative with disease progression (Katz et al., 2005;

Kim et al., 2003; Wei et al., 2005). KLF4 expression is frequently downregulated in bladder cancer (Ohnishi et al., 2003) and in esophageal squamous cell carcinomas; KLF4 expression is reduced or lost when compared to normal esophageal tissue (Wang et al.,

2002). In addition to tumors of the GI tract, decreased KLF4 expression has been shown in prostate (Foster et al., 2000) and lung cancers (Hu et al., 2009), adult T-cell leukemia

(Yasunaga et al., 2004) and glioma-associated vascular endothelial cells (Madden et al.,

2004). Several mechanisms have been described whereby both genetic and epigenetic alterations in colorectal, gastric and esophageal cancers, including loss of heterozygosity

(LOH), promoter hypermethylation, and point mutations that disrupt protein activity

(Kakinuma et al., 2004; Miura et al., 1995; Miura et al., 1996; Roth et al., 2001; Wei et al., 2005; Zhao et al., 2004), contribute to loss of KLF4 expression.

1.3.4.2 KLF4 as an oncogene

In contrast to the above studies, accumulating evidence suggests a pleiotropic role for

KLF4, acting as an oncogene in certain contexts (Rowland and Peeper, 2005). In the late

1990s, KLF4 was identified in a screen of E1A-immortalized rat kidney epithelial cells as a factor that could induced transformation, and produce tumors in xenografted mice

(Foster et al., 1999). In laryngeal squamous cell carcinoma, KLF4 overexpression is an early event in tumor progression and conditional expression of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch, resulting in hyperplasia, dysplasia, and eventual squamous cell carcinoma (Foster et al., 2005; Foster et al., 1999;

Huang et al., 2005a). It is interesting that KLF4 actually induces expression of several retinoic acid receptors including RXRα, which can then be activated by RXR selective agonists to prevent skin tumors in a KLF4-induced transgenic model of skin cancer

(Jiang et al., 2009)

1.3.4.3 The role of KLF4 in breast cancer and metastasis

The role of KLF4 in breast cancer biology is controversial. This may be due in large part to the fact that breast cancer is not a single, homogeneous disease, but rather consists of at least 5 distinct molecular subtypes associated with different clinical outcomes (Polyak,

2007). Initial studies examining the expression of KLF4 in breast cancer specimens revealed increased levels in ductal carcinomas in situ and invasive carcinoma when compared to uninvolved adjacent epithelium (Foster et al., 2000), suggesting KLF4 may act as an oncogene in the breast. In a separate study by the same group, analysis of the subcellular localization of KFL4 in 146 patients suggested that nuclear KLF4 was associated with an aggressive phenotype in early-stage breast cancer, although overall expression of KFL4 was not associated with clinical outcome (Pandya et al., 2004).

However, in vitro studies reveal decreased KLF4 DNA binding activity and protein levels in breast cancer cell lines compared with the non-transformed MCF-10A mammary epithelial cell line (Aslakson and Miller, 1992). While one study examining the effects of KLF4 knock-down in two ER+ breast cancer cell lines (MCF7 and MDA-

MB-134) determined that loss of KLF4 induces apoptosis (Rowland et al., 2005), another found that KLF4 abrogates estrogen-dependent MCF7 cell growth by inhibiting ER target gene transcription (Akaogi et al., 2009). Furthermore, interrogation of publically available human breast cancer gene expression data sets reveals that KLF4 expression significantly correlates with ERα positivity. KLF4 mRNA levels are also lower in breast carcinomas than normal tissue, and inversely correlated with increasing tumor grade

(Akaogi et al., 2009). Finally, a study analyzing differential gene expression among 18 primary human breast tumor cell lines, derived from African American and Caucasian patients, found decreased levels of KLF4 to be associated with the more metastatic cell lines and tumors derived from African American patients (Yancy et al., 2007).

In a simplified model, Rowland et al., conclude that the proliferative (oncogenic) versus anti-proliferative (tumor-suppressive) effects of KLF4, not only in breast, but in other types of tissue, is highly dependent upon the cellular status of two transcriptional targets of KFL4, p53 and p21, as well as proliferative stimuli or dysregulated growth factor signaling (Rowland et al., 2005; Rowland and Peeper, 2005). In light of recent findings identifying various KLF4 isoforms and their potential for differential modulation, it is likely there are multiple layers of yet unknown regulation that are highly context-specific.

1.3.4.4 Krüppel-like factors, EMT and metastasis

The involvement of Krüppel-like factors in breast cancer and EMT has been recently highlighted in the literature. KLF8 was initially identified as a transcriptional repressor and downstream effector of focal adhesion kinase (Zhao et al., 2003) and is overexpressed in several types of invasive human cancer (Wang and Zhao, 2007). As a transcriptional repressor of E-cadherin, KLF8 was shown to induce EMT in MCF-10A mammary epithelial cells (Wang et al., 2007). KLF17, a negative regulator of EMT, is downregulated in human breast cancer, and overexpression in the 4T1 tumor model was able to suppress lung metastasis in these mice (Gumireddy et al., 2009). KLF6 is a proposed tumor suppressor, due to mutational inactivation or loss of expression in a variety of tumors. While KLF6, similar to KLF8, plays a permissive role in TGF-β- induced EMT (Holian et al., 2008), its role in breast cancer is somewhat elusive. Like

KLF4, KLF6 also inhibits estrogen-mediated cell growth in breast cancer cells (Liu et al.,

2009) however nuclear KLF6 expression is found to correlate with HER2 overexpression in breast tumors (Gehrau et al., 2010), suggesting its role may be sub-type dependent.

As described in section 1.2.1, EMT is characterized by loss of intercellular adhesions (E- cadherin), down-regulation of epithelial markers (cytokeratins), up-regulation of mesenchymal markers (smooth muscle actin (SMA), vimentin), and acquisition of a fibroblast-like morphology, culminating in increased motility, invasiveness and metastatic capacity. As KLF4 is a known inhibitor of SMA, it would suggest an inhibitory role for KLF4 during EMT. However, recently it has been proposed that the epithelial-mesenchymal transition generates cells with properties of stem cells (Mani et al., 2008). One potential mechanism involves the E-cadherin suppressor ZEB1 and its ability to induce EMT and maintain stemness through de-repression of stem-like factors, including Sox2 and KLF4 via repression of stemness-inhibiting microRNAs (Wellner et al., 2009). Paradoxically, as will be discussed in Chapter II and Chapter III, we have shown that KFL4 inhibits EMT (Yori et al., 2010a) and mammary tumor progression

(Yori et al., 2010b). While the contradiction in these findings require further assessment, including what if any role KLF4 might play in the self-renewal of CSCs, support for

KFL4 as in inhibitor of EMT comes from Li et al. who also show that KLF4 is potent activator of E-cadherin and suppressor of the EMT-associated transcription factor Snail

(Li et al., 2010b). Interestingly, another microRNA, miR-10b, which has been found to initiate invasion and metastasis in breast cancer (Ma et al., 2010; Ma et al., 2007) also promotes migration and invasion in esophageal cancer cells through post-transcriptional downregulation of KLF4 (Tian et al., 2010), further supporting a potential role for KLF4

38 as an inhibitor of breast cancer progression. The relationship between KLF4 and the processes of EMT and MET is depicted in Figure 1.3.

1.4 Statement of Purpose

The functionality of KFL4 as both tumor suppressor and oncogene is well established, yet the paradoxical nature of its actions in breast cancer has not been mechanistically clarified. Furthermore, little is known regarding the role of KLF4 in metastatic progression for any type of cancer. The studies presented herein define a novel role for

KLF4 as a negative regulator of EMT, a process associated with tumor metastasis. The inhibition of E-cadherin gene expression, resulting from KLF4 ablation, is a direct effect of the loss of KFL4 binding and transactivation of the E-cadherin promoter (Chapter 2).

The decrease in breast cancer cell migration and invasion, as a result of forced KLF4 expression suggested a potential role for KLF4 in the regulation of breast tumor metastasis. Consistent with our in vitro findings, KFL4 was able to suppress the metastatic spread of breast cancer cells in a xenograft mouse model (Chapter 3).

Previous studies in our laboratory identified KLF4 as being lost in tumors arising from a mouse model of HER2/Neu induced mammary cancer, suggesting a potential role for

KLF4 as an inhibitor of primary tumor formation. The absence of conditionally-enforced

KLF4 expression in HER2/Neu tumors suggests that KLF4 is non-permissive for mammary tumor formation. Furthermore, forced expression of KLF4 in an orthotopic tumor model was sufficient to inhibit primary tumor growth and metastasis (Chapter 3).

Collectively, these studies provide both mechanistic and functional evidence for KLF4 as a negative regulator of EMT and tumor/metastasis-suppressor in the breast.

Figure 1.1 The link between normal mammary epithelial hierarchy, molecular subtypes, gene expression signatures and clinical markers of breast cancer.

Hierarchical clustering of microarray data segregates breast cancer into at least five molecularly distinct groups (Sorlie et al., 2001). The normal-like group has been excluded from this figure, as currently, it is thought that this original subtype is an artifact of having a high percentage of normal “contamination” in the tumor specimens (Parker et al., 2009). Recently a new claudin-low subtype has been identified (Herschkowitz et al.,

2007). Breast tumors may arise from the transformation of normal breast cells at various stages of differentiation, with gene expression signatures related to the normal cell type of origin. Alternatively, transformation of mammary stem cells (MaSCs) or progenitor cells may produce CSCs that in turn give rise to a tumor containing a heterogeneous population of cells (Prat and Perou, 2009). Furthermore, it has been proposed that a terminally differentiated cancer cell within a heterogeneous tumor may acquire stem-like features through an EMT (Hollier et al., 2009). Classification of breast cancer subtypes according to IHC marker profiles of data from more than 10,000 cases of breast cancer among 12 different studies (Blows et al., 2010).

Figure 1.1

Figure 1.2 Organization of the human KLF4 gene and protein. The KLF4 gene is located on the reverse strand of chromosome 9q31.2 and covers a 5.6 kb region. Red boxes denote the five exons with yellow representing the intronic regions. The four black boxes represent the open reading frame (ORF) within the first identified 2,931 bp cDNA

(Yet et al., 1998). Subsequently, 7 different transcripts, encoding 5 protein products have been identified (Ensembl and (Wei et al., 2010)). The KLF4 protein contains an activation domain (green) a repression domain (pink) and three C-terminal zinc fingers

(gray) which bind consensus elements in the promoter region of target genes. A putative

PEST sequence flanked by the two arrowheads may serve as target for ubiquitin- dependent degradation. Several other sites of post-translational modification have been identified and are discussed in the text of section 1.3.2.1. Numbering of amino-acid sequences for three of the most common annotated protein products is shown. Sequence

A is a 470 amino acid protein that was first described by Wei et al. Sequence B represents a 379 amino acid sequence from a 5‟-ATG in-frame start codon. Sequence C is a 513 amino acid product resulting from the inclusion of the third intron (102 base pairs, 34 amino acids) N-terminal to the nuclear localization signal (NLS), denoted by a light gray box.

Figure 1.2

Figure 1.3 KLF4 is a central player in the regulatory networks modulating EMT and MET during generation of iPSCs and CSCs. During the generation of iPS cells, the four “Yamanaka factors” known as Sox2, KLF4, Oct4 and c-Myc or (SKOM) are sufficient to reprogram a differentiated somatic cell to a stem-like state (Takahashi and

Yamanaka, 2006). Recently, the sequential order of the full reprogramming process was determined to first require conversion of fibroblasts into an intermediate epithelial cell

(initiation phase) which occurs through a mesenchymal-to-epithelial transition (MET) (Li et al., 2010b). During this phase, Sox2 and Oct4 repress several EMT-inducers including

Snail and members of the TGF-β pathway. Myc further prevents the EMT-program from being activated by inhibiting TGF-β, while KLF4 induces E-cadherin and suppresses

Snail to reinforce the epithelial program. Furthermore, due to the high levels of endogenous KLF4 in mammary epithelial cells, they could be reprogrammed into iPS cells in the absence of exogenous KLF4 (Li et al., 2010b). It has also been shown that the addition of BMP7 to the SKOM factors can increase the reprogramming efficiency by inhibiting the TGF-β pathway and activating the miR200 family of microRNAs

(Samavarchi-Tehrani et al., 2010). While this first phase appears to be reversible, a second maturation phase is associated with the expression of ES cell markers, including

Nanog, and appears to be irreversible.

Since MET is a crucial step towards a stem-like state during the generation of iPS cells, it is difficult to reconcile the findings that the induction of EMT in immortalized mammary epithelial cells (HMLEs) is able to generate cells with stem cell properties (Mani et al.,

2008). Furthermore, EMT promotes the generation of cancer stem cells (CSCs) from mammary tumor cells. One mechanism may be through ZEB1/2 inhibition of the

45 miR200 family, and de-repression of KLF4 and Sox2 (Wellner et al., 2009). How KLF4 might support both MET and EMT is unclear. BMP7, bone morphogenic protein; MEF, mouse embryonic fibroblast; Tgfb(R); transforming growth factor beta (receptor).

Figure 1.3

Table 1.1 Regulators and Targets of KLF4

Factor/Condition Expression Tissue (disease)/cell line References

Regulators of KLF4

Expression

LPS (Lipopolysaccharide) ↑ macrophages (Feinberg et al., 2005)

VSMC (vascular smooth muscle (Meng et al., 2009; Wang ATRA (all trans retinoic acid) ↑ cells) et al., 2008a)

HT-29 (colon adenocarcinoma (Chen et al., 2004; Shie et Butyrate ↑ cells) al., 2000b) COS-1 (kidney cells), CHO CDX2 (caudal-related (Dang et al., 2001; ↑ (Chinese hamster ovary cells); homeobox protein) Mahatan et al., 1999) RKO (colon carcinoma cells)

MEFs (mouse embryonic (Garrett-Sinha et al., Contact inhibition ↑ fibroblasts) 1996; Shields et al., 1996)

Endothelin-1 ↑ Cardiomyocytes (Cullingford et al., 2008) (Cullingford et al., 2008; Hydrogen Peroxide ↑ Cardiomyocytes, VSMC Nickenig et al., 2002) IBMX (isobutyl methyl ↑ adipocytes (Birsoy et al., 2008) xanthine)

colon carcinoma cells, (Chen et al., 2000; Interferon-γ macrophages Feinberg et al., 2005)

KLF4 ↑ COS-1, CHO (Dang et al., 2002)

KLF5 ↓ COS-1, CHO (Dang et al., 2002)

Methyl methanesulfonate ↑ MEFs (Zhang et al., 2000)

miR-10b ↓ esophageal cancer cells (Tian et al., 2010) hESCs (human embryonic stem miR-145 ↓ (Xu et al., 2009) cells)

miR-200c, miR-203 ↓ mPaCa, pancreatic cancer cells (Wellner et al., 2009)

Notch1 ↓ Goblet cells (Zheng et al., 2009b)

p53 MEFs (Zhang et al., 2000) POVPC (1-palmytoyl-2-(5-

oxovaleroyl)-sn-glycero-3- ↑ SMC (smooth muscle cells) (Pidkovka et al., 2007) phosphocholine (Chen et al., 2004; Serum deprivation ↑ Colon cancer / COS-1, CHO Mahatan et al., 1999)

HUVEC (human umbilical vein Shear stress (Hamik et al., 2007) endothelial cells) Colon cancer cells, 4T1 (breast Snail ↓ (De Craene et al., 2005) cancer cells)

Sp1/Sp3 ↑ COS-1, CHO (Mahatan et al., 1999) (Adam et al., 2000; TGF-β ↑/↓ VSMC; macrophages Feinberg et al., 2005; Li et al., 2010a) TNF-α (Tumor necrosis macrophages (Feinberg et al., 2005) factor-alpha) (Chen et al., 2004; Shie et Trichostatin A ↑ HT-29 al., 2000b) γ -irradiation ↑ HCT116 (colon carcinoma cells) (Yoon et al., 2003)

Targets of KFL4

A33 antigen ↑ SW122 colon carcinoma cells (Mao et al., 2003) HeLa (cervical cancer cells), B2R (Bradykinin receptor 2) ↑ (Saifudeen et al., 2005) IMCD kidney cells (Ghaleb et al., 2007b; Li Bax ↓ K562 leukemia cells et al., 2010c) Bcl-2 (B-cell lymphoma 2) ↑ K562 leukemia cells (Li et al., 2010c)

CD11b (Integrin, alphaM) ↓ leukocytes (Noti et al., 2005)

Cyclin B1 ↓ HCT116 (Yoon and Yang, 2004) (Shie et al., 2000a; Shie et Cyclin D1 ↓ HT-29 al., 2000b) Cyclin E ↓ HCT116 (Yoon et al., 2005)

CYP1A1 ↓ CHO (Zhang et al., 1998) esophageal squamous cell Cytokeratin 4 ↑ (Luo et al., 2004) carcinoma

fibroblasts, MCF-10A (Li et al., 2010b; Yori et E-cadherin ↑ (mammary epithelial cells), al., 2010a) ESSRB (Estrogen related ↑ ES cells (Jiang et al., 2008a) receptor beta)

FBXO15 (F-box protein 5) ↑ ES cells (Jiang et al., 2008a) FGF5 (Fibroblast growth ↓ ES cells (Jiang et al., 2008a) factor 5)

Histidine decarboxylase ↓ gastric adenocarcinoma cells (Ai et al., 2004)

HMGB1 (High mobility ↑ macrophages (Liu et al., 2008a) group protein B1) HSC70 (Heat shock cognate ↑ macrophages (Liu et al., 2008c) protein 70) SMVT (Sodium dependent HEK-293 (human embryonic ↑ (Reidling and Said, 2007) multivitamin transporter) kidney cells) HSP90 (Heat shock protein ↑ macrophages (Liu et al., 2010) 90)

IL-10 (Interleukin-10) ↑ macrophages (Liu et al., 2007) iNOS (inducible nitric oxide ↑ macrophages (Feinberg et al., 2005) synthase)

IAP (Intestinal alkaline (Chen et al., 2003; ↑ RKO phosphatase) Hinnebusch et al., 2004)

esophageal, pancreatic cancer (Brembeck and Rustgi, Keratin 19 ↑ cells 2000)

Keratin 4 ↑ esophageal squamous epithelium (Jenkins et al., 1998) (Jiang et al., 2008a; KLF2 ↓ ES cells Nakatake et al., 2006) Laminin α1 ↓ intestinal epithelial cells (Piccinni et al., 2004)

Laminin-α 3A ↑ MCF-10A (Miller et al., 2001)

Laminin-γ 1 ↑ mesangial cells (Higaki et al., 2002)

Lefty1 ↑ ES (embryonic stem cells), HeLa (Nakatake et al., 2006) (Jiang et al., 2008a; Nanog ↑ ES cells Nakatake et al., 2006) Nestin ↓ ES cells (Jiang et al., 2008a)

Oct4 ↑ ES cells (Jiang et al., 2008a)

Ornithine decarboxylase ↓ HT-29 (Chen et al., 2002) (Mahatan et al., 1999; p21Cip1 ↑ Cos-1/CHO; VSMC; MEFs Nickenig et al., 2002; Zhang et al., 2000) Kip1 p27 ↑ pancreatic cancer cells (Wei et al., 2008) V12 RAS -MEFs (mouse (Rowland et al., 2005; p53 ↓/↑ embryonic fibroblasts), VSMC Wassmann et al., 2007) Cip7 p57 ↑ RKO (Chen et al., 2003) PAI-1 (Plasminogen activator ↓ macrophages (Feinberg et al., 2005) inhibitor 1) PDGFβ (Platelet-derived ↓ VSMC (Zheng et al., 2009a) growth factor beta)

PKG-1α (Protein kinase G) ↑ aortic SMCs (Liu et al., 2003) RARα (Retinoic acid receptor ↓ VSMC (Zheng et al., 2009a) alpha)

Rb (Retinoblastoma) ↑ RKO (Chen et al., 2003) RXRα/RARγ (Retinoid X receptor/Retinoic acid receptor ↑ RK3E (rat kidney cells) (Jiang et al., 2009) gamma)

Smooth muscle alpha actin ↓ aortic SMCs (Liu et al., 2003)

SM22α (Transgelin) ↓ VSMCs (Adam et al., 2000) (Li et al., 2010b; Yori et Snail ↓ fibroblasts, MCF-10A, 4T1 al., 2010b) Sox2 ↑ ES cells (Jiang et al., 2008a) gastric cancer / gastric cancer Sp1 ↓ (Kanai et al., 2006) cell lines SPARC (secreted protein H322, A549 lung cancer cell ↓ (Zhou et al., 2010) acidic and rich in cysteine) lines SPRR1A, SPRR2A (Small esophageal squamous cell ↑ (Luo et al., 2004) proline-rich protein 1A/2A) carcinoma

TBX3 (T-box 3) ↑ ES cells (Jiang et al., 2008a) TCL1 (T-cell ↑ ES cells (Jiang et al., 2008a) leukemia/lymphoma 1) TGFBR1 (Transforming ↓ VSMC (Li et al., 2010a) growth factor beta receptor 1)

Thrombomodulin HUVEC (Hamik et al., 2007) u-PAR (Plasminogen -/- ↑ KLF4 colon / HCT116 (Wang et al., 2004) activator, urokinase receptor)

* Entries in bold were first identified in the studies presented herein.

CHAPTER 2

KRÜPPEL-LIKE FACTOR 4 (KLF4) INHIBITS EPITHELIAL-TO-MESENCHYMAL

TRANSITION THROUGH REGULATION OF E-CADHERIN GENE EXPRESSION

(Yori et al., Journal of Biological Chemistry, March 2010)

The Krüppel-like factor 4 (KLF4) is a transcriptional regulator of proliferation and differentiation in epithelial cells, both during development and tumorigenesis. While

KLF4 functions as a tumor suppressor in several tissues, including the colon, the role of KLF4 in breast cancer is less clear. Here, we show that KLF4 is necessary for maintenance of the epithelial phenotype in non-transformed MCF-10A mammary epithelial cells. KLF4 silencing led to alterations in epithelial cell morphology and migration, indicative of an epithelial-to-mesenchymal transition

(EMT). Consistent with these changes, decreased levels of KLF4 also resulted in the loss of E-cadherin protein and mRNA. Promoter/reporter analyses revealed decreased E-cadherin promoter activity with KLF4 silencing while chromatin immunoprecipitation (ChIP) identified endogenous KLF4 binding to the GC-rich/E- box region of this promoter. Furthermore, forced expression of KLF4 in the highly metastatic MDA-MB-231 breast tumor cell line was sufficient to restore E-cadherin expression and suppress migration and invasion. These findings identify E-cadherin as a novel transcriptional target of KLF4. The clear requirement for KLF4 to maintain E-cadherin expression and prevent EMT in mammary epithelial cells supports a metastasis suppressive role for KLF4 in breast cancer.

2.1 Introduction

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that was first identified in a screen for transcription factors involved in growth regulation (Shields et al., 1996).

KLF4 is primarily regarded as a negative regulator of the cell cycle, repressing a multitude of genes that promote proliferation while at the same time, upregulating inhibitors of proliferation (Chen et al., 2001). KLF4 also plays a crucial role in differentiation during organogenesis of various tissues such as the skin, colon and eye

(Katz et al., 2002; Segre et al., 1999; Swamynathan et al., 2007). With the advent of induced pluripotent stem (iPS) cells, KLF4 has gained recognition as one of the

“pluripotency genes” that can reprogram somatic cells into a stem cell-like state

(Takahashi and Yamanaka, 2006), acting in the capacity to maintain self-renewal (Jiang et al., 2008b).

Given its stem-cell promoting activity and its ability to regulate growth and differentiation during development, it is not surprising that KLF4 also plays various roles in tumorigenesis. The frequent loss of KLF4 expression in gastric and colorectal cancers has led to studies revealing a tumor suppressive role for this factor in these and other tissues (Dang et al., 2000; Dang et al., 2003; Ghaleb et al., 2007b; Ohnishi et al., 2003;

Yasunaga et al., 2004; Zhao et al., 2004). Conversely, overexpression of KLF4 in the skin leads to squamous cell carcinoma (Huang et al., 2005b). However, the role of KLF4 during the progression of breast cancer is not well defined. Immunohistochemical studies have revealed that KLF4 expression can be increased and undergo altered localization in

DCIS of the breast (Foster et al., 2000), suggesting that it may act as a oncogene in this tissue. This is further supported by the association of nuclear KFL4 with an aggressive breast cancer phenotype (Pandya et al., 2004). In contrast, Akaogi et al. reported that review of 9 independent, publicly available gene expression data sets revealed decreased

KLF4 mRNA expression in breast cancers when compared to normal breast. In addition,

KLF4 was inversely correlated with increasing tumor grade in 15 independent gene expression array analyses of breast cancer samples (Akaogi et al., 2009). Expression of

KLF4 protein is also relatively low in breast cancer cell lines compared to non-transformed mammary epithelial cells (Miller et al., 2001). Thus, there is evidence that KLF4 can act in a tumor promoting and tumor suppressive manner depending on the tissue of study. To reconcile these findings, Rowland et al. proposed a cell type specific functionality for KLF4, wherein its ability to act as a tumor suppressor versus oncogene is largely context-specific and depends upon the relative expression of p21 and p53

(Rowland et al., 2005). Of note, it remains unknown if KLF4 may regulate other properties of tumors, such as metastatic capacity. Furthermore, the specific function of

KLF4 in non-transformed mammary epithelium has not been previously addressed.

Recent studies identifying transcriptional targets of KLF4 reveal that it promotes the expression of epithelial-specific cytokeratins in colon cancer cells (Chen et al., 2003), suggesting that it may sustain an epithelial phenotype. Many of these keratins are suppressed during epithelial-to-mesenchymal transition (EMT) (Paccione et al., 2008), a process defined by a loss of epithelial-specific characteristics and the acquisition of a mesenchymal phenotype. While EMT is an essential step during development (Thiery

54 and Sleeman, 2006; Yang and Weinberg, 2008), loss of epithelial characteristics in tumors is associated with increased aggressiveness and poor prognosis, implicating EMT as a mechanism for tumor progression and metastasis (Thiery, 2002). A hallmark of

EMT is the loss of E-cadherin. In fact, silencing of E-cadherin alone in epithelial cells is sufficient to induce a full EMT (Lehembre et al., 2008). This loss is functionally significant because direct suppression of E-cadherin causes a decrease in cell-cell adhesion and increased invasion and motility (Thompson et al., 1994). E-cadherin is the prototypical cadherin, modulating intercellular adhesions and signaling involving catenins (Gottardi et al., 2001; Ireton et al., 2002), receptor tyrosine kinases (Bremm et al., 2008; Qian et al., 2004) and small GTPases (Braga et al., 1997; Wheelock and

Johnson, 2003). While much of the literature ascribes an anti-proliferative function to E- cadherin, there is also a requirement for sustained E-cadherin to maintain proliferation of normal epithelial cells (Boussadia et al., 2002; Fournier et al., 2008; Liu et al., 2006).

In breast cancer, loss of E-cadherin is correlated with a more invasive phenotype and metastatic disease progression. Early loss of E-cadherin is observed during tumor progression in lobular disease, while ductal breast carcinomas have more variable expression (Knudsen and Wheelock, 2005). Even when primary tumors maintain E- cadherin expression, loss of this protein in metastatic lesions has been observed (Wu et al., 2008). The identification of E-cadherin as a tumor and metastasis suppressor has fueled the discovery of multiple molecular mechanisms controlling E-cadherin expression (Baranwal and Alahari, 2009; Onder et al., 2008). Herein, we report for the first time that E-cadherin is a direct transcriptional target of KLF4 and that this regulation

55 is necessary to maintain the epithelial phenotype in mammary epithelial cells.

Furthermore, we show that forced expression of KLF4 in the highly metastatic

MDA-MB-231 breast cancer cell line restores E-cadherin expression, resulting in reduced migration and invasion. These data indicate that KLF4 may play an important role in restraining metastatic activity of breast cancers.

2.2 Materials and Methods

Cell culture. Human mammary epithelial MCF-10A cells (American Type Culture

Collection, ATCC) were cultured as either monolayers or in 3D cultures as previously described (Debnath and Brugge, 2005). MDA-MB-231 cells (ATCC) were cultured in

RPMI 1640 medium (MP Biomedicals, LLC.) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in atmosphere containing 5% CO2.

Generation of viral constructs and stable knock-downs. Short hairpin RNAs

(shRNAs) were expressed from either the Lentilox 3.7 (ATCC) lentivirus vector or the microRNA-based TMP-tTA retrovirus vector (kind gift from Dr. Ed Stavnezer). For lentiviral knockdown, we used the previously published and validated shRNA sequence

5‟-GGA CGG CTG TGG ATG GAA A-3‟ (Rowland et al., 2005) to generate the stable shKLF4 line or a scrambled sequence as the negative control (shNS). The sequence designated as shKLF4 targets nucleotides 1592-1610 in the human KLF4 cDNA sequence (GenBank™ accession number NM 004235.3). Lentiviral production and target cell transduction was performed as previously described with slight modification

(Johnson et al., 2004). 293FT cells were transfected simultaneously with Lipofectamine

2000 and ViraPower Lentiviral packaging kit (Invitrogen) according to the manufacturer‟s instructions. Transfected 293FT cells were cultured for 48h at 37°C after which time virus-containing media was harvested. Viral supernatant was filtered through a 0.45μm filter, supplemented with 4 mg/ml polybrene (Sigma-Aldrich) and used to transduce target cells at 32°C overnight. Stable MirKLF4 knockdowns were created using the replication-defective retroviral vector TMP-tTA as previously described (Zhang and Stavnezer, 2009). The shRNA sequences targeting human KLF4 were chosen using

RNAi Codex. The sequence designated as MirKLF4 targets nucleotides 1834-1852 in the human KLF4 cDNA sequence (GenBank™ accession number NM 004235.3). A scrambled shRNA (MirNS) sequence was used as the negative control.

For adenovirus KLF4 (AdKLF4), the 3XFlag.HA:KLF4 vector was created through two rounds of subcloning using the previous published pcDNA3-KLF4 construct (Feinberg et al., 2005). For the 1st round of cloning, pcDNA3-KLF4 was digested with BamHI and

BstXI. Using a set of primers (Oligo #1 and #2) containing a 5‟ BamHI linker, Kozak motif, NheI site and HA tag, the N-terminus of KLF4 was amplified. Both the vector and the amplified PCR fragment were digested with BamHI and BstXI and ligated to create the HA-KLF4 vector. For the 2nd round of subcloning, the 3XFlag DNA sequence was amplified from the p3XFlag-CMV vector (Sigma) with a second set of primers (Oligo #3 and #4). Both the HA vector containing KLF4 and the amplified 3XFlag DNA fragment were digested with BamHI and NheI and ligated in frame, resulting in the

3XFlag.HA:KLF4 construct. The authenticity of the 3XFlag.HA:KLF4 construct was

57 verified by DNA sequencing and verification of established target gene effects.

Adenoviral production and purification was performed by Welgen, Inc. The virus titer was calculated at 1012 vpu/ml. Empty vector control adenovirus (AdGFP) containing

GFP was also produced and supplied by Welgen, Inc. Cells were infected at MOIs ranging from 200-1000. Oligo #1: 5‟-ATG CGG ATC CGC CAC CAT GGC TAG

CTA CCC CTA CGA CGT GCC CGA CTA CGC CGC TGT CAG CGA CGC G-3‟,

Oligo #2: 5‟-AAT ACC AGG TCT GTG GCC ACG GT-3‟, Oligo #3: 5‟- ATG CGG

ATC CGC CAC CAT GGA CTA CAA GGA C-3‟, Oligo #4: 5‟-GTA CGC TAG CCT

TGT CAT CGT CGT C-3‟.

RNA preparation, cDNA Synthesis, and qRT-PCR. Total RNA was isolated from cells using Trizol Reagent (Invitrogen), and cDNA was synthesized from 1μg of total

RNA using Superscript II reverse transcriptase (Invitrogen) according to manufacturer‟s protocols. For real-time PCR, relative gene expression was determined using the

StepOne Plus RT-PCR System (Applied Biosystems) following the manufacturer‟s protocol. The following Taqman assays (Applied Biosystems) were used: KLF4

(Hs00358836_m1) and E-cadherin (Hs001704423_m1). RNA levels were normalized against human TATA box binding protein (TBP).

Immunoblotting and immunofluorescence. Cells were lysed in RIPA buffer and protein concentration was determined using the BioRad protein assay (BioRad). Lysates were run on a 12% SDS-PAGE gel and transferred to PVDF. Antibodies used for immunoblotting include: anti-rabbit KLF4 (Millipore), anti-β-actin (Sigma),

58 anti-E-cadherin and anti-p120 (BD Transduction Laboratories), anti-N-cadherin, anti-Cytokeratin 8/18 and anti-β-catenin (Cell Signaling). For immunofluorescence, cells were grown on glass coverslips, fixed in methanol at -20°C for 10 min, permeabilized and blocked in .05% Tween20/1% BSA/1x PBS for 15 min and incubated overnight at

4°C with mouse anti-E-cadherin. Samples were washed three times with PBS and incubated with a FITC conjugated secondary antibody (Jackson Immuno-Research) at

RT° for 1h. Coverslips were mounted with Vectashield hard set mounting media containing DAPI (Vector Laboratories) and fluorescence was visualized using a Leica inverted microscope.

Growth rate, proliferation and cell cycle analysis. To measure cell growth, cells were trypsinized at indicated times, and counted using a hemocytometer. Bromodeoxyuridine

(BrdU) incorporation was performed by incubating cells with 10μM BrdU (Sigma-

Aldrich) for 2h at 37◦C, prior to fixation and labeling with an anti-BrdU antibody (BD

Biosciences) according to manufacturer‟s protocol. Secondary detection with either goat- anti mouse Alexa594 (Molecular Probes) or FITC-conjugated secondary antibody

(Jackson Immuno-Research) was performed in the dark at RT° for 30 min. Mounting media containing DAPI (Vector Laboratories) was used to counterstain nuclei and cells were counted using a Leica inverted microscope. Cell cycle analysis using flow cytometry was carried out as previously described (Montanez-Wiscovich et al., 2009).

Transwell migration and invasion assays- Cell migration was performed using 8.0-μm pore size polycarbonate membrane transwell inserts in a 24 well plate (Costar). Non- migrated cells were removed with a cotton swab. Inserts were fixed and stained with

Hema3 (Fisher Scientific) and migrated nuclei were counted. Invasion assays were performed as above using BD MatrigelTM Invasion Chambers (BD Biosciences) per manufacturer‟s instructions.

Generation of E-cadherin reporter construct and luciferase reporter assays.

Genomic DNA was isolated from MCF-10A cells and the E-cadherin proximal promoter was PCR amplified using the following primers: forward, 5‟-[XhoI]GTG AAA GAG

TGA GCC CCA TC-3‟ and reverse, 5‟-[HindIII]CAC AGG TGC TTT GCA GTT C-3‟.

Both pGL3basic-luciferase reporter plasmid (Promega) and PCR products were digested with XhoI and HindIII and ligated together to form the (-359/+30) Ecad-Luc reporter.

Cells were transfected in 6 well dishes, using Lipofectamine 2000 (Invitrogen) according to manufacturer‟s protocol, with 0.2 μg of the Renilla luciferase reporter vector phRG-

TK (Promega) as a control and either 1.0 μg of pGL3basic or 1.0 μg of the Ecad-Luc reporter gene. Luciferase assays were performed using the Dual Luciferase Assay

System (Promega).

Chromatin immunoprecipitation (ChIP). ChIP assays were performed as previously described, with modification (Scacheri et al., 2006). Briefly MCF-10A or MDA-MB-231 cells were grown to near confluency on 150-mm dishes. Cells were crosslinked and lysed, followed by sonication (Vertis Versonic 300, Output 4, 25s, 6x) in 3 ml of lysis buffer. After centrifugation, samples were precleared with 100 μl protein A/G-agarose

(Calbiochem) and rotated for 2 h at 4°C. Precleared chromatin was then divided into aliquots and immunoprecipitated overnight with specific antibodies to KLF4 (H-180)

(Santa Cruz Biotechnology), anti-Flag M2 (Sigma) or control IgG (normal rabbit IgG or normal mouse IgG) (Jackson Immuno-Research Laboratories). Supernatant (1/5) from control IgG sample was processed as Input. After washing and reversal of crosslinks, followed by phenol-chloroform extraction and ethanol precipitation, DNA was resuspended in 65 μl sterile H20. PCR was performed using 5 μl of immunoprecipitated

DNA as template and the following gene-specific primers corresponding to –170/+10 of the human E-cadherin promoter, based on GenBank™ accession number

L34545.1; forward: (5‟-TAG AGG GTC ACC GCG TCT AT-3‟), reverse: (5‟-TCA

CAG GTG CTT TGC AGT TC-3‟).

Statistical analyses. Statistical analyses were performed using two-tailed Student‟s t-test with P-values less than 0.05 considered significant.

2.3 Results

KLF4 is required for the maintenance of mammary epithelial cell morphology. To directly assess the role of KLF4 in non-transformed mammary epithelial cells, we used lentiviral and retroviral vector mediated shRNA to create stable MCF-10A cell lines with suppressed KLF4 expression. As previously reported for other cell lines (Shields et al.,

1996), serum deprivation induced KLF4 expression (Figure 2.1A). However, KLF4 protein and mRNA were suppressed by greater than 60%, both in the presence or absence of serum (Figure 2.1A and 2.1B), using two different KLF4-specific RNAi approaches

(shRNA and mirRNA) that targeted different regions of the KLF4 mRNA. MCF-10A

61 cells grown in monolayer cultures form cobblestone clusters of expanding colonies that become more cuboidal and tightly packed as confluency increases (Imbalzano et al.,

2009). Likewise, cells expressing a non-silencing shRNA (shNS and MirNS) maintained a cobblestone-like morphology (Figure 2.1C). In contrast, KLF4 silencing (shKLF4 and

MirKLF4) resulted in an elongated, fibroblastic morphology reminiscent of EMT, a process that is also associated with increased migration. To assess this feature of EMT, we performed transwell migration assays. KLF4 silencing resulted in a greater than 2 fold increase in migration compared to both the parental and control shNS or MirNS cells

(Figure 2.1D).

KLF4 silencing results in loss of acinus formation and decreased proliferation of non-transformed MCF-10A mammary epithelial cells. In vivo, breast epithelium exists within a complex ductal structure that receives multiple inputs from both nearby epithelial cells as well as the adjacent stroma. This three-dimensional growth can be simulated in vitro using matrigel or other ECM supports. MCF-10A mammary epithelial cells grown in three dimensional cultures proliferate to form polarized acini, and by day

10, the inner cells have undergone apoptosis/autophagy to form a functional lumen

(Debnath et al., 2003). We used this 3D culture method to determine if loss of KLF4 would alter acinus formation and growth. While parental and shNS control cells formed multiple acini with hollow lumens, shKLF4 cells rarely formed clusters greater than 4-6 cells (Figure 2.2A). By day 4, when acinus formation was clearly visible in the parental and shNS control cells, fewer than 5% of the KLF4 knock-down cells had formed colonies beyond 4 cells (Figure 2.2B).

KLF4 has been shown to inhibit proliferation and promote differentiation of skin and colonic epithelium (Segre et al., 1999; Shie et al., 2000b). Thus it was surprising that loss of KLF4 led to a decrease in acinus formation and growth of MCF-10A cells (Figure

2.2A and 2.2B). A similar inhibition of growth was observed in monolayer cultures following KLF4 silencing (Figure 2.2C). To determine whether this effect was due to alterations in proliferation, or changes in apoptosis, we used BrdU incorporation assays to assess the proliferation rate as well as Propidium Iodide staining to perform FACS-based analysis of the cell cycle. In comparison to control shNS cells, BrdU incorporation was reduced over 40% in the shKLF4 and MirKLF4 cells (Figure 2.2D and data not shown), with no significant change in percentage of cells in the subG1 population (Figure 2.2E).

Rather, silencing of KLF4 induced a statistically significant G1-block in the cell cycle.

Together, these results identify a previously unspecified requirement for KLF4 in maintaining proliferation and G1/S phase progression of non-transformed mammary epithelial cells.

KLF4 is required for the expression of E-cadherin in mammary epithelial cells.

Cadherin mediated signaling is an integral component of tissue morphogenesis and homeostasis (Gumbiner, 2005). In the breast, organization of the mammary epithelium is dependent upon maintenance of adherens junctions through homotypic interactions between E-cadherin on adjacent cells (Knudsen and Wheelock, 2005). In addition, sustained E-cadherin expression is necessary to maintain basal proliferation of mammary epithelial cells (Boussadia et al., 2002; Liu et al., 2006). The inability of cells to organize into acini following KLF4 silencing, as well as the decreased proliferation and acquisition

63 of morphological and migratory changes that are indicative of an EMT, prompted us to evaluate whether expression of E-cadherin, as well as other adherens junction proteins, was altered in the KLF4-silenced cells. Indeed, both protein and mRNA levels of E- cadherin were markedly reduced in both shKLF4 and MirKLF4 cells compared to shNS and MirNS control cells (Figure 2.3A and 2.3B). Suppression of E-cadherin was further confirmed by the complete lack of membrane staining for this protein in the KLF4 knock- down cells (Figure 2.3C).

In addition to the loss of E-cadherin, KLF4 silencing resulted in a concomitant appearance of N-cadherin (Figure 2.3A). This process, known as “cadherin switching”, is often associated with EMT (Hazan et al., 2004; Maeda et al., 2005). Furthermore, β- catenin levels were decreased. There was also a switch in the overall isoform pattern of p120 expression, with appearance of the mesenchymal-specific isoform 1. Together, these changes suggest that KLF4 plays an important role in preventing EMT of mammary epithelial cells through maintenance of E-cadherin and the adherens junction complex.

KLF4 binds the proximal GC-rich region of the E-cadherin promoter to transcriptionally activate E-cadherin expression. The Krüppel-like family of transcription factors regulates a diverse set of genes through direct binding to GC-rich promoter regulatory regions containing a CACCC consensus sequence (Duyen et al.,

2000; Kaczynski et al., 2003). Furthermore, several Krüppel-like factors have previously been shown to regulate E-cadherin gene expression, including KLF6 and KLF8 (DiFeo et al., 2006; Wang et al., 2007). KLF4 silencing in MCF-10A cells greatly reduced

E-cadherin mRNA levels suggesting it may be necessary for sustaining E-cadherin transcription. To test this, the (-359/+30) proximal E-cadherin promoter, which contains several putative KLF4 binding sites, was linked to a luciferase reporter cassette (Ecad-

Luc) (Figure 2.4A) and transiently transfected into MCF-10A cells with and without

KLF4 silencing. Reporter activity was decreased more than 10 fold in shKLF4 cells compared to the shNS control cells. Conversely, overexpression of KLF4 in the MCF-

10A parental cells resulted in a greater than 2 fold induction of luciferase expression

(Figure 2.4B). To determine if KLF4 interacts with the endogenous E-cadherin promoter, we performed chromatin immunoprecipitation (ChIP) analyses. Primers flanking the GC-boxes of the E-cadherin promoter were used to PCR amplify chromatin fragments enriched by KLF4 binding to this region, relative to rIgG control (Figure

2.4C).

Forced expression of KLF4 in the highly metastatic MDA-MB-231 breast tumor cells restores E-cadherin expression and epithelial morphology, while inhibiting migration and invasion. We next asked whether KLF4 could modulate E-cadherin expression, migration and invasion in mesenchymal-like MDA-MB-231 breast cancer cells that lack expression of endogenous E-cadherin. These cells also express relatively low levels of KLF4 compared with MCF-10A cells (Figure 2.5A) (Miller et al., 2001).

Forced expression of KLF4 resulted in the induction of E-cadherin protein expression

(Figure 2.5B). In addition, KLF4 increased expression of cytokeratin 18 (Krt18), another phenotypic marker of epithelial cells. Furthermore, these cells acquired an epithelial morphology as early as 24h post transduction with AdKLF4, compared to

AdGFP control (Figure 2.5C). Similar to the MCF-10A cells, we found KLF4 regulation of E-cadherin in the MDA-MB-231 cells to be transcriptional, as forced expression of

KLF4 induced E-cadherin mRNA (Figure 2.6A), as well as increased activity of the

Ecad-Luc promoter (Figure 2.6B). While induction of this reporter by KLF4 suggested direct transcriptional regulation of the E-cadherin gene, this gene is hypermethylated in

MDA-MB-231 cells (data not shown) (Liu et al., 2005b). We therefore performed ChIP assays to determine if KLF4 could bind the endogenous E-cadherin promoter in this context. Following transduction with either control AdGFP or AdKLF4 (flag/HA- tagged), chromatin fragments were co-immunoprecipitated with an anti-Flag antibody and amplified by PCR with the primers described in Figure 2.4A. ChIP analysis revealed enrichment of the E-cadherin promoter in KLF4 overexpressing cells (AdKLF4) immunoprecipitated with flag/HA-tagged KLF4 when compared to the control (AdGFP) transduced cells (Figure 2.6C).

Restoration of E-cadherin alone is sufficient to reduce invasion of MDA-MB-231 cells in vitro, and forced expression of either Krt18 or E-cadherin inhibits primary tumor growth and metastasis (Buhler and Schaller, 2005; Mbalaviele et al., 1996). We postulated that forced expression of KLF4, and in turn induction of E-cadherin, as well as Krt18, would also suppress the aggressive, mesenchymal properties of these cells. Indeed, forced

KLF4 expression repressed both migration and invasion of MDA-MB-231 cells in vitro

(Figure 2.6D and 2.E), indicating that KLF4 activates a transcriptional program in breast tumor cells that elicits a less invasive, more differentiated epithelial phenotype.

2.4 Discussion

There is strong evidence for KLF4 as a tumor suppressor in several human cancers, including gastric and colorectal (Dang et al., 2000; Dang et al., 2003; Zhao et al., 2004), yet an obvious disparity exists among studies examining KLF4 expression during the progression of breast cancer (Akaogi et al., 2009; Foster et al., 2000; Pandya et al., 2004).

Moreover, the functional role of KLF4 in non-transformed mammary epithelial cells has not been examined. We have shown that loss of KLF4 in MCF-10A cells results in altered cell morphology, loss of E-cadherin and increased migration, all of which are canonical features of EMT. However, these changes did not result in transformation, as

KLF4-deficient cells were unable to form colonies in soft agar (data not shown). In fact, we found that KLF4-silenced cells were impaired in their ability to proliferate (Figure

2.2C and 2.2D).

Previous studies have demonstrated that KLF4 inhibits proliferation of colon cancer cells by blocking G1/S progression of the cell cycle (Chen et al., 2001). Paradoxically, the results herein indicate that KLF4 is coordinately required for maintenance of proliferation and E-cadherin expression in non-transformed mammary epithelial cells. It is possible that the decrease in proliferation observed with KLF4 silencing is due to the loss of

E-cadherin. Similar to KLF4, E-cadherin has both growth inhibitory (Perrais et al., 2007;

St.Croix et al., 1998) and growth promoting roles (Fournier et al., 2008; Liu et al., 2006).

These functions of E-cadherin are dependent upon its level of expression as well as cellular context. While KLF4 expression is relatively low in proliferating MCF-10A

67 cells, it appears necessary to maintain a basal proliferative rate. However, as observed in other cell types (Shie et al., 2000a; Shields et al., 1996), we also found that growth arrest, in response to serum deprivation, was accompanied by a concomitant increase in KLF4 protein (Figure 2.1A). These data suggest that like E-cadherin, KLF4 may be necessary both for proliferation and growth inhibition within the same cells, dependent upon its absolute level of expression.

The ability of MCF-10A cells to form functional acinar structures in three-dimensional culture has provided significant insights into stroma-epithelial interactions, mechanisms and pathways in the development of normal mammary tissue architecture and breast tumorigenesis. KLF4 silencing resulted in a 40% reduction in proliferation of monolayer cultures (Figure 2.2D) however, less than 10% of these cells were able to form acini in matrigel (Figure 2.2A). Even after more than 20 days in culture, by which time normal acini become senescent, relatively few KLF4 knock-down cells had progressed to form acini (data not shown). These results suggest that decreased proliferation, in response to reduced KLF4 expression, is not solely responsible for the complete lack of acinus formation and progression.

Several possibilities exist that may explain this outcome. First, KLF4 appears to play an integral role in the maintenance of the stem or pluripotent state (Takahashi and

Yamanaka, 2006; Wei et al., 2009). MCF-10A cells have been characterized as stem- like, because a subpopulation of these cells express cytokeratin 5/6 (Chua et al., 2006), a marker of breast epithelial progenitor cells (Boecker et al., 2002). In fact, mammosphere

68 formation is a well established model for assessing mammary stem/progenitor cells

(Dontu et al., 2003a). Therefore, KLF4 may be required to maintain the population of stem/progenitor cells that can differentiate into acini. Conversely, KLF4 is also required for the terminal differentiation of many cell types during development and the inability of

KLF4-silenced cells to form acini in 3D cultures may be associated with incomplete terminal differentiation of the mammary luminal phenotype. Lastly, the increased migratory capacity of the KLF4 knock-down cells (Figure 2.1D), in conjunction with the subsequent loss of E-cadherin (Figure 2.3), may also prevent these cells from generating the intercellular adhesions and signals required for polarized acinus formation.

E-cadherin regulates epithelial morphogenesis (Gumbiner, 2005), and disruption of acinus formation has been observed in several studies where E-cadherin levels are suppressed (Bennett et al., 2008; Chua et al., 2006) Thus, KLF4 silencing may block acinus formation through reduced cell-cell adhesions.

Several studies have recently shown a link between the acquisition of stem cell properties and the process of EMT (Mani et al., 2008; Morel et al., 2008). Hence, the induction of

EMT upon KLF4 knockdown suggests that KLF4 may also inhibit formation or self- renewal of mammary stem cells. This possibility contrasts with the established “stem- maintaining” function of KLF4 in iPS cells, as well as our findings that loss of KLF4 prevents mammary acinar formation. It is likely that the expression of other stem cell factors, in addition to KLF4, play important roles in mediating the relationship between

EMT and stem cell properties. Thus, further studies will be necessary to reveal the context-specific roles of KLF4 in regulating stem cell properties in the mammary gland.

KLF4 is highly expressed in epithelial cells, but only transiently or not at all in mesenchymal cells (Garrett-Sinha et al., 1996). Moreover, forced expression of KLF4 in colon cancer cells induces expression of several genes associated with the epithelial phenotype (Chen et al., 2003). We found that silencing of KLF4 induced EMT in MCF-

10A mammary epithelial cells as evidenced by morphological alterations, increased migration, and loss of E-cadherin (Figure 2.1 and 2.3). It is noteworthy that TGF-β, a well known inducer of EMT, represses KLF4 expression (Feinberg et al., 2005). TGF-β signals through both Smad-dependent and independent pathways to also inhibit E- cadherin expression during EMT (Medici et al., 2006; Nawshad et al., 2005; Peinado et al., 2003). In addition, KLF4 can bind to Smad3 and prevent activation of Smad- responsive promoters (Hu et al., 2007). Together, these data suggest that suppression of

KLF4 expression may be required for EMT of mammary epithelial cells. Recently it has been shown that another Krüppel-like factor, KLF17, also acts as a negative regulator of

EMT in breast cancer, although its ability to regulate E-cadherin transcription remains unknown (Gumireddy et al., 2009). It will be interesting to determine if these two factors cooperate to control the epithelial phenotype of breast cancer cells. The results presented herein demonstrate that KLF4 silencing induces EMT in the absence of any additional stimuli, suggesting that KLF4 may be necessary and sufficient to prevent EMT of non- transformed mammary epithelium.

The loss of E-cadherin mRNA observed with KLF4 silencing prompted us to examine the ability of KLF4 to transcriptionally regulate the E-cadherin gene. Using both reporter

70 and ChIP assays (Figure 2.4) we found that endogenous KLF4 binds to and activates the

E-cadherin promoter. While these results do not rule out the possibility that KLF4 indirectly activates the promoter through interactions with other transcription factors, it is likely this interaction is through binding to the KLF4 consensus elements in the proximal

(-170/+10) region of the promoter. Several reports have demonstrated that other Sp1 family members, including KLF6 and KLF8 regulate the E-cadherin promoter in ovarian carcinoma cells (SKOV-3) and MCF-10A cells, respectively (DiFeo et al., 2006; Wang et al., 2007). KLF6 activates E-cadherin transcription similar to KLF4 while KLF8 binding results in repression of the E-cadherin promoter. These data demonstrate the distinct abilities of individual Krüppel-like factors to modulate expression of the same target genes.

Repression of E-cadherin gene expression in MDA-MB-231 breast cancer cells is due, in part, to promoter hypermethylation as well as transcription factors such as Snail, Zeb1 and δEF that bind to the E-boxes flanking the putative KLF4 binding sites (Liu et al.,

2005b). These proteins modify chromatin structure through recruitment of repressor complexes and act as a major regulatory mechanism of E-cadherin suppression during

EMT (Peinado et al., 2007). In contrast to the above transcriptional suppressors, KLF4 can interact with chromatin remodeling proteins such as the histone acetyltransferase, p300 (Evans et al., 2007), to promote local unwinding of DNA. Hence, it is feasible that the transcriptional activation of E-cadherin by KLF4 is through cooperation with p300/CBP. Of note, Snail, a key inducer of EMT, suppresses KLF4 expression in colon cancer cells (De Craene et al., 2005), further suggesting that loss of KLF4 may be

71 necessary for epithelial cells to undergo mesenchymal changes that promote invasion and migration.

We have focused on KLF4‟s regulation of E-cadherin because silencing of E-cadherin alone in epithelial cells is sufficient to induce a full EMT (Lehembre et al., 2008). This would suggest that restoration of E-cadherin in the KLF4 knockdown cells could revert their mesenchymal phenotype. However, these cells were deficient in their ability to target exogenously expressed E-cadherin to the membrane (data not shown). Decreased levels of β-catenin in these cells (Figure 2.3A) could contribute to this mislocalization as

β-catenin is required for proper membrane targeting of E-cadherin (Chen et al., 1999). In a reciprocal fashion, membrane bound E-cadherin anchors β-catenin at the adherens junction and prevents nuclear localization or degradation in the cytoplasm (Orsulic et al.,

1999). Similarly, sustained p120 expression is necessary to stabilize E-cadherin at the plasma membrane (Ireton et al., 2002), while loss of E-cadherin during EMT causes mislocalization of p120 (Thoreson and Reynolds, 2002), resulting in increased migration.

The acquisition of the mesenchymal-specific p120 isoform 1 in the KLF4-silenced cells

(Figure 2.3A) supports an epithelial-to-mesenchymal transition and likely contributes to the increased migration of these cells (Mo and Reynolds, 1996; Yanagisawa et al., 2008).

Together, these data indicate that KLF4 is necessary to maintain several components of the adherens junction, in part through sustained E-cadherin gene expression.

Forced expression of epithelial proteins, such as E-cadherin and Krt18, block tumor growth and metastasis (Buhler and Schaller, 2005; Frixen et al., 1991). Krt18 belongs to

72 a large group of cytokeratins that are coordinately induced with forced expression of

KLF4 in colon cancer cells (Chen et al., 2003). In addition to restoring E-cadherin, we found that forced expression of KLF4 also increased Krt18 expression in MDA-MB-231 cells (Figure 2.5B). These molecular alterations support the conversion of the MDA-

MB-231 cells to a more epithelial morphology, with an inhibited migratory and invasive capacity (Figure 2.6D and E). Thus, we conclude that the absolute levels of KLF4 act as a rheostat in determining the epithelial or mesenchymal phenotype of breast cancer cells.

These findings suggest that reduced expression of KLF4 during breast tumorigenesis provides a pro-migratory and invasive foundation from which metastatic progression can occur. This is consistent with the reduced expression of KLF4 mRNA that occurs with increasing grade of breast tumors (Akaogi et al., 2009), which are intrinsically more metastatic. Furthermore, African American women have a higher rate of breast cancer related morbidity when compared to Caucasian women, due to increased metastases

(Yancy et al., 2007). In an effort to identify expression profiles that may be unique to metastatic breast cancer in African American women, Yancy et al. identified KLF4 as being decreased in cell lines derived from these tumors. These data, in conjunction with our findings that KLF4 regulates EMT in mammary cells, highlight the necessity for future studies directed at identifying the signals responsible for suppression of KLF4 during breast cancer progression and metastasis.

2.5 Acknowledgements

This work was supported by National Institutes of Health grants (CA090398) to RAK,

CURE supplement to (CA090398) to EJ, HL075427 and HL086548 (MKJ) and the Susan

G. Komen Foundation (BCTR108306) to RAK. JLY was the recipient of a Department of Defense (DOD) pre-doctoral fellowship (DAMD17-03-1-0302). We are grateful for assistance from the Case Comprehensive Cancer Center core facilities (P30 CA43703).

We thank Dr. Ed Stavnezer for providing the TMP-tTA retroviral vector and Dr. Peter

Scacheri for technical support in ChIP experiments.

Figure 2.1 KLF4 is required for the maintenance of mammary epithelial cell morphology. (A) Immunoblot analysis of KLF4 and β-actin in MCF-10A cells stably transduced with either non-specific shRNA control (shNS and MirNS) or shRNA against

KLF4 (shKLF4 and MirKLF4). Cells were cultured in complete growth medium, +/-

10% serum. (B) qRT-PCR analysis of KLF4 mRNA in shNS control and shKLF4 cells.

Relative KLF4 mRNA levels were normalized to the human TATA binding protein

(TBP). (C) Mesenchymal morphology induced by loss of KLF4 in MCF-10A cells.

Cells were plated at low (top row) and high (bottom row) densities. Cells were monitored for morphological changes using phase-contrast microscopy. (D) Increased migration of shKLF4 and MirKLF4 cells compared to shNS and MirNS controls. A total of 1x105 cells were suspended in 100 μl complete medium, seeded on transwell migration inserts, and allowed to migrate towards complete medium for 20h. Five fields per insert were counted. Panels (B) and (D) represent the averages and standard deviations from three independent experiments done in triplicate. *P<0.02, **P<0.002, ***P<0.0001.

Figure 2.1

Figure 2.2 KLF4 silencing results in loss of acinus formation and decreased proliferation of mammary epithelial cells. (A) For 3D cultures, 2.5x105 cells were plated using the overlay method (Debnath et al., 2003). Photomicrographs were taken after 10 days in culture. Insets are fluorescent images of the control and shKLF4 cells which also express GFP. (B) Quantitation of mammary acini formation. By day 4, the majority of the cells in shNS control cultures had formed visible clusters at least 8 cells, while less than 10% of shKLF4 cells formed clusters surpassing 4 cells in size. Error bars represent the standard deviations of two experiments done in duplicate. (C) Growth rate determination of shNS and shKLF4 cells. 1x104 cells were plated in complete medium.

At 24h time intervals, cells were trypsinized and counted. (D) BrdU incorporation of shNS and shKLF4 cells. Cells were plated at 50% confluency and allowed to grow for

72h prior to incubation with BrdU. Slides were processed for immunofluorescence and scored for % BrdU positivity. (E) Cell cycle analysis of shNS (black bars) and shKLF4

(gray bars) cells using propidium iodide uptake and flow cytometry. Cells were plated at

50% confluency and allowed to grow for 48h before being analyzed. Bars in panels (C)

- (E) represent the averages and standard deviations from three independent experiments performed in triplicate. *P<0.05, **P<0.005, ***P<5.0x10-5.

Figure 2.2

Figure 2.3 KLF4 is required to sustain E-cadherin expression in non-transformed mammary epithelial cells. (A) Western blot analysis of E-cadherin, N-cadherin, p120,

β-catenin, KLF4 and β-actin and (B) qRT-PCR analysis of E-cadherin mRNA in shNS and MirNS control vs. shKLF4 and MirKLF4 cells. Graph represents the average fold change and standard deviation from three independent experiments. *P<0.02,

**P<1.0x10-7. (C) Immunofluorescence staining for E-cadherin (green) in shNS control and shKLF4 knockdown cells. Nuclear DNA is counterstained with DAPI (blue).

Figure 2.3

Figure 2.4 KLF4 binds and activates the E-cadherin promoter. (A) Schematic representation of the (-359/+30) proximal E-cadherin luciferase construct (Ecad-Luc).

Black boxes (KLF4) represent GC-boxes containing putative KLF4 target sites.

Numbered boxes depict two E-boxes located near the transcriptional start site. Grey arrows depict the location of the forward and reverse primers used for ChIP PCR amplification. (B) E-cadherin promoter activity is lost in shKLF4 cells. The Ecad-Luc reporter plasmid or pGL3basic was cotransfected with a renilla expressing control

(phRG-TK) into MCF-10A shNS control and shKLF4 cells. For KLF4 over-expression, parental MCF-10A cells were transfected 24h prior to transduction with either AdGFP control or AdKLF4. Luciferase values, normalized to renilla levels, were expressed as fold change over shNS or AdGFP controls. Error bars represent standard deviations of three independent experiments performed in triplicate (*P<1.0x10-4, **P<1.0x10-5).

KLF4 antibody or rIgG serum was used to immunoprecipitate DNA-protein complexes from MCF-10A cells. Binding of KLF4 at the E-cadherin promoter was enriched over rIgG control. Representative amplification of PCR products, using the primers described in panel (A) is shown. A molecular weight ladder is shown in the far right lane.

Independent ChIP experiments were performed at least two times.

Figure 2.4

Figure 2.5 KLF4 induces expression of E-cadherin protein and a transition to epithelial morphology in the mesenchymal-like MDA-MB-231 breast cancer cells.

(A) Western blot analysis comparing KLF4 and E-cadherin levels in MCF-10A cells and

MDA-MB-231 cells. (B) Western blot of E-cadherin, KLF4, Krt18 and β-actin in

MDA-MB-231 cells 72h post transduction with empty vector control (AdGFP) or flag/HA-tagged KLF4 (AdKLF4) adenovirus. (C) Phase contrast images of MDA-MB-

231 cells transduced with AdGFP or AdKLF4 adenovirus. Images were captured 24h post transduction.

Figure 2.5

Figure 2.6 KLF4 transcriptional activation of E-cadherin results in decreased migration and invasion of MDA-MB-231 cells. (A) qRT-PCR of KLF4 and E- cadherin mRNA levels in MDA-MB-231 cells transduced with either AdGFP control or

AdKLF4 adenovirus. (B) Activation of the Ecad-Luc reporter in MDA-MB-231 cells overexpressing KLF4. The (-359/+30) Ecad-Luc reporter vector or pGL3 control was cotransfected with a renilla expressing control (phRG-TK) into MDA-MB-231 cells.

Cells were transduced with either AdGFP control or AdKLF4 12h later. Luciferase and renilla activities were quantified 48h post transfection. Luciferase values were normalized to renilla levels and expressed as fold change over AdGFP control cells. (C)

Chromatin immunoprecipitation of KLF4 at the E-cadherin promoter in MDA-MB-231 cells. A Flag antibody was used to immunoprecipitate DNA-protein complexes from both

AdGFP and flag/HA-tagged AdKLF4 transduced MDA-MB-231 cells. Chromatin fragments were PCR amplified with the same E-cadherin promoter primers described in

Figure 2.4A. AdGFP cells served as a negative control. (D) Migration and (E) invasion of AdGFP and AdKLF4/MDA-MB-231 cells. Cells were transduced with AdGFP or

AdKLF4 48h prior to trypsinization and plating onto transwell supports for migration, or matrigel-coated supports for invasion. Cells were allowed to migrate or invade through supports for 6 or 24 hrs, respectively. For panels (A) and (C), cells were either harvested for RNA or fixed for ChIP analysis at 72h post-transduction. Panel (A) is a representative of three independent experiments performed in triplicate. Error bars represent the standard deviations. For panel (B), error bars represent the standard deviations of three independent experiments performed in triplicate. For panels (D) and

(E), five fields were counted per insert. Data represent the average fold change and

85 standard deviations from three independent experiments performed in triplicate.

*P<.005, **P<5.0 x 10-7.

Figure 2.6

CHAPTER 3

KRÜPPEL-LIKE FACTOR 4 INHIBITS TUMORIGENIC PROGRESSION AND

METASTASIS IN A MOUSE MODEL OF BREAST CANCER

(Yori et al., Cancer Research (submitted), November 2010)

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that functions as an oncogene or tumor suppressor in a highly tissue-specific, cell dependent manner.

However, its precise role in breast cancer and metastasis is unknown. Here we show that KLF4 expression is lost in a mouse model of HER2/NEU/ERBB2 positive breast cancer. To determine whether enforced KLF4 expression could alter tumor latency in these mice, we used a doxycycline inducible expression model in the context of the

MMTV-Neu transgene. Surprisingly, tumors that developed in this model also lost

KLF4 expression, suggesting negative selection for sustained expression. In contrast, transient adenoviral expression of KLF4 in the 4T1 orthotopic mammary cancer model significantly attenuated primary tumor growth as well as micrometastases to the lungs and liver. These results can be attributed, in part, to decreased proliferation and increased apoptosis. We have previously reported that

KLF4 inhibits epithelial-to-mesenchymal transition (EMT), a preliminary step in metastatic progression. Overexpression of KLF4 in 4T1 cells led to a significant reduction in the expression of Snail, a key mediator of EMT and metastasis.

Conversely, KLF4 silencing increased Snail expression in the non-transformed

MCF-10A cell line. Collectively, these data demonstrate the first functional, in vivo evidence for KLF4 as a tumor suppressor in breast cancer cells. Furthermore, our

88 findings suggest an inhibitory role for KLF4 during breast cancer metastases that functions, in part, through repression of Snail.

3.1 Introduction

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that regulates a multitude of processes in normal tissue including proliferation, differentiation, apoptosis, tissue homeostasis and self-renewal (Garrett-Sinha et al., 1996; Ghaleb et al., 2007b;

Jiang et al., 2008a; Segre et al., 1999; Shields et al., 1996), to name a few. KLF4 can also have both tumor suppressive and oncogenic roles in cancer. KLF4 was first identified as a tumor suppressor, due to frequent loss of expression in colon, esophageal, gastric, bladder, prostate and lung cancers (Hu et al., 2009; Ohnishi et al., 2003; Schulz and Hatina, 2006; Wei et al., 2005; Yang et al., 2005a; Zhao et al., 2004). Subsequent studies however have suggested a role for KLF4 as an oncogene in other tissues, including breast (Foster et al., 2000; Rowland et al., 2005). Paradoxically, KLF4 was most recently found to suppress estrogen-dependent breast cancer cell growth, and its expression is inversely correlated with increasing tumor stage and grade (Akaogi et al.,

2009), further confounding its role in this disease.

In human mammary epithelial cells, KLF4 is a transcriptional activator of E-cadherin and suppressor of epithelial-to-mesenchymal transition (EMT) (Yori et al., 2010a). This process, whereby cells acquire a fibroblast-like morphology and altered molecular signature associated with reduced cellular adhesion and increased motility, is critical not

89 only during development, but may also be involved in cancer progression (Thiery et al.,

2009; Yang and Weinberg, 2008). While controversy still persists over the existence of

EMT during tumorigenesis and metastasis, compelling evidence supporting its relevance in human carcinomas, including breast, prostate, melanoma and others has been reported

(see review (Blick et al., 2008)). A hallmark of EMT is the loss of E-cadherin expression, which is transcriptionally suppressed by Snail (Cano et al., 2000). In breast cancer, both loss of E-cadherin and increased Snail expression are predictive of poor prognosis (Heimann et al., 2000) . Furthermore, Snail correlates with increasing tumor grade, lymph node status, and metastasis (Blanco et al., 2002).

Disease recurrence and distant metastases are the leading cause of death in breast cancer patients. One subtype of breast cancer that is highly metastatic involves the amplification and overexpression of the HER2/Neu gene in 15-30% of all breast cancer cases. Elevated expression of this receptor tyrosine kinase is associated with a higher risk of recurrence, as tumors which are initially responsive to trastuzumab (Herceptin) often become resistant. One mechanism by which Her2/Neu-dependent tumors can recur is through upregulation of Snail (Moody et al., 2005). However, the mechanisms by which Snail is induced are not well understood. Here we show that KLF4 expression is suppressed in a mouse model of HER2/Neu-induced tumorigenesis. We also demonstrate, in the clinically relevant, orthotopic 4T1 xenograft model, that forced expression of KLF4 inhibits primary tumor growth and metastases. In vitro analyses revealed that KLF4 is able to suppress Snail expression, suggesting a mechanism whereby loss of KLF4 leads to increased breast cancer metastasis.

3.2 Materials and Methods

Transgenic mice. All mice were housed in microisolator-plus units under pathogen- free conditions in a 12 hour light/dark cycle. Food and water were provided ad libitum.

The NeuN mice (FVB/N-TgN(MMTV-neu)202Mul) containing the rat proto-oncogene c- neu transgene (Guy et al., 1992), and the NeuT mice (FVB/N-Tg(MMTV-

Erbb2)NK1Mul/J) containing the activated c-neu transgene (Muller et al., 1988), both targeted to mammary epithelium by the MMTV-LTR promoter, were purchased from

Jackson Laboratories. The TRE-KLF4 and MMTV-rtTA (MTB) mice, both on the

FVB/N genetic background, have been previously characterized (Jaubert et al., 2003;

Segre et al., 1999) Mice were sacrificed once initial tumors had reached 1.5 cm by caliper measurement. All animal studies were approved by the Case Western Reserve

University Institutional Animal Care and Use Committee.

Microarray and expression analyses. Microarray analyses comparing age-matched wild type glands (AMWT), hyperplastic tissue adjacent to the tumor and NeuN tumors has been described in detail (Landis et al., 2005). Real time quantitative PCR (qRT-

PCR) was performed on RNA harvested from tissue and cells as previously described

(Landis et al., 2005; Yori et al., 2010a) using mouse and human gene-specific Taqman assays (Applied Biosystems). RNA levels were normalized against mouse Gapdh, mouse

β-actin or human TATA box binding protein (TBP) as indicated.

Cell culture and generation of 4T1-luciferase cells. The MCF-10A and 4T1 cells lines were purchased from ATCC. Generation and characterization of MCF-10A cells with stable knock-down of KLF4 (shKLF4), as well as non-specific control (shNS) cells has been reported (Yori et al., 2010a). The MCF-10A and 4T1 progression series were obtained from Dr. Fred Miller at Karmanos Cancer Institute. All MCF-10A lines, including the MCF-10AT1K, MCF-10CA1h and MCF-10CA1a were cultured in complete media (Debnath and Brugge, 2005). All 4T1 lines, including the 4T07 and

67NR cells were cultured in DMEM (Mediatech, Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

4T1-luciferase expressing cells (4T1-luc) were generated by subcloning the complete gene from the Luciferase T7 Control DNA (Promega) as a BamHI/SacI fragment into pBlueLZRS-2. Subsequent digestion yielded the luciferase containing BamH1/SfiI fragment which was inserted into the LZBOB-Hygro retroviral expression vector. Virus was generated using the Phoenix packaging cells as described (Johnson et al., 2004) with modifications. Vectors and Phoenix cells were kind gifts from Keith R. Johnson at

UNMC. Briefly, transfected Phoenix cells were cultured for 48 hours at 37°C before virus-containing media was harvested. 4T1 cells were plated overnight at low density and infected the next day with 0.45 μm filtered media containing virus and supplemented with 4 mg/ml polybrene (Sigma). Media was changed after overnight incubation at 32°C.

Infected 4T1 cells were cultured to 80% confluency and selected in 100 μg/ml hygromycin (Invitrogen).

Transwell migration and invasion assays. Assays were performed as previously described (Yori et al., 2010a). Briefly, 5.0x105 cells were seeded on transwell supports

(Costar) or BD MatrigelTM Invasion Chambers (BD Biosciences) and allowed to migrate or invade towards complete media for the indicated times. Five (20x) fields per transwell support were counted.

Bromodeoxyuridine incorporation and fluorescence-activated cell sorting analysis.

4T1 cells were infected with empty vector adenovirus control (AdGFP) or KLF4 expressing adenovirus (AdKLF4) (Yori et al., 2010a) for 48 hours prior to a 30 minute incubation with 10μmol/L bromodeoxyuridine (BrdU) (Sigma-Aldrich) at 37°C. Cells were fixed and labeled with anti-BrdU (BD Biosciences) and analyzed by fluorescence- activated cell sorting (FACS) as previously described (Yori et al., 2010a).

4T1 orthotopic tumor model. Twenty-four hours prior to injection, 4T1 or 4T1-luc cells were infected with AdGFP control or AdKFL4. The next day, cells were trypsinized, washed, and counted prior to resuspension in DMEM. Six to eight week old female BALB/c mice (Jackson Laboratory) were inoculated with 0.5x106 cells (50 μl) in the no. 9 mammary fat pad. Tumors were measured weekly by calipers. Mice injected with luciferase expressing cells were anesthetized with 2% Isoflurane and injected intraperitoneally with 200 μl of d-luciferin potassium salt at 12.5 mg/ml in PBS five minutes prior to imaging on a Xenogen IVIS 200 scanner (Caliper Sciences).

Morphologic and immunohistochemical analysis. Tissues were fixed overnight in 4% paraformaldehyde and embedded in paraffin. 5μm sections were dewaxed and stained with H&E. For immunostaining, cleared sections were rehydrated and antigen retrieval was performed using 10mmol/L sodium citrate buffer (pH 6.0) in a pressure cooker for

20 minutes at 125°C. Endogenous peroxidases were quenched in block (Dako EnVision+

System-HRP, Dako) containing 15μl/ml normal goat serum (Jackson Immunologicals), and sections were incubated overnight at 4°C with primary antibodies: anti-KLF4

(1:500, H-180, Santa Cruz Biotechnology), anti-Flag (1:500, M2, Sigma) or anti- cytokeratin 5/6 (1:20, Zymed). Slides were washed in TBS 0.05% Tween20, and incubated with labeled polymer-HRP anti-rabbit or anti-mouse (Dako) for 1 hour at room temperature. The signal was developed using 3,3‟-diaminobenzidine and slides were counterstained with hematoxylin and mounted. Images were captured using a Nikon microscope and Zeiss AxioCam HRc imaging system.

Identification of lung and liver metastases. Lung metastases were identified by in vivo bioluminescence imaging or H&E stained sections. Six sections per lung, collected at

100μm intervals, were evaluated. Two liver sections per mouse, separated by at least

100μm were stained with Cytokeratin 5/6 and counted to quantify tumor cell infiltrate and micrometastases.

Western Blot Analysis. Cells were lysed in radio-immunoprecipitation assay buffer as previously described (Yori et al., 2010a) and 50μg of whole-cell lysates were separated on 12% SDS-PAGE. Western blots were probed with primary antibodies directed to

KLF4 (Millipore), N-cadherin, Snail, cleaved Caspase-3, cleaved Caspase-7 (Cell

Signaling), E-cadherin and P120 (BD Transduction Laboratories), β-actin (Sigma), and

Vimentin (Santa Cruz Biotechnology). Peroxidase-conjugated secondary antibodies were from Santa Cruz, and blots were developed with Pierce ECL Western Blotting Substrate

(Thermo Scientific).

Statistical analyses. Statistical analyses were performed using two-tailed Student‟s t- test, unless otherwise stated in figure legends, with P values <0.05 considered statistically significant. Error bars represent standard deviations.

3.3 Results

KLF4 expression is suppressed in two mouse models of Her2/Neu positive breast cancer. We previously reported transcriptome analyses of MMTV-c-neu (NeuN) induced mouse mammary tumors (Landis et al., 2005). Further evaluation of these data revealed a progressive decrease in KLF4 expression with increasing tumor progression

(Figure 3.1A). Microarray data was further confirmed using qRT-PCR (Figure 3.1B).

In addition, we examined the expression of KLF4 in tumors arising in NeuT mice which express the activated rat Neu oncogene from the MMTV promoter (Muller et al., 1988).

Similar to mice that overexpress the wild-type Neu oncogene (NeuN), tumors from NeuT mice also displayed a dramatic loss of KLF4 mRNA (Figure 3.1B). To determine whether enforced expression of KLF4 would alter MMTV-NeuT induced tumor progression, we generated a tri-transgenic model which drives inducible overexpression

95 of KLF4 in the mammary gland (Figure 3.1C). A „tet-ON‟ transgenic line (MTB) expressing the reverse tetracycline transactivator (rtTA), under control of the mouse mammary tumor virus promoter (MMTV-rtTA), has been previously characterized and shown to express rtTA in the mammary glands of female virgin mice as early as 5 weeks of age (Gunther et al., 2002). Mice with a doxycycline-inducible transgene that expresses

KLF4 (TRE-KLF4) (Jaubert et al., 2003) were crossed with the MTB mice to yield the

MTB/TRE-KLF4 bi-transgenics. In the presence of doxycycline, these mice displayed elevated KLF4 in the mammary epithelium, compared to control littermates (Figure

3.1D). We next crossed control MTB and MTB/TRE-KLF4 mice with homozygous

MMTV-NeuT mice. Upon weaning, all mice were continuously administered doxycycline in the water for the remainder of the experiment. As expected, control

MTB/NeuT mice developed tumors with a mean tumor latency of 29 weeks.

Surprisingly, forced expression of KFL4 in the MTB/TRE-KLF4/NeuT tri-transgenics did not significantly alter mammary tumor latency (Figure 3.1E). However, when we examined KLF4 mRNA expression in these tumors compared with the controls, there was no evidence of KLF4 overexpression (Figure 3.1F). These data suggest that KLF4 expression is incompatible with Her2/Neu tumorigenesis, and that cells expressing KLF4 may undergo negative selection during formation of mammary tumors.

KLF4 expression is inversely correlated with metastatic progression and inhibits the migration and invasion of 4T1 tumor cells. To determine whether loss of KLF4 during mammary tumorigenesis is a general phenomenon that is not restricted to HER2/Neu tumors, we examined two cell line series that have been widely used to study the

96 molecular events during breast cancer development and metastasis. The MCF-10A series is comprised of at least four isogenic lines, representing normal breast epithelium (MCF-

10A), benign cells (MCF-10AT1K), carcinoma in situ (MCF-10ACA1h) and invasive carcinoma (MCF-10ACA1a), the latter of which form metastases to the lungs (Santner et al., 2001; Strickland et al., 2000). The 4T1 series consists of several lines which were isolated from a single spontaneously arising BALB/c mouse mammary tumor (Aslakson and Miller, 1992). When orthotopically injected into the mammary fat pad all lines form primary tumors with different metastatic propensities. We used the 4T1 line which is highly metastatic to multiple sites, the 4T07 line which form micrometastases at several sites, and the 67NR line which is non-metastatic. To determine if KLF4 expression is reduced with increasing metastatic potential we performed qRT-PCR on both series.

KLF4 expression decreases with increasing metastatic capacity in the MCF-10A series

(Figure 3.2A). Likewise, KLF4 is lowest in the most metastatic line of the 4T1 series, and is elevated in the less metastatic sibling lines (Figure 3.2B). We have previously shown that KLF4 represses migration and invasion of human non-transformed MCF-

10A cells and MDA-MB-231 mammary tumor cells (Yori et al., 2010a). To determine if

KLF4 also regulates these properties in 4T1 cells, they were transduced with an adenoviral KLF4 expression vector (AdKLF4). Expression of KLF4 significantly reduces migration (Figure 3.2C) and invasion (Figure 3.2D) of 4T1 cells when compared to adenovirus empty vector control (AdGFP). Thus, reduced KLF4 expression during metastatic progression and the ability of KLF4 to modulate the migratory and invasive capacities of breast cancer cells suggest a role for KLF4 in preventing the breast tumors metastasis.

KLF4 alters the growth of 4T1 cells by inhibiting proliferation and increasing apoptosis. KLF4 is an established regulator of proliferation through transcriptional control of multiple cell-cycle factors (Chen et al., 2003). Brightfield microscopy revealed decreased cell number in KLF4-overexprssing 4T1 cells when compared to

AdGFP infected controls (data not shown). FACS analyses of BrdU incorporation was used to determine if KLF4 overexpression resulted in decreased proliferation. The percentage of BrdU positive cells was reduced in the AdKLF4 transduced cells compared to the AdGFP control cells (Figure 3.3A and 3.3B). KLF4 also induces apoptosis in a variety of cancer cells including lymphoma (Guan et al.) gastric (Wei et al., 2005), bladder (Ohnishi et al., 2003), and colon (Chen et al., 2000) cancer cells. Hence, we determined whether forced expression of KLF4 also altered apoptosis in the 4T1 cells.

Propidium iodide staining and cell cycle analysis indicated a trend towards an increase in the sub-G1 population of the AdKLF4 cells (Figure 3.3C), while Western blot analyses revealed an increase in cleaved Caspase 3 and cleaved Caspase 7 (Figure 3.3D). These data demonstrate that, in addition to regulation of metastasis, KLF4 may also affect primary tumor growth of breast cancer cells by decreasing proliferation and increasing apoptosis.

Transient overexpression of KLF4 inhibits primary mammary tumor growth and decreases lung and liver micrometastases. Since stable, long term expression of KLF4 was incompatible with autochthonous NeuT tumorigenesis, we evaluated the ability of

KLF4 to regulate tumor growth and progression using a xenograft model and transient

98 overexpression of KLF4. Orthotopic injection of 4T1 cells into immunocompetent syngeneic mice, resulting in primary tumor formation and metastases, is a clinically relevant model of spontaneous breast cancer closely resembling many of the processes observed in the human disease (Pulaski and Ostrand-Rosenberg, 2001). These cells were transduced with either AdGFP or AdKLF4 and subsequently injected into the mammary fat pad of 6-8 week old BALB/c mice. An aliquot of the transduced cells was also replated and elevated KLF4 expression was confirmed for up to 7 days post-infection

(Figure 3.4A). As shown in Figure 3.4B, the growth of primary tumors arising from

AdKLF4 transduced 4T1 cells was reduced at day 13, compared with control AdGFP-

4T1 tumors. In a separate experimental cohort using luciferase expressing 4T1 cells

(4T1-luc), a similar reduction in tumor volume was observed in the AdKLF4 group compared to controls (Figure 3.4C). Neither AdGFP tumors nor AdKLF4 tumors were palpable until at least 8 days post-injection, however, in vivo bioluminescence imaging revealed a decrease in the AdKLF4 tumor size compared with the AdGFP controls at 5 days post-injection (Figure 3.4D). Three representative mice from each group are shown in Figure 3.4E. For mice imaged at 5 days, tumors were resected from half of the animals in each group and KLF4 expression was confirmed in tumor sections using a Flag antibody that recognizes exogenous, adenovirally-expressed, flag-KLF4 (Figure 3.4F).

Five days after implantation, the percentage of strongly expressing cells is greatly reduced compared with post-infection day 1, but similar to the percentage seen in day 5 cultures in vitro (data not shown). While KLF4-induced inhibition of growth was detectable at day 5 post implantation, no differences in proliferation (phospho-Histone

H3 staining), or apoptosis (TUNEL staining) were observed (data not shown), suggesting

99 that earlier changes in these processes must have occurred that lead to reduced tumor size at this later time point. This possibility is further supported by the greatly reduced number of cells still expressing KLF4.

4T1 tumors metastasize to various organs including lung and liver (Aslakson and Miller,

1992). Tumor cell dissemination occurs early in this model, and early resection of primary tumors does not preclude metastatic spread (Pulaski et al., 2000). We hypothesized, based on our in vitro migration and invasion studies that forced expression of KLF4 early during the tumorigenic process may prevent or delay metastases. To test this, we examined the lungs and livers from AdGFP control and AdKLF4 tumor bearing mice at 21 days post injection. Upon removal of the lungs, no macrometastases were visible from either control or AdKLF4 groups. However, bioluminescence imaging and histological examination of multiple lung sections revealed that over 60% of the mice in the control AdGFP group, compared with less than 10% of AdKLF4 tumor bearing mice, had developed lung micrometastases (Table 1 and Figure 3.5A1 and 3.5A2). To identify tumor cells in the liver, tissue sections were stained with anti-cytokeratin 5/6

(Figure 3.5A3-A6) and quantified. While all mice had developed micrometastases at this time, AdKLF4 reduced the number of micrometastases in the liver by 40% (Figure 3.5B).

Hence, transient restoration of KLF4 expression attenuates early metastatic progression in a clinically relevant mouse model of breast cancer.

KLF4 inhibits Snail expression in both MCF-10A mammary epithelial cells and 4T1 breast cancer cells. We have previously identified KLF4 as an inhibitor of EMT in

100 mammary epithelial cells, in part, through activation of E-cadherin gene expression (Yori et al., 2010a). In contrast, Snail plays an important role in the induction of EMT through transcriptional suppression of E-cadherin (Cano et al., 2000). Recently, Snail expression was found to be suppressed by KLF4 during the reprogramming of mouse fibroblasts into induced pluripotent stem (iPS) cells (Li et al., 2010b). Therefore, we postulated that, in addition to E-cadherin, Snail may be a target through which KLF4 inhibits metastasis of breast cancer cells. We first analyzed Snail expression in the non-transformed MCF-10A cells in which KLF4 expression has been stably silenced ((Yori et al., 2010a) and Figure

3.6B). Loss of KLF4 (shKLF4) resulted in increased Snail mRNA and protein (Figure

3.6A and 3.6B), as well as other molecular changes consistent with EMT, including decreased E-cadherin, a P120 isoform switch, and an increase in the mesenchymal marker, vimentin, when compared to the control cells (shNS). Conversely, KLF4 overexpression in 4T1 cells decreased both Snail transcript and protein (Figure 3.6C and

3.6D), while at the same time increasing E-cadherin levels. These results indicate that one mechanism whereby KLF4 regulates the migratory and invasive capacity of breast cancer cells, and in turn, tumor metastasis, may be through either direct or indirect modulation of Snail expression.

3.4 Discussion

This study demonstrates that KLF4 is lost during Her2/Neu-induced mammary tumorigenesis, while forced expression of KLF4 in the orthotopic 4T1 model is able to inhibit primary tumor growth and metastases of breast cancer cells. Taken together, these

101 findings provide the first in vivo evidence supporting a role for KLF4 as a tumor and metastases suppressor in the breast. These functional data are further supported by the ability of KLF4 to inhibit breast cancer cell proliferation, promote apoptosis and reduce expression of the metastasis inducer, Snail.

KLF4 has been recognized as a tumor suppressor in many types of cancer, and more recently, has been shown to suppress the migration and invasion of esophageal (Tian et al., 2010) and breast cancer (Yori et al., 2010a) cells, suggesting a potential role as a metastasis suppressor. However, conflicting data exists regarding the function of KLF4 in breast cancer. Initial studies employing immunohistochemical and in situ analyses of human breast tumors reported increased KLF4 expression relative to adjacent, uninvolved epithelium (Foster et al., 2000), while subsequent reports have shown that KLF4 mRNA levels are decreased in breast cancer, relative to normal tissue, and inversely correlated with increasing tumor grade (Akaogi et al., 2009). In vitro, the ability of KLF4 to act as tumor suppressor or tumor promoter has been shown to be largely cell type specific

(Rowland and Peeper, 2005), yet many studies examining the role of KLF4 in breast cancer have been conducted in fibroblast and other non-mammary epithelial cells. A recent study examining the role of KLF4 in the generation of induced pluripotent stem

(iPS) cells from fibroblasts versus epithelial cells (Li et al., 2010b) highlights the requirement of examining KLF4 in a cell-specific context. Even within the same breast cancer cell line, it has been reported that loss of KLF4 induces apoptosis in MCF7 cells

(Rowland et al., 2005), while others demonstrate that KLF4 inhibits estrogen-dependent proliferation in these same cells (Akaogi et al., 2009).

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Tumorigenic progression is a multi-step process. Here we show, in concordance with human breast cancer arrays, that KLF4 expression is progressively suppressed in a mouse model of metastatic breast cancer that is induced by HER2/Neu overexpression (Figure

3.1A). The specific loss of exogenously expressed KLF4 in these tumors suggests that

KLF4 precludes tumor formation. As the transgenic expression of NEU and KLF4 is not ubiquitous throughout the mammary epithelium, one possibility is that the cells expressing NEU, and not KLF4, are those capable of forming tumors. It is also is feasible that Her2/NEU signaling may post-transcriptionally inhibit KLF4 expression.

Prior to the study reported herein, little was known regarding the role of KLF4, if any, during metastatic progression. Recently, Tian et al. found that KLF4 was suppressed by

MicroRNA-10b, resulting in increased migration and invasion of esophageal cancer cells

(Tian et al., 2010). This becomes significant in the context of breast cancer as Ma et al. have shown that silencing of miR-10b inhibits metastasis of 4T1 mouse tumors (Ma et al., 2010). We found that KLF4 expression inhibits lung and liver micrometastases in this same model (Table 3.1 and Figure 3.5). Other evidence supporting such a role for

KLF4 is its ability to suppress EMT in mammary epithelial cells as well as support the mesenchymal-to-epithelial transition (MET) conversion during iPS cell generation from fibroblasts (Li et al., 2010b; Yori et al., 2010a).

Compelling evidence for EMT in breast cancer and its role in metastasis comes from recent studies examining a transcriptional driver of this process, Snail, and its elevated

103 expression in carcinomas associated with lymph node metastases, as well as its expression in distal metastases (Blanco et al., 2002; Come et al., 2006; Elloul et al.,

2005). We found that, in addition to other molecular changes associated with EMT,

KLF4 suppresses Snail expression in both mammary epithelial cells and breast tumor cells (Figure 3.6). Whether this effect occurs through direct regulation of the Snail gene by KLF4 remains to be determined. The concurrent inhibition of Snail and activation of

E-cadherin by KLF4 suggests that induction of MET is one mechanism whereby KLF4 inhibits metastasis. KLF4 and Snail appear to act in a yin and yang manner to modulate the epithelial phenotype. While KLF4 maintains expression of a multitude of epithelial specific genes (Chen et al., 2003), Snail transcriptionally suppresses several of the same targets. Of note, KLF4 is also repressed by Snail (De Craene et al., 2005). Thus it appears that a negative feedback loop exists between these two transcriptional regulators to control the epithelial or mesenchymal states.

It has been well established that breast cancer is not one, biologically distinct disease, but a heterogeneous group of at least 5 different molecular sub-types, each having different outcomes (Perou et al., 2000; Sorlie et al., 2001). While our findings begin to shed some light on the function of KLF4 in breast cancer and metastatic progression using two separate mouse models of mammary tumorigenesis, it is important that additional models, both in vitro and in vivo, representing various breast cancer sub-types, be examined. Molecular heterogeneity of breast cancer could, in part, be explained by the stem cell hypothesis (Stingl and Caldas, 2007), whereby tumors arise from mammary stem cells that are endowed with multi-lineage differentiation potential and self-renewal

104

(Dontu et al., 2003a). As KLF4 plays a role in the induction and maintenance of iPS cells, it will also be important to determine if similar regulation occurs in mammary and tumor stem cells.

In summary, loss of KLF4 during mammary tumorigenesis, in combination with ability of KLF4 to reduce tumor formation and metastasis, support a tumor and metastasis- suppressive role for KLF4 in breast cancer. While further studies are required to characterize the reciprocal regulation between Snail and KLF4, our findings provide new insights into a potential mechanism by which KLF4 inhibits breast cancer metastasis.

3.5 Acknowledgements

We would like to thank Dr. Julie Segre (National Human Genome Research Institute,

NIH) for her generous gift of the TRE-KLF4 transgenic mouse. This work was supported by National Institutes of Health grants (CA090398) to RAK and the Susan G.

Komen Foundation (BCTR108306) to RAK. JLY was the recipient of a Department of

Defense (DOD) pre-doctoral fellowship (DAMD17-03-1-0302). This research was supported by the Tissue Procurement, Histology, and Immunohistochemistry Core

Facility, the Imaging Research Core Facility, and the Cytometry & Imaging Microscopy

Core Facilities of the Case Comprehensive Cancer Center (P30 CA43703).

105

Table 3.1 Incidence of lung and liver micrometastases in AdGFP-4T1 and

AdKLF4-4T1 tumor bearing mice at 21 days post injection.

Number of Mice with Micrometastases

Mice Lung Liver

AdGFP (n=9) 6/9 9/9

AdKLF4 (n=9) 1/9* 9/9

*P = 0.025, Fisher Exact Test.

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Figure 3.1 Loss of KLF4 expression in Her2/Neu-induced mouse mammary tumors.

A, Microarray analysis of MMTV-Neu-induced tumors (NeuN) (Landis et al., 2005) reveals a significant reduction in KLF4 expression when compared with both age- matched wildtype (AMWT) and hyperplastic tissue (#, P<1.0E-4; &, P<2.0E-7). B, qRT-PCR confirms decreased expression of KLF4 in NeuN tumors compared to AMWT glands. The reduction in KLF4 expression is comparable between tumors from NeuN mice and tumors from transgenic mice expressing activated-Neu (NeuT). (AMWT, n=3;

NeuN, n=4; NeuT, n=3; *, P<0.05) C, Breeding paradigm used to generate triple transgenic mice expressing the mammary specific, reversible tetracycline transactivator

(rtTA) (MTB), activated-Neu (NeuT) and inducible KLF4 (TRE-KLF4). D, Doxycycline induction of KLF4 in the mammary epithelium of MTB/TRE-KLF4 mice. Both MTB control and MTB/TRE-KFL4 mice were administered water containing 5% sucrose and

2mg/ml doxycycline (Sigma) for 3 weeks prior to tissue harvest. Sections were stained with anti-KLF4. E, NeuT-induced tumor latency is not altered in mice harboring the

KLF4 expression cassette. All mice were administered doxycycline starting at weaning, and palpated twice weekly, starting at 15 weeks. (MTB/NeuT, n=16; MTB/NeuT/TRE-

KLF4, n=14). Statistical analysis and graphical representation of tumor development was generated by Kaplan–Meier survival analysis. Censored events, or animals that were removed for reasons unrelated to tumor development, are indicated with an “X”. Median tumor latencies between groups were not different (log rank test χ2 = .05, P=0.81). F,

Doxycycline-inducible KLF4 expression is lost in tumors from the MTB/TRE-

KLF4/NeuT mice. qRT-PCR showing comparable levels of KLF4 expression between

107 tumors that do not carry the inducible KLF4 transgene (MTB/NeuT, n=7) and those that do (MTB/TRE-KLF4/NeuT, n=6). (P=0.98).

108

Figure 3.1

109

Figure 3.2 KLF4 expression inversely correlates with metastatic progression. qRT-

PCR analysis of the A, MCF-10A and B, 4T1 progression series reveals decreasing KLF4 expression with increasing tumorigenicity and/or metastatic capacity. Total RNA was harvested from cells at 60-70% confluency. C, Transwell migration of 4T1 cells is inhibited by KLF4. D, KLF4 inhibits 4T1 cell invasion into matrigel. Cells were allowed to migrate or invade, towards complete media for 6 and 24 hours, respectively.

Experiments were performed at least three times in triplicate, (*, P<0.01; **, P<.001;

***, P<.0005; #, P<.0001). Overexpression of KLF4 in cells used for migration and invasion experiments is confirmed in Figure 3.4A.

110

Figure 3.2

111

Figure 3.3 KLF4 inhibits growth of 4T1 cells. A, Representative density plots from

FACS analysis of 4T1 cells infected with either AdGFP or AdKLF4 for 72 hours. Cells in the upper right quadrant are positive for BrdU incorporation. Cells in the bottom left quadrant represent cells in the subG1 phase. B, Quantitation of BrdU incorporation (*,

P<0.02), and C, sub-G1 population (P=0.08). D, Representative western blot showing increased levels of cleaved Caspase-3 and cleaved Caspase-7 in AdKLF4-4T1 cells compared to AdGFP-4T1 control cells. Experiments were performed at least three times in triplicate.

112

Figure 3.3

113

Figure 3.4 KLF4 inhibits primary tumor growth of 4T1 cells. A, Western blot shows maintained (in vitro) expression of virally-transduced KLF4 in 4T1 cells at days 4 and 7 post-infection. B, 4T1 (n=5 per group) and C, 4T1-luc (n=4 per group) tumors expressing AdKLF4 have reduced volume compared to AdGFP control tumors. Tumors were measured using calipers at various intervals over a 2 week period. D, AdKLF4 significantly reduces primary tumor formation determined by whole animal bioluminescence scanning at day 5 post-injection (AdGFP, n=9; AdKLF4, n=10).

Luminescence is expressed as photons/sec/ROI (region of interest). E, Bioluminescent images of 3 representative animals from each group in D. The relationship between color and light intensity in arbitrary units is given by the color bar to the right of each group.

F, Immunohistochemical staining of primary AdGFP-4T1 and AdKLF4-4T1 mammary tumors dissected at day 5 post-injection, using anti-flag to detect exogenous KLF4. (*,

P<0.01; **, P<0.005; ***, P<5.0E-6)

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Figure 3.4

115

Figure 3.5 KLF4 overexpression decreases lung and liver micrometastases. A1,

Representative micrometastasis from lung of AdGFP-4T1 mouse and A2, largest metastasis from the only AdKLF4-4T1 mouse that developed lung metastases. A3-A6,

Representative liver sections from AdGFP-4T1 mice and AdKLF4-4T1 mice, stained with anti-cytokeratin 5/6 (CK5/6) to detect metastatic mammary tumor cells in the liver

(arrows). B, Quantitation of CK5/6 staining. Livers from AdKLF4-4T1 mice had a significant reduction in micrometastases compared to AdGFP-4T1 mice (Mann-Whitney test, *, P<0.05).

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Figure 3.5

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Figure 3.6 KLF4 inhibits Snail expression in mammary epithelial cells and tumor cells. A, Stable knock-down of KLF4 (shKLF4) in MCF-10A cell results in increased

Snail mRNA expression, compared to control (shNS) cells, as detected by qRT-PCR. B,

Representative western blot showing increased Snail protein with loss of KLF4. Other changes associated with EMT are also observed such as increased vimentin and loss of E- cadherin. D and E, Forced expression of KLF4 (AdKLF4) in 4T1 cells inhibits Snail while increasing E-cadherin expression. Cells were plated in triplicate. Total RNA or protein was harvested at 48 hours post-infection. Experiments were performed at least 3 times (*, P<.05; **, P<2.0E-4).

118

Figure 3.6

119

CHAPTER 4

SUMMARY AND FUTURE DIRECTIONS

4.1 Summary

The goal of this thesis has been to gain insight into the functions of KLF4 during breast cancer progression and metastasis. In doing so, a novel inhibitory role for KLF4 during the process of EMT has been defined (Chapter 2). The discovery that KLF4 is necessary for maintenance of the mammary epithelial phenotype led to the identification of E- cadherin as a transcriptional target of KLF4. The inability of KLF4 deficient cells to form mammospheres implicates KLF4 in the maintenance of MaSCs. This is not surprising when considering the self-renewal role for KLF4 in iPS cell generation. In light of the CSC hypothesis and the potential contributions of EMT in promoting CSCs and metastasis, it will be important to determine how KLF4 directs these processes.

This dissertation builds on previous array studies examining gene expression changes in a mouse model of HER2+ breast cancer (Landis et al., 2005). The observation that KLF4 is downregulated in these tumors led to the hypothesis that KLF4 exerts a tumor suppressive function in the breast. In Chapter 3, this hypothesis was directly tested through the use of a transgenic mouse designed to maintain KLF4 expression in the context of HER2-induced tumorigenesis. However, the inability of HER2- overexpressing tumors to sustain KLF4 expression precluded a determination of whether

KLF4 could inhibit tumorigenesis and metastasis. This suggests that KLF4 is non-

120 permissive for HER2-initiated tumorigenesis, and that HER2 signaling may suppress

KLF4. However, a connection between the HER2 pathway and KLF4 has not yet been examined. Transient expression of KLF4 in a second mouse model of mammary tumorigenesis led us to the first in vivo evidence demonstrating that KLF4 not only inhibits primary tumor growth, but also attenuates metastasis. These studies described in

Chapter 3 not only confirm our hypothesis from Chapter 2, but also reveal another novel target of KFL4, the EMT-inducer Snail.

In summary, we have begun to uncover and define a tumor suppressive role for KLF4 in breast cancer. This body of work has generated many questions and hypotheses regarding the mechanisms whereby KLF4 modulates breast tumor growth and metastasis, including what role KLF4 may play in cancer stem cells. In section 4.2, I will outline several of these hypotheses and approaches to further explore these questions, beginning with how KLF4 may be pharmacologically targeted for therapy in breast cancer and metastasis.

4.2 Future directions

4.2.1 Pharmacological targeting of KLF4

The data presented herein support a role for KLF4 in inhibiting breast cancer growth and metastasis, suggesting that targeted therapies that increase KLF4 expression in breast tumors may prove to be a viable pharmacological option to treat and prevent metastatic

121 spread. The concept of a “druggable target”, which is used to describe proteins that favor interactions with drug-like chemical compounds, is one of several features examined during the initial identification and validation phase of drug discovery. Recently a simplified set of eight properties that are desirable for a target protein has been identified and include parameters such as membrane or extracellular localization, involvement in binding and signaling and, and lack of a PEST signal signifying a long half-life, to name a few (Bakheet and Doig, 2009). Based on these criteria, it would appear that KLF4 is not a particularly “druggable target”. In fact there have been no reports identifying small molecules that directly bind any KLF family members and modulate their function.

However, recent identification of post-translational modifications, resulting in altered protein activity, have been described for KLF4 (see section 1.3.2.1) and may provide a number of sites for potential pharmacological intervention using small molecules.

Ideally, one would like to employ the use of a small molecule therapeutic over a peptide or protein drug due to the unique demands imposed by the physicochemical and biological properties of peptides and protein drugs on routes of delivery. Unlike small molecules which generally display good oral bioavailability, there are several barriers to peptide/protein bioavailability after oral administration including membrane permeability, requirement for specialized mechanisms of transport, large size, short plasma half-life, susceptibility to proteolytic degradation, and decreased solubility. Even so, recent advances in protein and peptide drug delivery systems have considerably increased the efficacy of these types of drugs (Malik et al., 2007). In addition to treatment with small molecules to increase KLF4 expression and activity in breast cancer,

122 gene therapy to replace KLF4 and construction of KLF4 mimetics are several alternative approaches.

Engineered zinc-finger-based artificial transcription factor as a KLF4 mimetic. It has been estimated that up to 10% of the human genome encodes transcription factors, many of which have been identified as being dysregulated during cancer development and progression. The Cys2His2 zinc finger(s) found in KLF4 is the most common DNA- binding motif (Sera, 2009). Over the years, this relatively simple mode of DNA recognition has been used to engineer zinc-finger-based artificial transcription factors for gene regulation and genome modification (Sander et al., 2007). While transcription factors are not considered particularly “druggable targets”, recent studies have successfully demonstrated delivery and transcriptional regulation of specific target sequences by these artificial transcription factors both in vitro and in vivo (Sera, 2009).

Hence, this may present a possible therapeutic modality to restore KLF4 transcriptional function in breast tumor cells. As a protein drug, these artificial transcription factors are delivered using cell-penetrating peptides, and therefore could be targeted specifically to cancer cells.

Small molecules to induce KLF4 expression via regulation of either KLF4 or both upstream and downstream effectors. While many transcription factors translocate directly to the nucleus, some may remain in the cytoplasm until an appropriate signal is received. Here, cellular signaling can lead to post-translational modifications, often resulting in altered protein activity. These modifications offer potential sites for

123 pharmacological manipulation. For example, it has recently been shown that phosphorylation of KLF4, through the Smad and p38 MAPK pathways, mediates VSMC differentiation (Li et al., 2010a). While the phosphorylation state and it‟s affect on KLF4 activity in breast cancer cells has not yet been examined, one could anticipate that modulation of this modification through the use of kinase inhibitors directed at the TGF-β type I receptor or p38 MAPK. Other modifications, including acetylation, have also been described for KLF4. All-trans retinoic acid (ATRA), a ligand for the retinoic acid receptor, currently used to treat acute promyelocyitc leukemia, increases acetylation of

KLF4 and dissociation from HDAC2, in turn, increasing transcriptional activation of target genes (Meng et al., 2009). Post-translational modification studies have also revealed that KLF4 is targeted for ubiquitin-dependent proteolysis during cell cycle progression and in response to TGF-β signaling (Hu and Wan, 2010). Preclinical studies have demonstrated a selective susceptibility of transformed cells to proteasome inhibition-induced apoptosis and sensitization to the proapototic effects of conventional chemotherapeutics (Voorhees et al., 2003). While the proteasome inhibitor Bortezomib

(Velcade) has displayed limited activity in metastatic breast cancer patients, a new generation of proteasome inhibitors with more selective proteasome inhibition and efficacy in cancer therapy (Testa, 2009) make them an attractive avenue to pursue in an attempt to increase KLF4 levels in breast tumor cells.

Over the past few years, several natural products as well as synthetic compounds have been used clinically to target other signaling pathways that also induce expression of

KLF4. The natural ligand for the PPAR-γ nuclear hormone receptor 15-deoxy-

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Delta(12,14) prostaglandin J2 (15d-PGJ2) (Chen and Tseng, 2005) as well as thiazolidinediones (TZDs) (Rageul et al., 2009) and small molecule synthetic agonists such as betulin (Chintharlapalli et al., 2007), all increase expression of KLF4.

Interestingly, both 15d-PGJ2 and the TZD rosiglitazone have been shown to reduce the overall growth rate of breast cancer cells (Mueller et al., 1998) and, rosiglitazone was able to reduce the number of experimental lung metastases in a xenograft model of breast cancer (Magenta et al., 2008). In a recent pilot study, women with early stage breast cancer received short term treatment (2-6 weeks) with rosiglitazone, resulting in local and systemic effects on PPAR-γ signaling, however, tumor cell proliferation was not significantly altered (Yee et al., 2007). While a prior study reported that another TZD, trogltizone, failed to show any clinical benefits in patients with metastatic breast cancer

(Burstein et al., 2003), it has been shown that PPAR-γ immunoreactivity is significantly associated with improved clinical outcome in breast carcinoma patients(Suzuki et al.,

2006). Most informative will be retrospective studies on the long term use of TZDs as antidiabetic drugs. While it has not been determined if their long-term use alters the incidence or metastasis of breast cancer, preliminary findings suggest that they appear to reduce the risk of advanced lung cancer and improve survival among individuals with diabetes who also have lung cancer (Mazzone et al., 2010).

In addition to PPAR-γ agonists, the vasoprotective and cholesterol lowering class of drugs known as statins have also been shown to induce KLF4 expression and KLF4- induced gene expression via activation of Erk5 (Ohnesorge et al., 2010) Several dietary compounds such as sulforaphane which is derived from broccoli, and resveratrol, an

125 antioxidant found in the skin of red grapes, have also been identified as inducers of KLF4 expression (Traka et al., 2009; Villarreal et al., 2010), however their clinical efficacy has not yet been examined. Furthermore, resveratrol appears to possess several estrogenic properties, rendering it an unlikely candidate for treatment of estrogen receptor positive breast cancers.

As an alternative to targeting KLF4, a number of “druggable” transcription factors and cofactors have been identified that interact with KLF4, such as HDACs. ,At present, several small-molecule HDAC inhibitors are undergoing clinical trials as anticancer drugs (Shabason et al., 2010). The interaction between KLFs and nuclear receptors has also become important from a therapeutic standpoint. It is known, for example that

KLF5 interacts with both the RAR-RXR heterodimer and PPARδ (Oishi et al., 2008), while KLF13 interacts with the progesterone receptor (Zhang et al., 2003). Recently,

KLF4 has been shown to bind to the DNA binding region of ERα and inhibit its binding to estrogen response elements in the promoters of ER target genes (Akaogi et al., 2009).

Furthermore, KLF4 has been shown to complex with the thyroid hormone receptor to synergistically activate transcription upon thyroid hormone binding during enterocyte differentiation (Siddique et al., 2003), however the relationship between thyroid status and breast cancer is not clearly understood. It therefore may be possible to develop nuclear receptor ligands that selectively affect the interactions with and transactivation potential of KLF4.

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Considerations in the design of KLF4 therapeutics for treatment of breast cancer and metastasis. While the ultimate goal may be to increase the levels of KLF4 in breast cancers that have decreased or lost expression of this protein, pharmacokinetic analyses of potential compounds are essential for both design and optimization of effective therapies to target this factor. As mentioned previously, bioavailability is an important parameter when designing a new drug. In addition, the half-life of a drug, or the time it will take for levels to be reduce by half the original concentration in the plasma, is one of the most important factors that determines the selection of a dosing regimen. Most drugs are administered in a multiple dosing regimen at regular dosing intervals. An immediate rise in drug concentration is followed by a first order exponential decay that persists until the next dose is given. Accumulation of drug occurs when the administered dose is not eliminated in a single dose interval. Peak and trough concentration gradually rise upon repeated administration until a steady-state condition within the drug‟s therapeutic range has been reached. Steady state occurs when the amount of drug administered in a given time is equal to the amount of drug eliminated in that same period. The time to reach this steady-state level is specifically dependent on the half-life of a drug and generally occurs after 4 half-lives; however it is independent of dose. When the dosing interval is less than the half-life, a higher steady state occurs. If the dosing interval is greater than the half-life, the steady-state concentration is lower. An important consideration in designing a drug for KLF4 and in attaining therapeutic response will be the time necessary to reach a steady state, as a more rapid attainment of steady state might be able to reduce early progression of the disease. While a shorter half-life means more frequent dosing, which tends to reduce the likelihood of patient compliance, a drug with a long half life may be

127 unfavorable should toxicity or side effects arise, in addition to increasing time to steady- state. Furthermore, a drug with a shorter half-life may better help to maintain a drug concentration within the therapeutic window by reducing the large swings in peaks and troughs that you would have with each dose of a long half-life drug.

In addition to pharmacokinetic parameters, pharmacodynamic considerations such as efficacy, or intrinsic activity, are important in designing a drug. This is the ability of a drug to illicit a desired physiologic response, in this case, increase KLF4 expression.

While two drugs may be capable of producing the same effect, one may be able to do so at a much smaller dose, and thus be considered more potent. However, if induction of

KLF4 is not achieved, it doesn‟t matter how potent the drug is towards the target if ultimately it is not efficacious. Although the potency of a particular drug is an independent parameter of that drug‟s efficacy, potency does become an important issue in regards to in vivo efficacy testing, as large dose requirements become problematic for safety toxicology and clinical trials.

As master regulators of the cell, transcription factors are highly desirable targets for ligand discovery. However, unlike nuclear receptors which can be targeted through a ligand binding pocket, many transcription factors have expansive protein-protein interfaces, and generally lack potential hydrophobic binding pockets (Moellering et al.,

2009). Thus, the design of high-affinity, selective small molecules to target these factors remains challenging. As mentioned previously, there are currently no small molecules that have been identified which specifically target KLF4. While knowledge of the three

128 dimensional structure of KLF4 can provide insights into the structural determinant of selective regulation, the crystal structure of KLF4 has not yet been solved. Furthermore, only recently have specific post-translational modifications been identified that may facilitate the design and discovery of selective activators. The relatively high sequence homology among several domains of the KLF family members, as well as their ability to bind some of the same co-factors and sequences of DNA are also factors which inhibit selective design. Perhaps one of the most promising avenues regarding selective targeting of KLF4 is the development of the previously mentioned zinc-finger based artificial transcription factors. The ability to fuse other protein specific functional domains to the assembled zinc finger domains allows for selective modulation of endogenous gene expression (Sera, 2009). Future efforts aimed at 3D determination of

KLF4/co-factor complexes, which result in decreased KLF4 activity or signaling, will be important in the discovery and design of therapeutics that can disrupt these interactions.

Ultimately, pharmacological targeting of not only KLF4, but also KLF4-interacting proteins, downstream targets such as those identified herein, including Snail and E- cadherin, as well as pathways which downregulate KLF4 expression during breast tumorigenesis and progression will likely be the most effective means in treating breast cancers that have lost expression of KLF4. One such pathway, the TGF-β signaling pathway, will be discussed in the next several sections.

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4.2.2 Does TGF-β signaling regulate KLF4 expression during EMT?

Identification of KLF4 as a transcriptional activator of E-cadherin and repressor of EMT was described in Chapter 2. These findings suggest that signaling pathways which induce EMT are likely to require suppression of KLF4. Several studies have suggested

KLF4 regulation by TGF-β, however it is unclear whether TGF-β inhibits or induces

KLF4 expression. (Feinberg et al., 2005; Hu et al., 2007; Li et al., 2010a). As TGF-β is a potent EMT-promoting cytokine in non-transformed mammary epithelial cells, we were interested in understanding if TGF-β treatment of MCF-10A cells would affect KLF4 expression levels. In fact, treatment of these cells with TGF-β over a four day time course resulted in a progressive suppression of KLF4 protein as determined by western blot analysis (data not shown). One would predict that TGF-β would transcriptionally mediate KLF4 inhibition, as it has been shown that Snail, a downstream target of TGF-β signaling, suppresses KLF4 expression (De Craene et al., 2005). However, KLF4 mRNA was not consistently found to be decreased upon TGF-β treatment of MCF-10A cells.

This would suggest a post-transcriptional mechanism of regulation. To test the effects of

TGF-β on KLF4 protein stability, KLF4 protein levels could be evaluated at various time points of cyclohexamide treatment in the presence and absence of TGF-β. As described in Chapter 1, post-translational modifications to KLF4 have been shown to target KLF4 for degradation. Recently it has been shown that TGF-β promotes PIAS1 mediated sumoylation and subsequent degradation of KLF4 during phenotypic switching of smooth muscle cells (Kawai-Kowase et al., 2009). Thus we could also examine the role of

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PIAS1 as potential mechanism in TGF-β-mediated downregulation of KLF4 during

EMT.

4.2.3 Can forced expression of KLF4 block TGF-β-induced EMT?

The ability of KLF4 to induce a mesenchymal to epithelial transition (MET) in the highly aggressive MDA-MB-231 breast cancer cell line (Figure 2.6) suggests that KLF4 may be sufficient to prevent TGF-β-induced EMT. However, forced expression of exogenous

KLF4 was unable to prevent the apparent morphologic EMT that accompanied TGF-β treatment of human MCF-10A or murine NMuMG mammary epithelial cells, despite verification of mRNA overexpression (data not shown). However, it has not yet been determined whether KLF4 protein levels were maintained. If exogenous KLF4 protein is suppressed, this would further support the hypothesis that TGF-β signaling inhibits KLF4 protein stability. Alternatively, if EMT occurs in the presence of elevated KLF4 expression, this would indicate that KLF4 is not sufficient to prevent TGF-β-induced

EMT. As noted in section 1.3.4.4, several other Krüppel-like factors have also been shown to regulate EMT. In contrast to KLF4, KLF8 inhibits transcription of E-cadherin to induce EMT. Furthermore, KLF8 was shown to be induced by TGF-β, however, this induction was independent of Snail (Wang et al., 2007). It is possible that KLF8 induction by TGF-β may be sufficient to prevent KLF4 inhibition of EMT in mammary epithelial cells, as KLF8 binds and suppresses the KLF4 promoter (Wei et al., 2006).

Thus, future studies will be directed at evaluating the expression of KLF8 during TGF-β-

131 induced EMT as well as determining what effects, if any KLF8 has on the expression of

KLF4.

4.2.4 Does the TGF-β “switch” during tumorigenesis alter its effect on the regulation of

KLF4?

Research defining the complex roles of TGF-β in normal versus tumor tissue has clearly demonstrated that cancer cells have developed mechanisms to subvert the powerful growth inhibitory signal of TGF-β. This loss of growth inhibition, with concomitant increased TGF-β expression, is not only associated with malignant progression of breast cancer but also melanoma, glioma, ovarian, cervical and gastric cancers (Derynck et al.,

2001; Schmierer and Hill, 2007). It is now recognized that both the Smad-dependent and independent signaling responses to TGF-β are influenced by proteomic composition as well as cellular context (Derynck and Zhang, 2003; Massague and Chen, 2000). In light of our findings and others that TGF-β can suppress KLF4 (Feinberg et al., 2005; Hu et al., 2007), it is interesting that TGF-β can also upregulate KLF4 in the context of

VSMCs, mediating its growth inhibitory and differentiation effects through a

KLF4/Smad2 complex (Li et al., 2010a). These findings reinforce the context specific functions of KLF4, and suggest that in normal tissue, KLF4 may cooperate with TGF-β to inhibit growth. However, KLF4 may directly oppose the oncogenic and metastasis promoting properties of TGF-β that are observed during tumorigenic progression. In

Figure 3.2, we have shown that KLF4 expression is lowest in the most transformed and metastatic sibling lines of the MCF-10A and 4T1 progression series. Using these cell

132 line series, both of which respond to TGF-β by undergoing EMT, we can potentially delineate the response of KLF4 to normal versus oncogenic TGF-β signaling.

4.2.5 What are the mechanisms by which KLF4 suppresses Snail?

The Snail family of transcription factors has been described as potent mediators of EMT and metastasis. As downstream effectors of TGF-β signaling, they contribute to EMT through transcriptional suppression of the E-cadherin gene (Peinado et al., 2007).

Recently Snail was identified as a cofactor for Smad3/4 during TGF-β-induced EMT

(Vincent et al., 2009). Interestingly, KLF4 can also bind to Smad3 and interfere with

TGF-β signaling (Feinberg et al., 2005; Hu et al., 2007). Snail has also been shown to have tumor promoting effects. It is required for tumor growth of MDA-MB-231 cells

(Olmeda et al., 2007), and has been shown to drive tumor recurrence in a HER2-induced mouse model breast cancer (Moody et al., 2005). Furthermore, inhibition of pathways that activate Snail lead to reduced tumorigenesis and metastasis in 4T1 orthotopic mouse mammary tumor models (Holland et al., 2010; Huber et al., 2010). We have also shown that inhibition of tumor growth and metastatic progression through overexpression of

KLF4 is accompanied by downregulation of both Snail mRNA and protein in 4T1 breast cancer cells (Figures 3.4-3.6). In addition, knock-down of KLF4 in the MCF-10A cells results in increased levels of Snail. Collectively, these data suggest a mechanism whereby KLF4 inhibits EMT and metastasis through suppression of Snail. Subsequent interrogation of the proximal Snail promoter has revealed several putative KLF4 binding

133 sites and future studies could employ both luciferase assays and ChIP analysis to determine if KLF4 binds to and suppresses activity of the Snail promoter.

Several lines of evidence point to a competitive, feedback mechanism of regulation between KLF4 and Snail, as Snail has also been shown to suppress KLF4 expression (De

Craene et al., 2005). We have identified several putative Snail E-box binding sequences in the KLF4 promoter which can be analyzed for Snail binding in 4T1 tumor cells. We could also use RNAi-mediated depletion of Snail in these cells to determine if loss of

Snail is sufficient to induce KLF4 expression.

4.2.6 Does HER2 signaling regulate KLF4 expression?

Loss of exogenously-expressed KLF4 in the tumors of MMTV-Neu mice suggests that

KLF4 expression is incompatible with HER2/Neu-mediated tumorigenesis (Figure 3.1).

As discussed in section 3.4, one possibility is that cells expressing both KLF4 and NEU are not capable of forming tumors. A second possibility is that HER2-signaling may suppress KLF4 expression, though little is known regarding the regulation of KLF4 by

HER2 signaling. In fact, preliminary data from our lab indicates that HER2 may actually increase KLF4 expression in human HER2 dependent cell lines. However, as discussed in section 1.3.4, the role of KLF4 appears highly dependent upon the functionality of both p21 and p53 (Rowland and Peeper, 2005). In breast cancer, inactive or cytoplasmic p21 correlates with HER2 expression (Zhou et al., 2001). Thus, it is likely that the growth arresting effects of KLF4, via p21Cip1/Waf1 activation, would be muted in the

134 context of HER2 signaling. While future studies will be required to determine the mechanism by which HER2 regulates KLF4, the lack of a positive correlation between

KLF4 expression and HER2 status in breast tumors (Pandya et al., 2004) argues against a tumor-promoting role for KLF4 in HER2 breast cancers. As mentioned previously, luminal B tumors express both ER and HER2. Not only does cross-talk between these two pathways confer resistance to endocrine therapy (Bender and Nahta, 2008), but recently, it has been suggested that the ER pathway may confer resistance to anti-HER2 therapy (Loi, 2010). Interestingly, analysis of the Sorlie et al. data set indicates that both basal and luminal B tumors subtypes have the lowest expression of KLF4. In view of recent findings that KLF4 suppresses estrogen-dependent breast cancer growth (Akaogi et al., 2009), it is possible that KLF4 may be regulated through cross talk between these two pathways in ER+/HER2+ tumors. In conclusion, in vitro interrogation of the HER2 signaling pathway will be required to determine if HER2 activation suppresses KLF4.

While our in vivo model of inducible KLF4 expression was unable to directly asses the ability of KLF4 to inhibit tumor initiation, it may be possible to address the role of KLF4 in tumor progression and metastasis by delaying induction of KLF4 expression until after tumors have formed.

4.2.7 What is the role of KLF4 in the maintenance and self-renewal of MaSCs and

CSCs?

In addition to tumorigenesis, KLF4 plays an important role during stem cell maintenance.

In contrast to its role as an inducer of differentiation in epithelial cells, KLF4 functions to

135 maintain self-renewal and block differentiation of ES cells (Chan et al., 2009; Li et al.,

2004). The ability of human mammary epithelial cells to form mammospheres in three dimensional culture indicates the presence of a population of MaSCs (Dontu et al.,

2003a). Furthermore, these mammospheres are enriched for MaSCs, and can form a functional mouse mammary gland de novo (Li and Rosen, 2005). The induction of EMT in mammary epithelial cells results in the enrichment of a stem-like population and increased mammosphere formation (Mani et al., 2008). It is therefore difficult to reconcile the findings that loss of KLF4 and subsequent induction of EMT results in the loss of mammosphere/acinus formation in MCF-10A cells (Figure 2.2). On one hand, the requirement of KLF4 for maintaining self-renewal supports the loss of mammosphere formation in KLF4 knock-down cells. However, loss of KLF4 also results in EMT, which based on the findings of Weinberg and colleagues, should increase mammosphere potential. While we cannot rule out the possibility that reduced mammosphere formation was a result of decreased proliferation or increased migration, further studies will be needed to directly assess whether KLF4-null MCF-10A cells are less “stem-like”. This can be accomplished by labeling cells for defined stem cell markers such as CD44 and

CD24 and determining whether loss of KFL4 reduces the percentage of these cells.

KLF4 has also been identified as one of the four “Yamanaka Factors” able to convert a somatic cell back to a stem cell-like state, or an induced pluripotent stem (iPS) cell

(Takahashi and Yamanaka, 2006). This cellular reprogramming requires a mesenchymal to epithelial transition (MET) which is the opposite of EMT, and part of this MET is initiated by KLF4 induction of E-cadherin (Li et al., 2010b). These studies recapitulate

136 our findings and further support a role for KLF4 as a global inhibitor of EMT. In the context of cancer, this is significant, as in recent years, there has been increasing support of the hypothesis that tumors arise primarily from tumor initiating cells (TICs) or cancer stem cells (CSCs). These cancer stem cells express molecular markers associated with cells that have undergone an EMT, enabling them to disseminate, self-renew and seed new tumors. However, the finding that EMT promotes CSCs while MET promotes generation of iPS cells highlight intrinsic differences between these cells. Primarily,

CSCs have not been shown to be pluripotent.

Both normal stem cells and breast cancer stem cells express low levels of the miR-200 family of micro-RNAs which are negative regulators of the E-cadherin repressor ZEB1

(Shimono et al., 2009). In a negative feedback pathway, ZEB1 inhibits the miR-200 family to induce EMT (Burk et al., 2008). The miR-200 family also appears to target stemness factors including Sox2 and KLF4, suggesting a link between ZEB1 induced

EMT-activation and stemness maintenance. The study by Wellner et al. showed inhibition of KLF4 mRNA by miR-200c, however the effect on protein levels was not determined. Interestingly, these studies were performed in pancreatic cancer cells, which have increased levels of KLF4α, one of four recently identified KLF4 isoforms (Wei et al., 2010). It is likely that these KLF4 splice variants will add yet another layer of complexity in understanding the pleiotropic functions of KLF4 as tumor suppressor and oncogene. For example, the predominantly cytoplasmic localization of KLF4α suggests it may antagonize the function of full-length KLF4 given the findings that a nucleus- localization-deficient mutant KLF4 acts as a dominant-negative inhibitor (Shie and

137

Tseng, 2001). Thus, future studies assessing individual isoform composition, rather than total mRNA and protein expression will be required to more clearly define the role of

KLF4 in disease and stem-cell self-renewal.

Summary

The objective of these studies has been to gain insight into the function of KLF4 during breast cancer and metastasis. We have identified E-cadherin and Snail as two novel targets of KLF4. Through regulation of these factors, KLF4 inhibits both EMT and metastasis. Furthermore, loss of KLF4 in HER2+ tumors, in conjunction with its inhibition of primary tumor growth reveals a tumor suppressive function for KLF4 in the breast. These findings, together with the recently described relationship between EMT and cancer stem cells underscore the need for future studies examining the role of KLF4 in these cells. Determining whether KLF4 regulates the ability of CSCs to promote tumorigeneis and metastasis will be an important first step in unraveling the molecular mechanisms that control the transitions from resectable primary tumor to lethal metastatic disease.

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REFERENCES

(EBCTCG), E.B.C.T.C.G. (2005). Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet 365, 1687-1717.

Adam, P.J., Regan, C.P., Hautmann, M.B., and Owens, G.K. (2000). Positive- and negative-acting Kruppel-like transcription factors bind a transforming growth factor beta control element required for expression of the smooth muscle cell differentiation marker SM22alpha in vivo. J Biol Chem 275, 37798-37806.

Ai, W., Liu, Y., Langlois, M., and Wang, T.C. (2004). Kruppel-like factor 4 (KLF4) represses histidine decarboxylase gene expression through an upstream Sp1 site and downstream gastrin responsive elements. The Journal of biological chemistry 279, 8684- 8693.

Ai, W., Zheng, H., Yang, X., Liu, Y., and Wang, T.C. (2007). Tip60 functions as a potential corepressor of KLF4 in regulation of HDC promoter activity. Nucleic Acids Research, gkm656.

Aigner, K., Dampier, B., Descovich, L., Mikula, M., Sultan, A., Schreiber, M., Mikulits, W., Brabletz, T., Strand, D., Obrist, P., et al. (2007). The transcription factor ZEB1 (deltaEF1) promotes tumour cell dedifferentiation by repressing master regulators of epithelial polarity. Oncogene 26, 6979-6988.

Akaogi, K., Nakajima, Y., Ito, I., Kawasaki, S., Oie, S.H., Murayama, A., Kimura, K., and Yanagisawa, J. (2009). KLF4 suppresses estrogen-dependent breast cancer growth by inhibiting the transcriptional activity of ERalpha. Oncogene 28, 2894-2902.

Aslakson, C.J., and Miller, F.R. (1992). Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res 52, 1399-1405.

Aylon, Y., and Oren, M. (2007). Living with p53, dying of p53. Cell 130, 597-600.

Bachelder, R.E., Yoon, S.O., Franci, C., de Herreros, A.G., and Mercurio, A.M. (2005). Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial-mesenchymal transition. J Cell Biol 168, 29-33.

139

Bakheet, T.M., and Doig, A.J. (2009). Properties and identification of human protein drug targets. Bioinformatics (Oxford, England) 25, 451-457.

Baranwal, S., and Alahari, S.K. (2009). Molecular mechanisms controlling E-cadherin expression in breast cancer. Biochem Biophys Res Commun 384, 6-11.

Behr, R., and Kaestner, K.H. (2002). Developmental and cell type-specific expression of the zinc finger transcription factor Kruppel-like factor 4 (Klf4) in postnatal mouse testis. Mechanisms of development 115, 167-169.

Bender, L.M., and Nahta, R. (2008). Her2 cross talk and therapeutic resistance in breast cancer. Front Biosci 13, 3906-3912.

Bennett, H.L., Brummer, T., Jeanes, A., Yap, A.S., and Daly, R.J. (2008). Gab2 and Src co-operate in human mammary epithelial cells to promote growth factor independence and disruption of acinar morphogenesis. Oncogene 27, 2693-2704.

Berx, G., Cleton-Jansen, A.M., Strumane, K., de Leeuw, W.J., Nollet, F., van Roy, F., and Cornelisse, C. (1996). E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain. Oncogene 13, 1919-1925.

Berx, G., Raspe, E., Christofori, G., Thiery, J.P., and Sleeman, J.P. (2007). Pre-EMTing metastasis? Recapitulation of morphogenetic processes in cancer. Clinical & experimental metastasis 24, 587-597.

Bhargava, R., Beriwal, S., Striebel, J.M., and Dabbs, D.J. (2010). Breast cancer molecular class ERBB2: preponderance of tumors with apocrine differentiation and expression of basal phenotype markers CK5, CK5/6, and EGFR. Appl Immunohistochem Mol Morphol 18, 113-118.

Bhargava, R., Striebel, J., Beriwal, S., Flickinger, J.C., Onisko, A., Ahrendt, G., and Dabbs, D.J. (2009). Prevalence, morphologic features and proliferation indices of breast carcinoma molecular classes using immunohistochemical surrogate markers. International journal of clinical and experimental pathology 2, 444-455.

Bieker, J.J. (2001). Kruppel-like Factors: Three Fingers in Many Pies. Journal of Biological Chemistry 276, 34355-34358.

140

Birsoy, K., Chen, Z., and Friedman, J. (2008). Transcriptional regulation of adipogenesis by KLF4. Cell metabolism 7, 339-347.

Blanchon, L., Nores, R., Gallot, D., Marceau, G., Borel, V., Yang, V.W., Bocco, J.L., Lemery, D., Panzetta-Dutari, G., and Sapin, V. (2006). Activation of the human pregnancy-specific glycoprotein PSG-5 promoter by KLF4 and Sp1. Biochem Biophys Res Commun 343, 745-753.

Blanco, M.J., Moreno-Bueno, G., Sarrio, D., Locascio, A., Cano, A., Palacios, J., and Nieto, M.A. (2002). Correlation of Snail expression with histological grade and lymph node status in breast carcinomas. Oncogene 21, 3241-3246.

Blick, T., Widodo, E., Hugo, H., Waltham, M., Lenburg, M.E., Neve, R.M., and Thompson, E.W. (2008). Epithelial mesenchymal transition traits in human breast cancer cell lines. Clinical & experimental metastasis 25, 629-642.

Blows, F.M., Driver, K.E., Schmidt, M.K., Broeks, A., van Leeuwen, F.E., Wesseling, J., Cheang, M.C., Gelmon, K., Nielsen, T.O., Blomqvist, C., et al. (2010). Subtyping of Breast Cancer by Immunohistochemistry to Investigate a Relationship between Subtype and Short and Long Term Survival: A Collaborative Analysis of Data for 10,159 Cases from 12 Studies. PLoS Med 7, e1000279.

Bocker, W., Moll, R., Poremba, C., Holland, R., van Diest, P.J., Dervan, P., Burger, H., Wai, D., Ina Diallo, R., Brandt, B., et al. (2002). Common Adult Stem Cells in the Human Breast Give Rise to Glandular and Myoepithelial Cell Lineages: A New Cell Biological Concept. Laboratory investigation; a journal of technical methods and pathology 82, 737-746.

Boecker, W., Moll, R., Dervan, P., Buerger, H., Poremba, C., Diallo, R.I., Herbst, H., Schmidt, A., Lerch, M.M., and Buchwalow, I.B. (2002). Usual ductal hyperplasia of the breast is a committed stem (progenitor) cell lesion distinct from atypical ductal hyperplasia and ductal carcinoma in situ. J Pathol 198, 458-467.

Bolender, D.L., and Markwald, R.R. (1979). Epithelial-mesenchymal transformation in chick atrioventricular cushion morphogenesis. Scanning electron microscopy, 313-321.

Bourillot, P.-Y., and Savatier, P. (2010). Kruppel-like transcription factors and control of pluripotency. BMC Biology 8, 125.

141

Boussadia, O., Kutsch, S., Hierholzer, A., Delmas, V., and Kemler, R. (2002). E-cadherin is a survival factor for the lactating mouse mammary gland. MechDev 115, 53-62.

Braga, V.M., Machesky, L.M., Hall, A., and Hotchin, N.A. (1997). The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts. J Cell Biol 137, 1421-1431.

Brembeck, F.H., and Rustgi, A.K. (2000). The tissue-dependent keratin 19 gene transcription is regulated by GKLF/KLF4 and Sp1. J Biol Chem 275, 28230-28239.

Bremm, A., Walch, A., Fuchs, M., Mages, J., Duyster, J., Keller, G., Hermannstadter, C., Becker, K.F., Rauser, S., Langer, R., et al. (2008). Enhanced activation of epidermal growth factor receptor caused by tumor-derived E-cadherin mutations. Cancer Research 68, 707-714.

Briegel, K.J. (2006). Embryonic transcription factors in human breast cancer. IUBMB life 58, 123-132.

Bruce, W.R., and Van Der Gaag, H. (1963). A Quantitative Assay for the Number of Murine Lymphoma Cells Capable of Proliferation in Vivo. Nature 199, 79-80.

Buhler, H., and Schaller, G. (2005). Transfection of keratin 18 gene in human breast cancer cells causes induction of adhesion proteins and dramatic regression of malignancy in vitro and in vivo. Mol Cancer Res 3, 365-371.

Burk, U., Schubert, J., Wellner, U., Schmalhofer, O., Vincan, E., Spaderna, S., and Brabletz, T. (2008). A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells. EMBO reports 9, 582-589.

Burstein, H.J., Demetri, G.D., Mueller, E., Sarraf, P., Spiegelman, B.M., and Winer, E.P. (2003). Use of the peroxisome proliferator-activated receptor (PPAR) gamma ligand troglitazone as treatment for refractory breast cancer: a phase II study. Breast Cancer Res Treat 79, 391-397.

Cano, A., Perez-Moreno, M.A., Rodrigo, I., Locascio, A., Blanco, M.J., del Barrio, M.G., Portillo, F., and Nieto, M.A. (2000). The transcription factor snail controls epithelial- mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2, 76-83.

142

Carey, L.A., Dees, E.C., Sawyer, L., Gatti, L., Moore, D.T., Collichio, F., Ollila, D.W., Sartor, C.I., Graham, M.L., and Perou, C.M. (2007). The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes. Clin Cancer Res 13, 2329 - 2334.

Carey, L.A., Perou, C.M., Livasy, C.A., Dressler, L.G., Cowan, D., Conway, K., Karaca, G., Troester, M.A., Tse, C.K., Edmiston, S., et al. (2006). Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA 295, 2492 - 2502.

Carraway, K.L., Price-Schiavi, S.A., Komatsu, M., Jepson, S., Perez, A., and Carraway, C.A. (2001). Muc4/sialomucin complex in the mammary gland and breast cancer. J Mammary Gland Biol Neoplasia 6, 323-337.

Chan, K.K., Zhang, J., Chia, N.Y., Chan, Y.S., Sim, H.S., Tan, K.S., Oh, S.K., Ng, H.H., and Choo, A.B. (2009). KLF4 and PBX1 directly regulate NANOG expression in human embryonic stem cells. Stem Cells 27, 2114-2125.

Chao, Y.L., Shepard, C.R., and Wells, A. (2010). Breast carcinoma cells re-express E- cadherin during mesenchymal to epithelial reverting transition. Mol Cancer 9, 179.

Cheang, M.C., Chia, S.K., Voduc, D., Gao, D., Leung, S., Snider, J., Watson, M., Davies, S., Bernard, P.S., Parker, J.S., et al. (2009). Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. J Natl Cancer Inst 101, 736-750.

Chen, X., Johns, D.C., Geiman, D.E., Marban, E., Dang, D.T., Hamlin, G., Sun, R., and Yang, V.W. (2001). Kruppel-like factor 4 (gut-enriched Kruppel-like factor) inhibits cell proliferation by blocking G1/S progression of the cell cycle. J BiolChem 276, 30423- 30428.

Chen, X., Whitney, E.M., Gao, S.Y., and Yang, V.W. (2003). Transcriptional Profiling of Krnppel-like Factor 4 Reveals a Function in Cell Cycle Regulation and Epithelial Differentiation. Journal of Molecular Biology 326, 665-677.

Chen, Y.T., Stewart, D.B., and Nelson, W.J. (1999). Coupling assembly of the E- cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells. J Cell Biol 144, 687-699.

Chen, Z.Y., Rex, S., and Tseng, C.C. (2004). Kruppel-like factor 4 is transactivated by butyrate in colon cancer cells. The Journal of nutrition 134, 792-798.

143

Chen, Z.Y., Shie, J., and Tseng, C. (2000). Up-regulation of gut-enriched kruppel-like factor by interferon-gamma in human colon carcinoma cells. FEBS Lett 477, 67-72.

Chen, Z.Y., Shie, J.L., and Tseng, C.C. (2002). Gut-enriched Kruppel-like factor represses ornithine decarboxylase gene expression and functions as checkpoint regulator in colonic cancer cells. J Biol Chem 277, 46831-46839.

Chen, Z.Y., and Tseng, C.C. (2005). 15-Deoxy-{Delta}12,14 Prostaglandin J2 Up- Regulates Kruppel-Like Factor 4 Expression Independently of Peroxisome Proliferator- Activated Receptor {gamma} by Activating the Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signal Transduction Pathway in HT-29 Colon Cancer Cells. Molecular Pharmacology 68, 1203-1213.

Chen, Z.Y., Wang, X., Zhou, Y., Offner, G., and Tseng, C.C. (2005). Destabilization of Kruppel-Like Factor 4 Protein in Response to Serum Stimulation Involves the Ubiquitin- Proteasome Pathway. Cancer Research 65, 10394-10400.

Chiambaretta, F., De Graeve, F., Turet, G., Marceau, G., Gain, P., Dastugue, B., Rigal, D., and Sapin, V. (2004). Cell and tissue specific expression of human Kruppel-like transcription factors in human ocular surface. Molecular vision 10, 901-909.

Chintharlapalli, S., Papineni, S., Liu, S., Jutooru, I., Chadalapaka, G., Cho, S.-d., Murthy, R.S., You, Y., and Safe, S. (2007). 2-Cyano-lup-1-en-3-oxo-20-oic acid, a cyano derivative of betulinic acid, activates peroxisome proliferator-activated receptor Î³ in colon and pancreatic cancer cells, pp. 2337-2346.

Cho, K.B., Cho, M.K., Lee, W.Y., and Kang, K.W. (2010). Overexpression of c-myc induces epithelial mesenchymal transition in mammary epithelial cells. Cancer letters 293, 230-239.

Christoffersen, N.R., Silahtaroglu, A., Orom, U.A., Kauppinen, S., and Lund, A.H. (2007). miR-200b mediates post-transcriptional repression of ZFHX1B. RNA (New York, NY 13, 1172-1178.

Chua, H.L., Bhat-Nakshatri, P., Clare, S.E., Morimiya, A., Badve, S., and Nakshatri, H. (2006). NF-[kappa]B represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2. Oncogene 26, 711-724.

144

Cohnheim, V. (1875). Congenitales, quergestreiftes muskelsarkom der nieren. Virchows Arch Pathol Anat Physiol Klin Med 65, 64-69.

Come, C., Magnino, F., Bibeau, F., De Santa Barbara, P., Becker, K.F., Theillet, C., and Savagner, P. (2006). Snail and slug play distinct roles during breast carcinoma progression. Clin Cancer Res 12, 5395-5402.

Conery, A.R., Cao, Y., Thompson, E.A., Townsend, C.M., Jr., Ko, T.C., and Luo, K. (2004). Akt interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis. Nat Cell Biol 6, 366-372.

Conkright, M.D., Wani, M.A., Anderson, K.P., and Lingrel, J.B. (1999). A gene encoding an intestinal-enriched member of the Kruppel-like factor family expressed in intestinal epithelial cells. Nucleic Acids Res 27, 1263-1270.

Cook, T., Gebelein, B., Mesa, K., Mladek, A., and Urrutia, R. (1998). Molecular cloning and characterization of TIEG2 reveals a new subfamily of transforming growth factor- beta-inducible Sp1-like zinc finger-encoding genes involved in the regulation of cell growth. The Journal of biological chemistry 273, 25929-25936.

Creighton, C.J., Li, X., Landis, M., Dixon, J.M., Neumeister, V.M., Sjolund, A., Rimm, D.L., Wong, H., Rodriguez, A., Herschkowitz, J.I., et al. (2009). Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci USA 106, 13820 - 13825.

Cullingford, T.E., Butler, M.J., Marshall, A.K., Tham el, L., Sugden, P.H., and Clerk, A. (2008). Differential regulation of Kruppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: effects of endothelin-1, oxidative stress and cytokines. Biochimica et biophysica acta 1783, 1229-1236.

Dalerba, P., Cho, R.W., and Clarke, M.F. (2007). Cancer stem cells: models and concepts. Annual review of medicine 58, 267-284.

Dang, D.T., Bachman, K.E., Mahatan, C.S., Dang, L.H., Giardiello, F.M., and Yang, V.W. (2000). Decreased expression of the gut-enriched Kruppel-like factor gene in intestinal adenomas of multiple intestinal neoplasia mice and in colonic adenomas of familial adenomatous polyposis patients. FEBS Lett 476, 203-207.

145

Dang, D.T., Chen, X., Feng, J., Torbenson, M., Dang, L.H., and Yang, V.W. (2003). Overexpression of Kruppel-like factor 4 in the human colon cancer cell line RKO leads to reduced tumorigenecity. Oncogene 22, 3424-3430.

Dang, D.T., Mahatan, C.S., Dang, L.H., Agboola, I.A., and Yang, V.W. (2001). Expression of the gut-enriched Kruppel-like factor (Kruppel-like factor 4) gene in the human colon cancer cell line RKO is dependent on CDX2. Oncogene 20, 4884-4890.

Dang, D.T., Zhao, W., Mahatan, C.S., Geiman, D.E., and Yang, V.W. (2002). Opposing effects of Kruppel-like factor 4 (gut-enriched Kruppel-like factor) and Kruppel-like factor 5 (intestinal-enriched Kruppel-like factor) on the promoter of the Kruppel-like factor 4 gene. Nucleic Acids Res 30, 2736-2741.

Davies, J.A. (1996). Mesenchyme to epithelium transition during development of the mammalian kidney tubule. Acta anatomica 156, 187-201.

Dawood, S.S., Buzdar, A., Buchholz, T.A., Hortobagyi, G.N., and Gonzalez-Angulo, A.M. (2010). Evidence for change in prognostic stratification of breast tumor subtypes. J Clin Oncol (Meeting Abstracts) 28, 602-.

De Craene, B., Gilbert, B., Stove, C., Bruyneel, E., van Roy, F., and Berx, G. (2005). The transcription factor snail induces tumor cell invasion through modulation of the epithelial cell differentiation program. Cancer Research 65, 6237-6244.

Debnath, J., and Brugge, J.S. (2005). Modelling glandular epithelial cancers in three- dimensional cultures. Nat Rev Cancer 5, 675-688.

Debnath, J., Muthuswamy, S.K., and Brugge, J.S. (2003). Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30, 256-268.

Dent, R., Trudeau, M., Pritchard, K.I., Hanna, W.M., Kahn, H.K., Sawka, C.A., Lickley, L.A., Rawlinson, E., Sun, P., and Narod, S.A. (2007). Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 13, 4429-4434.

Derynck, R., Akhurst, R.J., and Balmain, A. (2001). TGF-[beta] signaling in tumor suppression and cancer progression. Nature genetics 29, 117-129.

146

Derynck, R., and Zhang, Y.E. (2003). Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425, 577-584.

DiFeo, A., Narla, G., Camacho-Vanegas, O., Nishio, H., Rose, S.L., Buller, R.E., Friedman, S.L., Walsh, M.J., and Martignetti, J.A. (2006). E-cadherin is a novel transcriptional target of the KLF6 tumor suppressor. Oncogene 25, 6026-6031.

Dominguez, D., Montserrat-Sentis, B., Virgos-Soler, A., Guaita, S., Grueso, J., Porta, M., Puig, I., Baulida, J., Franci, C., and Garcia de Herreros, A. (2003). Phosphorylation regulates the subcellular location and activity of the snail transcriptional repressor. Mol Cell Biol 23, 5078-5089.

Dontu, G., Abdallah, W.M., Foley, J.M., Jackson, K.W., Clarke, M.F., Kawamura, M.J., and Wicha, M.S. (2003a). In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17, 1253-1270.

Dontu, G., Al-Hajj, M., Abdallah, W.M., Clarke, M.F., and Wicha, M.S. (2003b). Stem cells in normal breast development and breast cancer. Cell proliferation 36 Suppl 1, 59- 72.

Dontu, G., El-Ashry, D., and Wicha, M.S. (2004). Breast cancer, stem/progenitor cells and the estrogen receptor. Trends in Endocrinology & Metabolism 15, 193-197.

Du, C., Zhang, C., Hassan, S., Biswas, M.H., and Balaji, K.C. (2010a). Protein kinase D1 suppresses epithelial-to-mesenchymal transition through phosphorylation of snail. Cancer Res 70, 7810-7819.

Du, J.X., McConnell, B.B., and Yang, V.W. (2010b). A small ubiquitin-related modifier- interacting motif functions as the transcriptional activation domain of Kruppel-like factor 4. The Journal of biological chemistry 285, 28298-28308.

Dunn, B.K., and Ryan, A. (2009). Phase 3 trials of aromatase inhibitors for breast cancer prevention: following in the path of the selective estrogen receptor modulators. Annals of the New York Academy of Sciences 1155, 141-161.

Duyen, T.D., Pevsner, J., and Yang, V.W. (2000). The biology of the mammalian Kruppel-like transcription factors. The International Journal of Biochemistry & Cell Biology 32, 1103-1121.

147

Ehlermann, J., Pfisterer, P., and Schorle, H. (2003). Dynamic expression of Kruppel-like factor 4 (Klf4), a target of transcription factor AP-2alpha during murine mid- embryogenesis. The anatomical record 273, 677-680.

Elloul, S., Elstrand, M.B., Nesland, J.M., Trope, C.G., Kvalheim, G., Goldberg, I., Reich, R., and Davidson, B. (2005). Snail, Slug, and Smad-interacting protein 1 as novel parameters of disease aggressiveness in metastatic ovarian and breast carcinoma. Cancer 103, 1631-1643.

Esteller, M., Silva, J.M., Dominguez, G., Bonilla, F., Matias-Guiu, X., Lerma, E., Bussaglia, E., Prat, J., Harkes, I.C., Repasky, E.A., et al. (2000). Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst 92, 564 - 569.

Esteva, F.J., Valero, V., Booser, D., Guerra, L.T., Murray, J.L., Pusztai, L., Cristofanilli, M., Arun, B., Esmaeli, B., Fritsche, H.A., et al. (2002). Phase II Study of Weekly Docetaxel and Trastuzumab for Patients With HER-2Â–Overexpressing Metastatic Breast Cancer. Journal of Clinical Oncology 20, 1800-1808.

Evans, P.M., Chen, X., Zhang, W., and Liu, C. (2010). KLF4 Interacts with {beta}- Catenin/TCF4 and Blocks p300/CBP Recruitment by {beta}-Catenin, pp. 372-381.

Evans, P.M., and Liu, C. (2006). KLF4 interacts with the histone acetyltransferase p300 and this interaction is important for KLF4-mediated transactivation. AACR Meeting Abstracts 2006, 410-441c.

Evans, P.M., Zhang, W., Chen, X., Yang, J., Bhakat, K., and Liu, C. (2007). Kruppel-like factor 4 is acetylated by p300 and regulates gene transcription via modulation of histone acetylation. Journal of Biological Chemistry 282, 33994-34002.

Feinberg, A.P. (2007). Phenotypic plasticity and the epigenetics of human disease. Nature 447, 433-440.

Feinberg, M.W., Cao, Z.X., Wara, A.K., Lebedeva, M.A., SenBanerjee, S., and Jain, M.K. (2005). Kruppel-like factor 4 is a mediator of proinflammatory signaling in macrophages. Journal of Biological Chemistry 280, 38247-38258.

Feng, W., Orlandi, R., Zhao, N., Carcangiu, M.L., Tagliabue, E., Xu, J., Bast, R.C., Jr., and Yu, Y. (2010). Tumor suppressor genes are frequently methylated in lymph node metastases of breast cancers. BMC cancer 10, 378.

148

Filipowicz, W., Bhattacharyya, S.N., and Sonenberg, N. (2008). Mechanisms of post- transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9, 102-114.

Foster, K.W., Frost, A.R., McKie-Bell, P., Lin, C.Y., Engler, J.A., Grizzle, W.E., and Ruppert, J.M. (2000). Increase of GKLF Messenger RNA and Protein Expression during Progression of Breast Cancer. Cancer Research 60, 6488-6495.

Foster, K.W., Liu, Z., Nail, C.D., Li, X., Fitzgerald, T.J., Bailey, S.K., Frost, A.R., Louro, I.D., Townes, T.M., Paterson, A.J., et al. (2005). Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia. Oncogene 24, 1491-1500.

Foster, K.W., Ren, S., Louro, I.D., Lobo-Ruppert, S.M., McKie-Bell, P., Grizzle, W., Hayes, M.R., Broker, T.R., Chow, L.T., and Ruppert, J.M. (1999). Oncogene expression cloning by retroviral transduction of adenovirus E1A-immortalized rat kidney RK3E cells: transformation of a host with epithelial features by c-MYC and the zinc finger protein GKLF. Cell Growth Differ 10, 423-434.

Foulkes, W.D. (2004). BRCA1 functions as a breast stem cell regulator. Journal of Medical Genetics 41, 1-5.

Fournier, A.K., Campbell, L.E., Castagnino, P., Liu, W.F., Chung, B.M., Weaver, V.M., Chen, C.S., and Assoian, R.K. (2008). Rac-dependent cyclin D1 gene expression regulated by cadherin- and integrin-mediated adhesion. Journal of Cell Science 121, 226- 233.

Frixen, U.H., Behrens, J., Sachs, M., Eberle, G., Voss, B., Warda, A., Lochner, D., and Birchmeier, W. (1991). E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J Cell Biol 113, 173-185.

Fukasawa, K. (2005). Centrosome amplification, chromosome instability and cancer development. Cancer letters 230, 6-19.

Fulford, L.G., Reis-Filho, J.S., Ryder, K., Jones, C., Gillett, C.E., Hanby, A., Easton, D., and Lakhani, S.R. (2007). Basal-like grade III invasive ductal carcinoma of the breast: patterns of metastasis and long-term survival. Breast Cancer Res 9, R4.

Gal, A., Sjoblom, T., Fedorova, L., Imreh, S., Beug, H., and Moustakas, A. (2008). Sustained TGF beta exposure suppresses Smad and non-Smad signalling in mammary

149 epithelial cells, leading to EMT and inhibition of growth arrest and apoptosis. Oncogene 27, 1218-1230.

Gammill, L.S., and Bronner-Fraser, M. (2003). Neural crest specification: migrating into genomics. Nature reviews 4, 795-805.

Garrett-Sinha, L.A., Eberspaecher, H., Seldin, M.F., and de Crombrugghe, B. (1996). A Gene for a Novel Zinc-finger Protein Expressed in Differentiated Epithelial Cells and Transiently in Certain Mesenchymal Cells. Journal of Biological Chemistry 271, 31384- 31390.

Gehrau, R.C., D'Astolfo, D.S., Dumur, C.I., Bocco, J.L., and Koritschoner, N.s.P. (2010). Nuclear Expression of KLF6 Tumor Suppressor Factor Is Highly Associated with Overexpression of ERBB2 Oncoprotein in Ductal Breast Carcinomas. PloS one 5, e8929.

Geiman, D.E., Ton-That, H., Johnson, J.M., and Yang, V.W. (2000). Transactivation and growth suppression by the gut-enriched Kruppel-like factor (Kruppel-like factor 4) are dependent on acidic amino acid residues and protein-protein interaction. Nucleic Acids Res 28, 1106-1113.

Gerhard, D.S., Wagner, L., Feingold, E.A., Shenmen, C.M., Grouse, L.H., Schuler, G., Klein, S.L., Old, S., Rasooly, R., Good, P., et al. (2004). The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome research 14, 2121-2127.

Ghaleb, A.M., Katz, J.P., Kaestner, K.H., Du, J.X., and Yang, V.W. (2007a). Kruppel- like factor 4 exhibits antiapoptotic activity following gamma-radiation-induced DNA damage. Oncogene 26, 2365-2373.

Ghaleb, A.M., McConnell, B.B., Nandan, M.O., Katz, J.P., Kaestner, K.H., and Yang, V.W. (2007b). Haploinsufficiency of Kruppel-like factor 4 promotes adenomatous polyposis coli dependent intestinal tumorigenesis. Cancer Research 67, 7147-7154.

Godmann, M., Kromberg, I., Mayer, J., and Behr, R. (2005). The mouse Kruppel-like Factor 4 (Klf4) gene: four functional polyadenylation sites which are used in a cell- specific manner as revealed by testicular transcript analysis and multiple processed pseudogenes. Gene 361, 149-156.

150

Gottardi, C.J., Wong, E., and Gumbiner, B.M. (2001). E-cadherin suppresses cellular transformation by inhibiting beta-catenin signaling in an adhesion-independent manner. J Cell Biol 153, 1049-1060.

Gregory, P.A., Bert, A.G., Paterson, E.L., Barry, S.C., Tsykin, A., Farshid, G., Vadas, M.A., Khew-Goodall, Y., and Goodall, G.J. (2008). The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 10, 593-601.

Guan, H., Xie, L., Leithauser, F., Flossbach, L., Moller, P., Wirth, T., and Ushmorov, A. KLF4 is a tumor suppressor in B-cell non-Hodgkin lymphoma and in classic Hodgkin lymphoma. Blood 116, 1469-1478.

Gumbiner, B.M. (2005). Regulation of cadherin-mediated adhesion in morphogenesis. Nat Rev Mol Cell Biol 6, 622-634.

Gumireddy, K., Li, A., Gimotty, P.A., Klein-Szanto, A.J., Showe, L.C., Katsaros, D., Coukos, G., Zhang, L., and Huang, Q. (2009). KLF17 is a negative regulator of epithelial-mesenchymal transition and metastasis in breast cancer. Nat Cell Biol 11, 1297-1304.

Gunther, E.J., Belka, G.K., Wertheim, G.B., Wang, J., Hartman, J.L., Boxer, R.B., and Chodosh, L.A. (2002). A novel doxycycline-inducible system for the transgenic analysis of mammary gland biology. Faseb J 16, 283-292.

Gupta, G.P., Perk, J., Acharyya, S., de Candia, P., Mittal, V., Todorova-Manova, K., Gerald, W.L., Brogi, E., Benezra, R., and Massague, J. (2007). ID genes mediate tumor reinitiation during breast cancer lung metastasis. Proceedings of the National Academy of Sciences of the United States of America 104, 19506-19511.

Gupta, P.B., Chaffer, C.L., and Weinberg, R.A. (2009). Cancer stem cells: mirage or reality? Nat Med 15, 1010-1012.

Gusterson, B. (2009). Do 'basal-like' breast cancers really exist? Nat Rev Cancer 9, 128- 134.

Guy, C.T., Webster, M.A., Schaller, M., Parsons, T.J., Cardiff, R.D., and Muller, W.J. (1992). Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. ProcNatlAcadSciUSA 89, 10578-10582.

151

Hagen, G., Muller, S., Beato, M., and Suske, G. (1992). Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes. Nucleic Acids Res 20, 5519-5525.

Hamik, A., Lin, Z., Kumar, A., Balcells, M., Sinha, S., Katz, J., Feinberg, M.W., Gerzsten, R.E., Edelman, E.R., and Jain, M.K. (2007). Kruppel-like factor 4 regulates endothelial inflammation. The Journal of biological chemistry 282, 13769-13779.

Hazan, R.B., Qiao, R., Keren, R., Badano, I., and Suyama, K. (2004). Cadherin switch in tumor progression. AnnNYAcad Sci 1014, 155-163.

He, L., and Hannon, G.J. (2004). MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5, 522-531.

Heimann, R., Lan, F., McBride, R., and Hellman, S. (2000). Separating favorable from unfavorable prognostic markers in breast cancer: the role of E-cadherin. Cancer Res 60, 298-304.

Herschkowitz, J.I., Simin, K., Weigman, V.J., Mikaelian, I., Usary, J., Hu, Z., Rasmussen, K.E., Jones, L.P., Assefnia, S., Chandrasekharan, S., et al. (2007). Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome Biol 8, R76.

Higaki, Y., Schullery, D., Kawata, Y., Shnyreva, M., Abrass, C., and Bomsztyk, K. (2002). Synergistic activation of the rat laminin gamma1 chain promoter by the gut- enriched Kruppel-like factor (GKLF/KLF4) and Sp1. Nucleic Acids Res 30, 2270-2279.

Hinnebusch, B.F., Siddique, A., Henderson, J.W., Malo, M.S., Zhang, W., Athaide, C.P., Abedrapo, M.A., Chen, X., Yang, V.W., and Hodin, R.A. (2004). Enterocyte differentiation marker intestinal alkaline phosphatase is a target gene of the gut-enriched Kruppel-like factor. Am J Physiol Gastrointest Liver Physiol 286, G23-30.

Hlubek, F., LÃ¶hberg, C., Meiler, J., Jung, A., Kirchner, T., and Brabletz, T. (2001). Tip60 Is a Cell-Type-Specific Transcriptional Regulator, pp. 635-641.

Holian, J., Qi, W., Kelly, D.J., Zhang, Y., Mreich, E., Pollock, C.A., and Chen, X.M. (2008). Role of Kruppel-like factor 6 in transforming growth factor-beta1-induced epithelial-mesenchymal transition of proximal tubule cells. American journal of physiology 295, F1388-1396.

152

Holland, S.J., Pan, A., Franci, C., Hu, Y., Chang, B., Li, W., Duan, M., Torneros, A., Yu, J., Heckrodt, T.J., et al. (2010). R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer. Cancer Research 70, 1544-1554.

Hollier, B.G., Evans, K., and Mani, S.A. (2009). The epithelial-to-mesenchymal transition and cancer stem cells: a coalition against cancer therapies. J Mammary Gland Biol Neoplasia 14, 29-43.

Howell, A. (2008). The endocrine prevention of breast cancer. Best practice & research 22, 615-623.

Hu, B., Wu, Z., Liu, T., Ullenbruch, M.R., Jin, H., and Phan, S.H. (2007). Gut-enriched Kruppel-like factor interaction with Smad3 inhibits myofibroblast differentiation. Am J RespirCell Mol Biol 36, 78-84.

Hu, D., and Wan, Y. (2010). Regulation of Kruppel-like factor 4(KLF4) by APC pathway is involved in TGF-{beta} signaling. The Journal of biological chemistry.

Hu, W., Hofstetter, W.L., Li, H., Zhou, Y., He, Y., Pataer, A., Wang, L., Xie, K., Swisher, S.G., and Fang, B. (2009). Putative tumor-suppressive function of Kruppel-like factor 4 in primary lung carcinoma. Clin Cancer Res 15, 5688-5695.

Huang, C.C., Liu, Z., Li, X., Bailey, S.K., Nail, C.D., Foster, K.W., Frost, A.R., Ruppert, J.M., and Lobo-Ruppert, S.M. (2005a). KLF4 and PCNA identify stages of tumor initiation in a conditional model of cutaneous squamous epithelial neoplasia. Cancer biology & therapy 4, 1401-1408.

Huang, C.C., Liu, Z., Li, X., Bailey, S.K., Nail, C.D., Foster, K.W., Frost, A.R., Ruppert, J.M., and Lobo-Ruppert, S.M. (2005b). KLF4 and PCNA identify stages of tumor initiation in a conditional model of cutaneous squamous epithelial neoplasia. Cancer Biol Ther 4, 1401-1408.

Huber, M.A., Maier, H.J., Alacakaptan, M., Wiedemann, E., Braunger, J.r., Boehmelt, G., Madwed, J.B., Young, E.R.R., Marshall, D.R., Pehamberger, H., et al. (2010). BI 5700, a Selective Chemical Inhibitor of IÎºB Kinase 2, Specifically Suppresses Epithelial- Mesenchymal Transition and Metastasis in Mouse Models of Tumor Progression. Genes & Cancer 1, 101-114.

153

Hurteau, G.J., Carlson, J.A., Roos, E., and Brock, G.J. (2009). Stable expression of miR- 200c alone is sufficient to regulate TCF8 (ZEB1) and restore E-cadherin expression. Cell cycle (Georgetown, Tex 8, 2064-2069.

Ikenouchi, J., Matsuda, M., Furuse, M., and Tsukita, S. (2003). Regulation of tight junctions during the epithelium-mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail. J Cell Sci 116, 1959-1967.

Imbalzano, K., Tatarkova, I., Imbalzano, A., and Nickerson, J. (2009). Increasingly transformed MCF-10A cells have a progressively tumor-like phenotype in three- dimensional basement membrane culture. Cancer Cell International 9, 7.

Ireton, R.C., Davis, M.A., van Hengel, J., Mariner, D.J., Barnes, K., Thoreson, M.A., Anastasiadis, P.Z., Matrisian, L., Bundy, L.M., Sealy, L., et al. (2002). A novel role for p120 catenin in E-cadherin function. J Cell Biol 159, 465-476.

Jaubert, J., Cheng, J., and Segre, J.A. (2003). Ectopic expression of kruppel like factor 4 (Klf4) accelerates formation of the epidermal permeability barrier. Development 130, 2767-2777.

Jenkins, T.D., Opitz, O.G., Okano, J.i., and Rustgi, A.K. (1998). Transactivation of the Human Keratin 4 and Epstein-Barr Virus ED-L2 Promoters by Gut-enriched Kruppel-like Factor. Journal of Biological Chemistry 273, 10747-10754.

Jiang, J., Chan, Y.S., Loh, Y.H., Cai, J., Tong, G.Q., Lim, C.A., Robson, P., Zhong, S., and Ng, H.H. (2008a). A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat Cell Biol 10, 353-360.

Jiang, J., Chan, Y.S., Loh, Y.H., Cai, J., Tong, G.Q., Lim, C.A., Robson, P., Zhong, S., and Ng, H.H. (2008b). A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat Cell Biol 10, 353-360.

Jiang, W., Deng, W., Bailey, S.K., Nail, C.D., Frost, A.R., Brouillette, W.J., Muccio, D.D., Grubbs, C.J., Ruppert, J.M., and Lobo-Ruppert, S.M. (2009). Prevention of KLF4- mediated tumor initiation and malignant transformation by UAB30 rexinoid. Cancer biology & therapy 8, 289-298.

Johnson, E., Theisen, C.S., Johnson, K.R., and Wheelock, M.J. (2004). R-cadherin influences cell motility via Rho family GTPases. J Biol Chem 279, 31041-31049.

154

Kaczynski, J., Cook, T., and Urrutia, R. (2003). Sp1- and Kruppel-like transcription factors. Genome Biol 4, 206.201-206.206.

Kaczynski, J., Zhang, J.S., Ellenrieder, V., Conley, A., Duenes, T., Kester, H., van Der Burg, B., and Urrutia, R. (2001). The Sp1-like protein BTEB3 inhibits transcription via the basic transcription element box by interacting with mSin3A and HDAC-1 co- repressors and competing with Sp1. The Journal of biological chemistry 276, 36749- 36756.

Kadonaga, J.T., Carner, K.R., Masiarz, F.R., and Tjian, R. (1987). Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 51, 1079-1090.

Kakinuma, N., Kohu, K., Sato, M., Yamada, T., Nakajima, M., Akiyama, T., Ohwada, S., and Shibanaka, Y. (2004). Candidate regions of tumor suppressor locus on chromosome 9q31.1 in gastric cancer. Int J Cancer 109, 71-75.

Kanai, M., Wei, D., Li, Q., Jia, Z., Ajani, J., Le, X., Yao, J., and Xie, K. (2006). Loss of Kruppel-like factor 4 expression contributes to Sp1 overexpression and human gastric cancer development and progression. Clin Cancer Res 12, 6395-6402.

Katz, J.P., Perreault, N., Goldstein, B.G., Actman, L., McNally, S.R., Silberg, D.G., Furth, E.E., and Kaestner, K.H. (2005). Loss of Klf4 in mice causes altered proliferation and differentiation and precancerous changes in the adult stomach. Gastroenterology 128, 935-945.

Katz, J.P., Perreault, N., Goldstein, B.G., Lee, C.S., Labosky, P.A., Yang, V.W., and Kaestner, K.H. (2002). The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon. Development 129, 2619-2628.

Kaufmann, M., and Pusztai, L. (2010). Use of standard markers and incorporation of molecular markers into breast cancer therapy: consensus recommendations from an International Expert Panel. Cancer.

Kawai-Kowase, K., Ohshima, T., Matsui, H., Tanaka, T., Shimizu, T., Iso, T., Arai, M., Owens, G.K., and Kurabayashi, M. (2009). PIAS1 mediates TGFbeta-induced SM alpha- actin gene expression through inhibition of KLF4 function-expression by protein sumoylation. Arteriosclerosis, thrombosis, and vascular biology 29, 99-106.

155

Kim, B., Bang, S., Lee, S., Kim, S., Jung, Y., Lee, C., Choi, K., Lee, S.G., Lee, K., Lee, Y., et al. (2003). Expression profiling and subtype-specific expression of stomach cancer. Cancer Res 63, 8248-8255.

Kleinsmith, L.J., and Pierce, G.B., Jr. (1964). Multipotentiality of Single Embryonal Carcinoma Cells. Cancer Res 24, 1544-1551.

Klymkowsky, M.W., and Savagner, P. (2009). Epithelial-mesenchymal transition: a cancer researcher's conceptual friend and foe. Am J Pathol 174, 1588-1593.

Knudsen, K.A., and Wheelock, M.J. (2005). Cadherins and the mammary gland. J Cell Biochem 95, 488-496.

Kondo, M., Wagers, A.J., Manz, M.G., Prohaska, S.S., Scherer, D.C., Beilhack, G.F., Shizuru, J.A., and Weissman, I.L. (2003). Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annual review of immunology 21, 759- 806.

Korkaya, H., Paulson, A., Iovino, F., and Wicha, M.S. (2008). HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion. Oncogene 27, 6120-6130.

Korpal, M., Lee, E.S., Hu, G., and Kang, Y. (2008). The miR-200 family inhibits epithelial-mesenchymal transition and cancer cell migration by direct targeting of E- cadherin transcriptional repressors ZEB1 and ZEB2. The Journal of biological chemistry 283, 14910-14914.

Kowalski, P.J., Rubin, M.A., and Kleer, C.G. (2003). E-cadherin expression in primary carcinomas of the breast and its distant metastases. Breast Cancer Res 5, R217-222.

Landis, M.D., Seachrist, D.D., Montanez-Wiscovich, M.E., Danielpour, D., and Keri, R.A. (2005). Gene expression profiling of cancer progression reveals intrinsic regulation of transforming growth factor-beta signaling in ErbB2/Neu-induced tumors from transgenic mice. Oncogene 24, 5173-5190.

Lehembre, F., Yilmaz, M., Wicki, A., Schomber, T., Strittmatter, K., Ziegler, D., Kren, A., Went, P., Derksen, P.W.B., Berns, A., et al. (2008). NCAM-induced focal adhesion assembly: a functional switch upon loss of E-cadherin. EMBO J 27, 2603-2615.

156

Li, H.X., Han, M., Bernier, M., Zheng, B., Sun, S.G., Su, M., Zhang, R., Fu, J.R., and Wen, J.K. (2010a). Kruppel-like factor 4 promotes differentiation by transforming growth factor-beta receptor-mediated Smad and p38 MAPK signaling in vascular smooth muscle cells. The Journal of biological chemistry 285, 17846-17856.

Li, R., Liang, J., Ni, S., Zhou, T., Qing, X., Li, H., He, W., Chen, J., Li, F., Zhuang, Q., et al. (2010b). A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell stem cell 7, 51-63.

Li, X., Lewis, M.T., Huang, J., Gutierrez, C., Osborne, C.K., Wu, M.F., Hilsenbeck, S.G., Pavlick, A., Zhang, X., Chamness, G.C., et al. (2008). Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst 100, 672 - 679.

Li, Y., McClintick, J., Zhong, L., Edenberg, H.J., Yoder, M.C., and Chan, R.J. (2004). Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4. Blood.

Li, Y., and Rosen, J.M. (2005). Stem/progenitor cells in mouse mammary gland development and breast cancer. J Mammary Gland Biol Neoplasia 10, 17-24.

Li, Z., Zhao, J., Li, Q., Yang, W., Song, Q., Li, W., and Liu, J. (2010c). KLF4 promotes hydrogen-peroxide-induced apoptosis of chronic myeloid leukemia cells involving the bcl-2/bax pathway. Cell stress & chaperones 15, 905-912.

Lin, Y., Wu, Y., Li, J., Dong, C., Ye, X., Chi, Y.I., Evers, B.M., and Zhou, B.P. (2010). The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1. Embo J 29, 1803-1816.

Liu, J., Du, T., Yuan, Y., He, Y., Tan, Z., and Liu, Z. (2009). KLF6 inhibits estrogen receptor-mediated cell growth in breast cancer via a c-Src-mediated pathway. Molecular and Cellular Biochemistry 335, 29-35.

Liu, J., Liu, Y., Zhang, H., Chen, G., Wang, K., and Xiao, X. (2008a). KLF4 promotes the expression, translocation, and releas eof HMGB1 in RAW264.7 macrophages in response to LPS. Shock (Augusta, Ga 30, 260-266.

Liu, J., Zhang, H., Liu, Y., Wang, K., Feng, Y., Liu, M., and Xiao, X. (2007). KLF4 regulates the expression of interleukin-10 in RAW264.7 macrophages. Biochemical and Biophysical Research Communications 362, 575-581.

157

Liu, S., Ginestier, C., Charafe-Jauffret, E., Foco, H., Kleer, C.G., Merajver, S.D., Dontu, G., and Wicha, M.S. (2008b). BRCA1 regulates human mammary stem/progenitor cell fate. Proceedings of the National Academy of Sciences of the United States of America 105, 1680-1685.

Liu, S.V. (2008). iPS cells: a more critical review. Stem cells and development 17, 391- 397.

Liu, W.F., Nelson, C.M., Pirone, D.M., and Chen, C.S. (2006). E-cadherin engagement stimulates proliferation via Rac1. J Cell Biol 173, 431-441.

Liu, Y., Liu, M., Liu, J., Zhang, H., Tu, Z., and Xiao, X. (2010). KLF4 is a novel regulator of the constitutively expressed HSP90. Cell stress & chaperones 15, 211-217.

Liu, Y., Sinha, S., McDonald, O.G., Shang, Y., Hoofnagle, M.H., and Owens, G.K. (2005a). Kruppel-like factor 4 abrogates myocardin-induced activation of smooth muscle gene expression. The Journal of biological chemistry 280, 9719-9727.

Liu, Y., Sinha, S., and Owens, G. (2003). A transforming growth factor-beta control element required for SM alpha-actin expression in vivo also partially mediates GKLF- dependent transcriptional repression. J BiolChem 278, 48004-48011.

Liu, Y., Zhao, J., Liu, J., Zhang, H., Liu, M., and Xiao, X. (2008c). Upregulation of the constitutively expressed HSC70 by KLF4. Cell stress & chaperones 13, 337-345.

Liu, Y.N., Lee, W.W., Wang, C.Y., Chao, T.H., Chen, Y., and Chen, J.H. (2005b). Regulatory mechanisms controlling human E-cadherin gene expression. Oncogene 24, 8277-8290.

Livasy, C.A., Karaca, G., Nanda, R., Tretiakova, M.S., Olopade, O.I., Moore, D.T., and Perou, C.M. (2006). Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol 19, 264-271.

Loi, S. (2010). Molecular and clinically distinct phenotypes in HER2-overexpressing breast cancer (HER2+ BC) correspond to estrogen receptor (ER) status. Paper presented at: IMPAKT Breast Cancer Conference.

158

Lu, Y., Zi, X., Zhao, Y., Mascarenhas, D., and Pollak, M. (2001). Insulin-Like Growth Factor-I Receptor Signaling and Resistance to Trastuzumab (Herceptin). Journal of the National Cancer Institute 93, 1852-1857.

Luo, A., Kong, J., Hu, G., Liew, C.C., Xiong, M., Wang, X., Ji, J., Wang, T., Zhi, H., Wu, M., et al. (2004). Discovery of Ca2+-relevant and differentiation-associated genes downregulated in esophageal squamous cell carcinoma using cDNA microarray. Oncogene 23, 1291-1299.

Ma, L., Reinhardt, F., Pan, E., Soutschek, J., Bhat, B., Marcusson, E.G., Teruya- Feldstein, J., Bell, G.W., and Weinberg, R.A. (2010). Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model. Nature biotechnology 28, 341- 347.

Ma, L., Teruya-Feldstein, J., and Weinberg, R.A. (2007). Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449, 682-688.

Ma, X.J., Wang, Z., Ryan, P.D., Isakoff, S.J., Barmettler, A., Fuller, A., Muir, B., Mohapatra, G., Salunga, R., Tuggle, J.T., et al. (2004). A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen. Cancer Cell 5, 607-616.

Madden, S.L., Cook, B.P., Nacht, M., Weber, W.D., Callahan, M.R., Jiang, Y., Dufault, M.R., Zhang, X., Zhang, W., Walter-Yohrling, J., et al. (2004). Vascular gene expression in nonneoplastic and malignant brain. Am J Pathol 165, 601-608.

Maeda, M., Johnson, K.R., and Wheelock, M.J. (2005). Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to-mesenchyme transition. Journal of Cell Science 118, 873-887.

Magenta, G., Borenstein, X., Rolando, R., and Jasnis, M.A. (2008). Rosiglitazone inhibits metastasis development of a murine mammary tumor cell line LMM3. BMC cancer 8, 47.

Mahatan, C.S., Kaestner, K.H., Geiman, D.E., and Yang, V.W. (1999). Characterization of the structure and regulation of the murine gene encoding gut-enriched Kruppel-like factor (Kruppel-like factor 4). Nucleic Acids Res 27, 4562-4569.

Makino, S. (1956). Further evidence favoring the concept of the stem cell in ascites tumors of rats. Annals of the New York Academy of Sciences 63, 818-830.

159

Malik, D.K., Baboota, S., Ahuja, A., Hasan, S., and Ali, J. (2007). Recent advances in protein and peptide drug delivery systems. Current drug delivery 4, 141-151.

Mani, S.A., Guo, W., Liao, M.J., Eaton, E.N., Ayyanan, A., Zhou, A.Y., Brooks, M., Reinhard, F., Zhang, C.C., Shipitsin, M., et al. (2008). The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 704-715.

Mani, S.A., Yang, J., Brooks, M., Schwaninger, G., Zhou, A., Miura, N., Kutok, J.L., Hartwell, K., Richardson, A.L., and Weinberg, R.A. (2007). Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proceedings of the National Academy of Sciences of the United States of America 104, 10069-10074.

Mao, Z., Song, S., Zhu, Y., Yi, X., Zhang, H., Shang, Y., and Tong, T. (2003). Transcriptional regulation of A33 antigen expression by gut-enriched Kruppel-like factor. Oncogene 22, 4434-4443.

Massague, J., and Chen, Y.G. (2000). Controlling TGF-beta signaling. Genes Dev 14, 627-644.

Mazzone, P.J., Rai, H.S., Beukemann, M., Xu, M., Abdallah, R., and Sasidhar, M. (2010). The Effect of Metformin and Thiazolidinedione Use on Lung Cancer Paper presented at: CHEST 2010, the76th annual meeting of the American College of Chest Physicians (Cleveland Clinic, Cleveland, OH, Chest ).

Mbalaviele, G., Dunstan, C.R., Sasaki, A., Williams, P.J., Mundy, G.R., and Yoneda, T. (1996). E-Cadherin Expression in Human Breast Cancer Cells Suppresses the Development of Osteolytic Bone Metastases in an Experimental Metastasis Model. Cancer Research 56, 4063-4070.

McCormick, S.M., Eskin, S.G., McIntire, L.V., Teng, C.L., Lu, C.M., Russell, C.G., and Chittur, K.K. (2001). DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells. Proceedings of the National Academy of Sciences of the United States of America 98, 8955-8960.

Medici, D., Hay, E.D., and Goodenough, D.A. (2006). Cooperation between snail and LEF-1 transcription factors is essential for TGF-beta1-induced epithelial-mesenchymal transition. Mol Biol Cell 17, 1871-1879.

160

Meissner, A., Wernig, M., and Jaenisch, R. (2007). Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol 25, 1177-1181.

Meng, F., Han, M., Zheng, B., Wang, C., Zhang, R., Zhang, X.-h., and Wen, J.-k. (2009). All-trans retinoic acid increases KLF4 acetylation by inducing HDAC2 phosphorylation and its dissociation from KLF4 in vascular smooth muscle cells. Biochemical and Biophysical Research Communications 387, 13-18.

Miller, I.J., and Bieker, J.J. (1993). A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kr•ppel family of nuclear proteins. Molecular and Cellular Biology 13, 2776-2786.

Miller, K.A., Eklund, E.A., Peddinghaus, M.L., Cao, Z., Fernandes, N., Turk, P.W., Thimmapaya, B., and Weitzman, S.A. (2001). Kruppel-like factor 4 regulates laminin alpha 3A expression in mammary epithelial cells. J BiolChem 276, 42863-42868.

Miura, K., Okita, K., Furukawa, Y., Matsuno, S., and Nakamura, Y. (1995). Deletion mapping in squamous cell carcinomas of the esophagus defines a region containing a tumor suppressor gene within a 4-centimorgan interval of the distal long arm of chromosome 9. Cancer Res 55, 1828-1830.

Miura, K., Suzuki, K., Tokino, T., Isomura, M., Inazawa, J., Matsuno, S., and Nakamura, Y. (1996). Detailed deletion mapping in squamous cell carcinomas of the esophagus narrows a region containing a putative tumor suppressor gene to about 200 kilobases on distal chromosome 9q. Cancer Res 56, 1629-1634.

Mo, Y.Y., and Reynolds, A.B. (1996). Identification of murine p120 isoforms and heterogeneous expression of p120cas isoforms in human tumor cell lines. Cancer Research 56, 2633-2640.

Moellering, R.E., Cornejo, M., Davis, T.N., Del Bianco, C., Aster, J.C., Blacklow, S.C., Kung, A.L., Gilliland, D.G., Verdine, G.L., and Bradner, J.E. (2009). Direct inhibition of the NOTCH transcription factor complex. Nature 462, 182-188.

Moll, R., Mitze, M., Frixen, U.H., and Birchmeier, W. (1993). Differential loss of E- cadherin expression in infiltrating ductal and lobular breast carcinomas. Am J Pathol 143, 1731-1742.

161

Montanez-Wiscovich, M.E., Seachrist, D.D., Landis, M.D., Visvader, J., Andersen, B., and Keri, R.A. (2009). LMO4 is an essential mediator of ErbB2/HER2/Neu-induced breast cancer cell cycle progression. Oncogene 28, 3608-3618.

Moody, S.E., Perez, D., Pan, T.C., Sarkisian, C.J., Portocarrero, C.P., Sterner, C.J., Notorfrancesco, K.L., Cardiff, R.D., and Chodosh, L.A. (2005). The transcriptional repressor Snail promotes mammary tumor recurrence. Cancer Cell 8, 197-209.

Morel, A.P., Lievre, M., Thomas, C., Hinkal, G., Ansieau, S., and Puisieux, A. (2008). Generation of breast cancer stem cells through epithelial-mesenchymal transition. PloS one 3, e2888.

Morris, G.J., Naidu, S., Topham, A.K., Guiles, F., Xu, Y., McCue, P., Schwartz, G.F., Park, P.K., Rosenberg, A.L., Brill, K., et al. (2007). Differences in breast carcinoma characteristics in newly diagnosed African-American and Caucasian patients: a single- institution compilation compared with the National Cancer Institute's Surveillance, Epidemiology, and End Results database. Cancer 110, 876-884.

Moustakas, A., and Heldin, C.H. (2007). Signaling networks guiding epithelial- mesenchymal transitions during embryogenesis and cancer progression. Cancer science 98, 1512-1520.

Mueller, E., Sarraf, P., Tontonoz, P., Evans, R.M., Martin, K.J., Zhang, M., Fletcher, C., Singer, S., and Spiegelman, B.M. (1998). Terminal differentiation of human breast cancer through PPAR gamma. Molecular cell 1, 465-470.

Muller, W.J., Sinn, E., Pattengale, P.K., Wallace, R., and Leder, P. (1988). Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene. Cell 54, 105-115.

Murphy, C.G., and Fornier, M. (2010). HER2-positive breast cancer: beyond trastuzumab. Oncology (Williston Park, NY 24, 410-415.

Nagata, Y., Lan, K.H., Zhou, X., Tan, M., Esteva, F.J., Sahin, A.A., Klos, K.S., Li, P., Monia, B.P., Nguyen, N.T., et al. (2004). PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 6, 117-127.

Nakatake, Y., Fukui, N., Iwamatsu, Y., Masui, S., Takahashi, K., Yagi, R., Yagi, K., Miyazaki, J., Matoba, R., Ko, M.S., et al. (2006). Klf4 cooperates with Oct3/4 and Sox2

162 to activate the Lefty1 core promoter in embryonic stem cells. Mol Cell Biol 26, 7772- 7782.

Narayan, M., Wilken, J.A., Harris, L.N., Baron, A.T., Kimbler, K.D., and Maihle, N.J. (2009). Trastuzumab-Induced HER Reprogramming in "Resistant" Breast Carcinoma Cells. Cancer Research 69, 2191-2194.

Nawshad, A., LaGamba, D., Polad, A., and Hay, E.D. (2005). Transforming growth factor-beta signaling during epithelial-mesenchymal transformation: implications for embryogenesis and tumor metastasis. Cells TissuesOrgans 179, 11-23.

Nguyen, D.X., Bos, P.D., and Massague, J. (2009). Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer 9, 274-284.

Nickenig, G., Baudler, S., Muller, C., Werner, C., Werner, N., Welzel, H., Strehlow, K., and Bohm, M. (2002). Redox-sensitive vascular smooth muscle cell proliferation is mediated by GKLF and Id3 in vitro and in vivo. Faseb J 16, 1077-1086.

Noti, J.D., Johnson, A.K., and Dillon, J.D. (2005). The leukocyte integrin gene CD11d is repressed by gut-enriched Kruppel-like factor 4 in myeloid cells. The Journal of biological chemistry 280, 3449-3457.

O'Brien, N.A., Browne, B.C., Chow, L., Wang, Y., Ginther, C., Arboleda, J., Duffy, M.J., Crown, J., O'Donovan, N., and Slamon, D.J. (2010). Activated Phosphoinositide 3- Kinase/AKT Signaling Confers Resistance to Trastuzumab but not Lapatinib. Molecular Cancer Therapeutics 9, 1489-1502.

Ohnesorge, N., Viemann, D., Schmidt, N., Czymai, T., Spiering, D.s.e., Schmolke, M., Ludwig, S., Roth, J., Goebeler, M., and Schmidt, M. (2010). Erk5 Activation Elicits a Vasoprotective Endothelial Phenotype via Induction of Kruppel-like Factor 4 (KLF4). 285, 26199-26210.

Ohnishi, S., Laub, F., Matsumoto, N., Asaka, M., Ramirez, F., Yoshida, T., and Terada, M. (2000). Developmental expression of the mouse gene coding for the Kruppel-like transcription factor KLF5. Dev Dyn 217, 421-429.

Ohnishi, S., Ohnami, S., Laub, F., Aoki, K., Suzuki, K., Kanai, Y., Haga, K., Asaka, M., Ramirez, F., and Yoshida, T. (2003). Downregulation and growth inhibitory effect of epithelial-type Kruppel-like transcription factor KLF4, but not KLF5, in bladder cancer. BiochemBiophysRes Commun 308, 251-256.

163

Oishi, Y., Manabe, I., Tobe, K., Ohsugi, M., Kubota, T., Fujiu, K., Maemura, K., Kubota, N., Kadowaki, T., and Nagai, R. (2008). SUMOylation of Kruppel-like transcription factor 5 acts as a molecular switch in transcriptional programs of lipid metabolism involving PPAR-[delta]. Nat Med 14, 656-666.

Olmeda, D., Moreno-Bueno, G., Flores, J.M., Fabra, A., Portillo, F., and Cano, A. (2007). SNAI1 is required for tumor growth and lymph node metastasis of human breast carcinoma MDA-MB-231 cells. Cancer Research 67, 11721-11731.

Onder, T.T., Gupta, P.B., Mani, S.A., Yang, J., Lander, E.S., and Weinberg, R.A. (2008). Loss of E-Cadherin Promotes Metastasis via Multiple Downstream Transcriptional Pathways. Cancer Research 68, 3645-3654.

Orsulic, S., Huber, O., Aberle, H., Arnold, S., and Kemler, R. (1999). E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. Journal of Cell Science 112 ( Pt 8), 1237-1245.

Ostler, J., Jones S, Zhao W, Yearsley K, Ye Y, and Barsky, S. (2008). Resistance to Her- 2/neu targeting in human bresat cancer may be mediated by epitelial-mesenchymal transition. Paper presented at: Proc Amer Assoc Cancer Res.

Ota, T., Suzuki, Y., Nishikawa, T., Otsuki, T., Sugiyama, T., Irie, R., Wakamatsu, A., Hayashi, K., Sato, H., Nagai, K., et al. (2004). Complete sequencing and characterization of 21,243 full-length human cDNAs. Nature genetics 36, 40-45.

Paccione, R.J., Miyazaki, H., Patel, V., Waseem, A., Gutkind, J.S., Zehner, Z.E., and Yeudall, W.A. (2008). Keratin down-regulation in vimentin-positive cancer cells is reversible by vimentin RNA interference, which inhibits growth and motility. Molecular Cancer Therapeutics 7, 2894-2903.

Paik, S., Shak, S., Tang, G., Kim, C., Baker, J., Cronin, M., Baehner, F.L., Walker, M.G., Watson, D., Park, T., et al. (2004). A multigene assay to predict recurrence of tamoxifen- treated, node-negative breast cancer. N Engl J Med 351, 2817-2826.

Pandya, A.Y., Talley, L.I., Frost, A.R., Fitzgerald, T.J., Trivedi, V., Chakravarthy, M., Chhieng, D.C., Grizzle, W.E., Engler, J.A., Krontiras, H., et al. (2004). Nuclear Localization of KLF4 Is Associated with an Aggressive Phenotype in Early-Stage Breast Cancer. Clinical Cancer Research 10, 2709-2719.

164

Panigada, M., Porcellini, S., Sutti, F., Doneda, L., Pozzoli, O., Consalez, G.G., Guttinger, M., and Grassi, F. (1999). GKLF in thymus epithelium as a developmentally regulated element of thymocyte-stroma cross-talk. MechDev 81, 103-113.

Park, C.H., Bergsagel, D.E., and McCulloch, E.A. (1971). Mouse myeloma tumor stem cells: a primary cell culture assay. J Natl Cancer Inst 46, 411-422.

Park, S.M., Gaur, A.B., Lengyel, E., and Peter, M.E. (2008). The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev 22, 894-907.

Parker, J., Mullins, M., Cheang, M., Leung, S., Voduc, D., Vickery, T., Davies, S., Fauron, C., He, X., Hu, Z., et al. (2009). Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol 27, 1160 - 1167.

Pece, S., Tosoni, D., Confalonieri, S., Mazzarol, G., Vecchi, M., Ronzoni, S., Bernard, L., Viale, G., Pelicci, P.G., and Di Fiore, P.P. (2010). Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content. Cell 140, 62-73.

Peinado, H., Olmeda, D., and Cano, A. (2007). Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 7, 415-428.

Peinado, H., Portillo, F., and Cano, A. (2004). Transcriptional regulation of cadherins during development and carcinogenesis. The International journal of developmental biology 48, 365-375.

Peinado, H., Quintanilla, M., and Cano, A. (2003). Transforming growth factor beta-1 induces snail transcription factor in epithelial cell lines: mechanisms for epithelial mesenchymal transitions. J Biol Chem 278, 21113-21123.

Perou, C.M., Sorlie, T., Eisen, M.B., van de Rijn, M., Jeffrey, S.S., Rees, C.A., Pollack, J.R., Ross, D.T., Johnsen, H., Akslen, L.A., et al. (2000). Molecular portraits of human breast tumours. Nature 406, 747-752.

Perrais, M., Chen, X., Perez-Moreno, M., and Gumbiner, B.M. (2007). E-Cadherin Homophilic Ligation Inhibits Cell Growth and Epidermal Growth Factor Receptor Signaling Independently of Other Cell Interactions. Molecular Biology of the Cell 18, 2013-2025.

165

Piccinni, S.A., Bolcato-Bellemin, A.L., Klein, A., Yang, V.W., Kedinger, M., Simon- Assmann, P., and Lefebvre, O. (2004). Kruppel-like factors regulate the Lama1 gene encoding the laminin alpha1 chain. The Journal of biological chemistry 279, 9103-9114.

Pidkovka, N.A., Cherepanova, O.A., Yoshida, T., Alexander, M.R., Deaton, R.A., Thomas, J.A., Leitinger, N., and Owens, G.K. (2007). Oxidized phospholipids induce phenotypic switching of vascular smooth muscle cells in vivo and in vitro. Circulation research 101, 792-801.

Polyak, K. (2007). Breast cancer: origins and evolution. The Journal of clinical investigation 117, 3155-3163.

Polyak, K., and Weinberg, R.A. (2009). Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer 9, 265-273.

Prat, A., Parker, J., Karginova, O., Fan, C., Livasy, C., Herschkowitz, J., He, X., and Perou, C. (2010). Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Research 12, R68.

Prat, A., and Perou, C.M. (2009). Mammary development meets cancer genomics. Nat Med 15, 842.

Pulaski, B.A., and Ostrand-Rosenberg, S. (2001). Mouse 4T1 breast tumor model. Current protocols in immunology / edited by John E Coligan [et al Chapter 20, Unit 20 22.

Pulaski, B.A., Terman, D.S., Khan, S., Muller, E., and Ostrand-Rosenberg, S. (2000). Cooperativity of Staphylococcal aureus enterotoxin B superantigen, major histocompatibility complex class II, and CD80 for immunotherapy of advanced spontaneous metastases in a clinically relevant postoperative mouse breast cancer model. Cancer Res 60, 2710-2715.

Qian, X., Karpova, T., Sheppard, A.M., McNally, J., and Lowy, D.R. (2004). E-cadherin- mediated adhesion inhibits ligand-dependent activation of diverse receptor tyrosine kinases. EMBO J 23, 1739-1748.

Rageul, J., Mottier, S., Jarry, A., Shah, Y., Théoleyre, S., Masson, D., Gonzalez, F.J., Laboisse, C.L., and Denis, M.G. (2009). KLF4-dependent, PPARγ-induced expression of GPA33 in colon cancer cell lines. International Journal of Cancer 125, 2802-2809.

166

Reidling, J.C., and Said, H.M. (2007). Regulation of the human biotin transporter hSMVT promoter by KLF-4 and AP-2: confirmation of promoter activity in vivo. Am J Physiol Cell Physiol 292, C1305-1312.

Roth, M.J., Hu, N., Emmert-Buck, M.R., Wang, Q.H., Dawsey, S.M., Li, G., Guo, W.J., Zhang, Y.Z., and Taylor, P.R. (2001). Genetic progression and heterogeneity associated with the development of esophageal squamous cell carcinoma. Cancer Res 61, 4098- 4104.

Rowland, B.D., Bernards, R., and Peeper, D.S. (2005). The KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene. NatCell Biol.

Rowland, B.D., and Peeper, D.S. (2005). KLF4, p21 and context-dependent opposing forces in cancer. NatRevCancer.

Saifudeen, Z., Dipp, S., Fan, H., and El-Dahr, S.S. (2005). Combinatorial control of the bradykinin B2 receptor promoter by p53, CREB, KLF-4, and CBP: implications for terminal nephron differentiation. American journal of physiology 288, F899-909.

Samavarchi-Tehrani, P., Golipour, A., David, L., Sung, H.K., Beyer, T.A., Datti, A., Woltjen, K., Nagy, A., and Wrana, J.L. (2010). Functional genomics reveals a BMP- driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell stem cell 7, 64-77.

Sander, J.D., Zaback, P., Joung, J.K., Voytas, D.F., and Dobbs, D. (2007). Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic acids research 35, W599-605.

Santisteban, M., Reiman, J.M., Asiedu, M.K., Behrens, M.D., Nassar, A., Kalli, K.R., Haluska, P., Ingle, J.N., Hartmann, L.C., Manjili, M.H., et al. (2009). Immune-induced epithelial to mesenchymal transition in vivo generates breast cancer stem cells. Cancer Res 69, 2887-2895.

Santner, S.J., Dawson, P.J., Tait, L., Soule, H.D., Eliason, J., Mohamed, A.N., Wolman, S.R., Heppner, G.H., and Miller, F.R. (2001). Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells. Breast Cancer Res Treat 65, 101-110.

Scacheri, P.C., Davis, S., Odom, D.T., Crawford, G.E., Perkins, S., Halawi, M.J., Agarwal, S.K., Marx, S.J., Spiegel, A.M., Meltzer, P.S., et al. (2006). Genome-wide

167 analysis of menin binding provides insights into MEN1 tumorigenesis. PLoSGenet 2, e51.

Scaltriti, M., Rojo, F., OcaÃ±a, A., Anido, J., Guzman, M., Cortes, J., Di Cosimo, S., Matias-Guiu, X., Ramon y Cajal, S., Arribas, J., et al. (2007). Expression of p95HER2, a Truncated Form of the HER2 Receptor, and Response to Anti-HER2 Therapies in Breast Cancer. Journal of the National Cancer Institute 99, 628-638.

Scaltriti, M., Verma, C., Guzman, M., Jimenez, J., Parra, J.L., Pedersen, K., Smith, D.J., Landolfi, S., Ramon y Cajal, S., Arribas, J., et al. (2009). Lapatinib, a HER2 tyrosine kinase inhibitor, induces stabilization and accumulation of HER2 and potentiates trastuzumab-dependent cell cytotoxicity. Oncogene 28, 803-814.

Schmierer, B., and Hill, C.S. (2007). TGF[beta]-SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol 8, 970-982.

Schulz, W.A., and Hatina, J. (2006). Epigenetics of prostate cancer: beyond DNA methylation. Journal of cellular and molecular medicine 10, 100-125.

Segre, J.A., Bauer, C., and Fuchs, E. (1999). Klf4 is a transcription factor required for establishing the barrier function of the skin. NatGenet 22, 356-360.

Sera, T. (2009). Zinc-finger-based artificial transcription factors and their applications. Advanced Drug Delivery Reviews 61, 513-526.

Shabason, J.E., Tofilon, P.J., and Camphausen, K. (2010). HDAC inhibitors in cancer care. Oncology (Williston Park, NY 24, 180-185.

Shattuck, D.L., Miller, J.K., Carraway, K.L., and Sweeney, C. (2008). Met Receptor Contributes to Trastuzumab Resistance of Her2-Overexpressing Breast Cancer Cells. Cancer Research 68, 1471-1477.

Shie, J.L., Chen, Z.Y., Fu, M., Pestell, R.G., and Tseng, C.C. (2000a). Gut-enriched Kruppel-like factor represses cyclin D1 promoter activity through Sp1 motif. Nucleic Acids Res 28, 2969-2976.

Shie, J.L., Chen, Z.Y., O'Brien, M.J., Pestell, R.G., Lee, M.E., and Tseng, C.C. (2000b). Role of gut-enriched Kruppel-like factor in colonic cell growth and differentiation. AmJ Physiol GastrointestLiver Physiol 279, G806-G814.

168

Shie, J.L., and Tseng, C.C. (2001). A nucleus-localization-deficient mutant serves as a dominant-negative inhibitor of gut-enriched Kruppel-like factor function. Biochem Biophys Res Commun 283, 205-208.

Shields, J.M., Christy, R.J., and Yang, V.W. (1996). Identification and Characterization of a Gene Encoding a Gut-enriched Kruppel-like Factor Expressed during Growth Arrest. Journal of Biological Chemistry 271, 20009-20017.

Shields, J.M., and Yang, V.W. (1997). Two potent nuclear localization signals in the gut- enriched Kruppel-like factor define a subfamily of closely related Kruppel proteins. The Journal of biological chemistry 272, 18504-18507.

Shimono, Y., Zabala, M., Cho, R.W., Lobo, N., Dalerba, P., Qian, D., Diehn, M., Liu, H., Panula, S.P., Chiao, E., et al. (2009). Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells. Cell 138, 592-603.

Shipitsin, M., Campbell, L., Argani, P., Weremowicz, S., Bloushtain-Qimron, N., Yao, J., Nikolskaya, T., Serebryiskaya, T., Beroukhim, R., Hu, M., et al. (2007). Molecular definition of breast tumor heterogeneity. Cancer Cell 11, 259 - 273.

Shipitsin, M., and Polyak, K. (2008). The cancer stem cell hypothesis: in search of definitions, markers, and relevance. Laboratory investigation; a journal of technical methods and pathology 88, 459-463.

Siddique, A., Malo, M., Ocuin, L., Hinnebusch, B., Abedrapo, M., Henderson, J., Zhang, W., Mozumder, M., Yang, V., and Hodin, R. (2003). Convergence of the thyroid hormone and gut-enriched Krüppel-like factor pathways in the context of enterocyte differentiation. Journal of Gastrointestinal Surgery 7, 1053-1061.

Sihto, H., Lundin, J., LehtimÃ¤ki, T., Sarlomo-Rikala, M., BÃ¼tzow, R., Holli, K., Sailas, L., Kataja, V., Lundin, M., Turpeenniemi-Hujanen, T., et al. (2008). Molecular Subtypes of Breast Cancers Detected in Mammography Screening and Outside of Screening. Clinical Cancer Research 14, 4103-4110.

Simmen, R.C., Pabona, J.M., Velarde, M.C., Simmons, C., Rahal, O., and Simmen, F.A. (2010). The emerging role of Kruppel-like factors in endocrine-responsive cancers of female reproductive tissues. The Journal of endocrinology 204, 223-231.

Singh, A., and Settleman, J. (2010). EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29, 4741-4751.

169

Slamon, D.J., Godolphin, W., Jones, L.A., Holt, J.A., Wong, S.G., Keith, D.E., Levin, W.J., Stuart, S.G., Udove, J., Ullrich, A., et al. (1989). Studies of the HER-2/neu protooncogene in human breast and ovarian cancer. Science 244, 707-712.

Smith, A.P., Verrecchia, A., Faga, G., Doni, M., Perna, D., Martinato, F., Guccione, E., and Amati, B. (2009). A positive role for Myc in TGFbeta-induced Snail transcription and epithelial-to-mesenchymal transition. Oncogene 28, 422-430.

Sogawa, K., Kikuchi, Y., Imataka, H., and Fujii-Kuriyama, Y. (1993). Comparison of DNA-binding properties between BTEB and Sp1. Journal of biochemistry 114, 605-609.

Sorlie, T., Perou, C.M., Tibshirani, R., Aas, T., Geisler, S., Johnsen, H., Hastie, T., Eisen, M.B., van de Rijn, M., Jeffrey, S.S., et al. (2001). Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proceedings of the National Academy of Sciences of the United States of America 98, 10869-10874.

Sorlie, T., Tibshirani, R., Parker, J., Hastie, T., Marron, J.S., Nobel, A., Deng, S., Johnsen, H., Pesich, R., Geisler, S., et al. (2003). Repeated observation of breast tumor subtypes in independent gene expression data sets. Proceedings of the National Academy of Sciences of the United States of America 100, 8418 - 8423.

Sotiriou, C., and Piccart, M.J. (2007). Taking gene-expression profiling to the clinic: when will molecular signatures become relevant to patient care? Nat Rev Cancer 7, 545- 553.

Sotiriou, C., Wirapati, P., Loi, S., Harris, A., Fox, S., Smeds, J., Nordgren, H., Farmer, P., Praz, V., Haibe-Kains, B., et al. (2006). Gene expression profiling in breast cancer: understanding the molecular basis of histologic grade to improve prognosis. J Natl Cancer Inst 98, 262-272.

Spaderna, S., Schmalhofer, O., Wahlbuhl, M., Dimmler, A., Bauer, K., Sultan, A., Hlubek, F., Jung, A., Strand, D., Eger, A., et al. (2008). The transcriptional repressor ZEB1 promotes metastasis and loss of cell polarity in cancer. Cancer Res 68, 537-544.

St.Croix, B., Sheehan, C., Rak, J.W., Florenes, V.A., Slingerland, J.M., and Kerbel, R.S. (1998). E-Cadherin-dependent Growth Suppression is Mediated by the Cyclin-dependent Kinase Inhibitor p27KIP1. The Journal of Cell Biology 142, 557-571.

Stingl, J., and Caldas, C. (2007). Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nat Rev Cancer 7, 791-799.

170

Stockinger, A., Eger, A., Wolf, J., Beug, H., and Foisner, R. (2001). E-cadherin regulates cell growth by modulating proliferation-dependent beta-catenin transcriptional activity. J Cell Biol 154, 1185-1196.

Stone, C.D., Chen, Z.Y., and Tseng, C.C. (2002). Gut-enriched Kruppel-like factor regulates colonic cell growth through APC/beta-catenin pathway. FEBS Lett 530, 147- 152.

Storci, G., Sansone, P., Trere, D., Tavolari, S., Taffurelli, M., Ceccarelli, C., Guarnieri, T., Paterini, P., Pariali, M., Montanaro, L., et al. (2008). The basal-like breast carcinoma phenotype is regulated by SLUG gene expression. J Pathol 214, 25-37.

Strickland, L.B., Dawson, P.J., Santner, S.J., and Miller, F.R. (2000). Progression of premalignant MCF10AT generates heterogeneous malignant variants with characteristic histologic types and immunohistochemical markers. Breast Cancer Res Treat 64, 235- 240.

Suske, G., Bruford, E., and Philipsen, S. (2005). Mammalian SP/KLF transcription factors: bring in the family. Genomics 85, 551-556.

Suzuki, T., Hayashi, S., Miki, Y., Nakamura, Y., Moriya, T., Sugawara, A., Ishida, T., Ohuchi, N., and Sasano, H. (2006). Peroxisome proliferator-activated receptor gamma in human breast carcinoma: a modulator of estrogenic actions. Endocrine-related cancer 13, 233-250.

Swamynathan, S.K., Katz, J.P., Kaestner, K.H., Ashery-Padan, R., Crawford, M.A., and Piatigorsky, J. (2007). Conditional Deletion of the Mouse Klf4 Gene Results in Corneal Epithelial Fragility, Stromal Edema, and Loss of Conjunctival Goblet Cells. Molecular and Cellular Biology 27, 182-194.

Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676.

Tamimi, R., Baer, H., Marotti, J., Galan, M., Galaburda, L., Fu, Y., Deitz, A., Connolly, J., Schnitt, S., Colditz, G., et al. (2008). Comparison of molecular phenotypes of ductal carcinoma in situ and invasive breast cancer. Breast Cancer Research 10, R67.

Testa, U. (2009). Proteasome inhibitors in cancer therapy. Current drug targets 10, 968- 981.

171

Thiery, J.P. (2002). Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2, 442-454.

Thiery, J.P. (2003). Epithelial-mesenchymal transitions in development and pathologies. Current opinion in cell biology 15, 740-746.

Thiery, J.P., Acloque, H., Huang, R.Y., and Nieto, M.A. (2009). Epithelial-mesenchymal transitions in development and disease. Cell 139, 871-890.

Thiery, J.P., and Sleeman, J.P. (2006). Complex networks orchestrate epithelial- mesenchymal transitions. Nat Rev Mol Cell Biol 7, 131-142.

Thompson, E.W., Torri, J., Sabol, M., Sommers, C.L., Byers, S., Valverius, E.M., Martin, G.R., Lippman, M.E., Stampfer, M.R., and Dickson, R.B. (1994). Oncogene-induced basement membrane invasiveness in human mammary epithelial cells. ClinExpMetastasis 12, 181-194.

Thoreson, M.A., and Reynolds, A.B. (2002). Altered expression of the catenin p120 in human cancer: implications for tumor progression. Differentiation 70, 583-589.

Tian, Y., Luo, A., Cai, Y., Su, Q., Ding, F., Chen, H., and Liu, Z. (2010). MicroRNA-10b promotes migration and invasion through KLF4 in human esophageal cancer cell lines. The Journal of biological chemistry 285, 7986-7994.

Traka, M.H., Chambers, K.F., Lund, E.K., Goodlad, R.A., Johnson, I.T., and Mithen, R.F. (2009). Involvement of KLF4 in sulforaphane- and iberin-mediated induction of p21(waf1/cip1). Nutrition and cancer 61, 137-145.

Trelstad, R.L., Hay, E.D., and Revel, J.D. (1967). Cell contact during early morphogenesis in the chick embryo. Dev Biol 16, 78-106.

Trelstad, R.L., Hayashi, A., Hayashi, K., and Donahoe, P.K. (1982). The epithelial- mesenchymal interface of the male rate Mullerian duct: loss of basement membrane integrity and ductal regression. Dev Biol 92, 27-40.

Trimboli, A.J., Fukino, K., de Bruin, A., Wei, G., Shen, L., Tanner, S.M., Creasap, N., Rosol, T.J., Robinson, M.L., Eng, C., et al. (2008). Direct evidence for epithelial- mesenchymal transitions in breast cancer. Cancer Res 68, 937-945.

172

Tsuda, H., Takarabe, T., Hasegawa, F., Fukutomi, T., and Hirohashi, S. (2000). Large, central acellular zones indicating myoepithelial tumor differentiation in high-grade invasive ductal carcinomas as markers of predisposition to lung and brain metastases. The American journal of surgical pathology 24, 197-202.

Turksen, K., and Troy, T.-C. (2002). Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 129, 1775-1784.

Turner, J., and Crossley, M. (1999). Basic Kruppel-like factor functions within a network of interacting haematopoietic transcription factors. Int J Biochem Cell Biol 31, 1169- 1174.

Turner, N., Tutt, A., and Ashworth, A. (2004). Hallmarks of 'BRCAness' in sporadic cancers. Nat Rev Cancer 4, 814-819.

Turner, N.C., and Reis-Filho, J.S. (2006). Basal-like breast cancer and the BRCA1 phenotype. Oncogene 25, 5846-5853.

Turner, N.C., Reis-Filho, J.S., Russell, A.M., Springall, R.J., Ryder, K., Steele, D., Savage, K., Gillett, C.E., Schmitt, F.C., Ashworth, A., et al. (2007). BRCA1 dysfunction in sporadic basal-like breast cancer. Oncogene 26, 2126-2132.

Valdes, F., Alvarez, A.M., Locascio, A., Vega, S., Herrera, B., Fernandez, M., Benito, M., Nieto, M.A., and Fabregat, I. (2002). The epithelial mesenchymal transition confers resistance to the apoptotic effects of transforming growth factor Beta in fetal rat hepatocytes. Mol Cancer Res 1, 68-78. van 't Veer, L.J., Dai, H., van de Vijver, M.J., He, Y.D., Hart, A.A., Mao, M., Peterse, H.L., van der Kooy, K., Marton, M.J., Witteveen, A.T., et al. (2002). Gene expression profiling predicts clinical outcome of breast cancer. Nature 415, 530-536.

Van Dyke, T., and Jacks, T. (2002). Cancer modeling in the modern era: progress and challenges. Cell 108, 135-144.

Vandewalle, C., Comijn, J., De Craene, B., Vermassen, P., Bruyneel, E., Andersen, H., Tulchinsky, E., Van Roy, F., and Berx, G. (2005). SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell-cell junctions. Nucleic Acids Res 33, 6566- 6578.

173

Villarreal, G., Jr., Zhang, Y., Larman, H.B., Gracia-Sancho, J., Koo, A., and Garcia- Cardena, G. (2010). Defining the regulation of KLF4 expression and its downstream transcriptional targets in vascular endothelial cells. Biochemical and biophysical research communications 391, 984-989.

Vincent, T., Neve, E.P.A., Johnson, J.R., Kukalev, A., Rojo, F., Albanell, J., Pietras, K., Virtanen, I., Philipson, L., Leopold, P.L., et al. (2009). A SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-[beta] mediated epithelial-mesenchymal transition. Nat Cell Biol 11, 943-950.

Visvader, J.E., and Lindeman, G.J. (2008). Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 8, 755-768.

Vogel, V.G., Costantino, J.P., Wickerham, D.L., Cronin, W.M., Cecchini, R.S., Atkins, J.N., Bevers, T.B., Fehrenbacher, L., Pajon, E.R., Wade, J.L., 3rd, et al. (2010). Update of the National Surgical Adjuvant Breast and Bowel Project Study of Tamoxifen and Raloxifene (STAR) P-2 Trial: Preventing breast cancer. Cancer prevention research (Philadelphia, Pa 3, 696-706.

Vogelstein, B., Lane, D., and Levine, A.J. (2000). Surfing the p53 network. Nature 408, 307-310.

Voorhees, P.M., Dees, E.C., O'Neil, B., and Orlowski, R.Z. (2003). The proteasome as a target for cancer therapy. Clin Cancer Res 9, 6316-6325.

Wang, C., Han, M., Zhao, X.M., and Wen, J.K. (2008a). Kruppel-like factor 4 is required for the expression of vascular smooth muscle cell differentiation marker genes induced by all-trans retinoic acid. Journal of biochemistry 144, 313-321.

Wang, H., Yang, L., Jamaluddin, M.S., and Boyd, D.D. (2004). The Kruppel-like KLF4 transcription factor, a novel regulator of urokinase receptor expression, drives synthesis of this binding site in colonic crypt luminal surface epithelial cells. The Journal of biological chemistry 279, 22674-22683.

Wang, N., Liu, Z.H., Ding, F., Wang, X.Q., Zhou, C.N., and Wu, M. (2002). Down- regulation of gut-enriched Kruppel-like factor expression in esophageal cancer. World J Gastroenterol 8, 966-970.

Wang, S.E., Xiang, B., Guix, M., Olivares, M.G., Parker, J., Chung, C.H., Pandiella, A., and Arteaga, C.L. (2008b). Transforming Growth Factor {beta} Engages TACE and

174

ErbB3 To Activate Phosphatidylinositol-3 Kinase/Akt in ErbB2-Overexpressing Breast Cancer and Desensitizes Cells to Trastuzumab. Molecular and Cellular Biology 28, 5605- 5620.

Wang, X., and Zhao, J. (2007). KLF8 transcription factor participates in oncogenic transformation. Oncogene 26, 456-461.

Wang, X., Zheng, M., Liu, G., Xia, W., McKeown-Longo, P.J., Hung, M.C., and Zhao, J. (2007). Kruppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion. Cancer Research 67, 7184-7193.

Wang, Y., Klijn, J.G., Zhang, Y., Sieuwerts, A.M., Look, M.P., Yang, F., Talantov, D., Timmermans, M., Meijer-van Gelder, M.E., Yu, J., et al. (2005). Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet 365, 671-679.

Wassmann, S., Wassmann, K., Jung, A., Velten, M., Knuefermann, P., Petoumenos, V., Becher, U., Werner, C., Mueller, C., and Nickenig, G. (2007). Induction of p53 by GKLF is essential for inhibition of proliferation of vascular smooth muscle cells. J Mol Cell Cardiol 43, 301-307.

Wei, D., Gong, W., Kanai, M., Schlunk, C., Wang, L., Yao, J.C., Wu, T.T., Huang, S., and Xie, K. (2005). Drastic down-regulation of Kruppel-like factor 4 expression is critical in human gastric cancer development and progression. Cancer Research 65, 2746- 2754.

Wei, D., Kanai, M., Jia, Z., Le, X., and Xie, K. (2008). Kruppel-like factor 4 induces p27Kip1 expression in and suppresses the growth and metastasis of human pancreatic cancer cells. Cancer Res 68, 4631-4639.

Wei, D., Wang, L., Kanai, M., Jia, Z., Le, X., Li, Q., Wang, H., and Xie, K. (2010). KLF4[alpha] Up-Regulation Promotes Cell Cycle Progression and Reduces Survival Time of Patients With Pancreatic Cancer. Gastroenterology In Press, Uncorrected Proof.

Wei, H., Wang, X., Gan, B., Urvalek, A.M., Melkoumian, Z.K., Guan, J.L., and Zhao, J. (2006). Sumoylation delimits KLF8 transcriptional activity associated with the cell cycle regulation. The Journal of biological chemistry 281, 16664-16671.

Wei, X., Xu, H., and Kufe, D. (2007). Human Mucin 1 Oncoprotein Represses Transcription of the p53 Tumor Suppressor Gene. Cancer Research 67, 1853-1858.

175

Wei, Z., Yang, Y., Zhang, P., Andrianakos, R., Hasegawa, K., Lyu, J., Chen, X., Bai, G., Liu, C., Pera, M., et al. (2009). Klf4 Directly Interacts with Oct4 and Sox2 to Promote Reprogramming. Stem Cells 9999.

Weigelt, B., and Reis-Filho, J.S. (2009). Histological and molecular types of breast cancer: is there a unifying taxonomy? Nat Rev Clin Oncol 6, 718-730.

Wellings, S.R., and Jensen, H.M. (1973). On the origin and progression of ductal carcinoma in the human breast. J Natl Cancer Inst 50, 1111-1118.

Wellner, U., Schubert, J., Burk, U.C., Schmalhofer, O., Zhu, F., Sonntag, A., Waldvogel, B., Vannier, C., Darling, D., zur Hausen, A., et al. (2009). The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol 11, 1487-1495.

Welm, B., Behbod, F., Goodell, M.A., and Rosen, J.M. (2003). Isolation and characterization of functional mammary gland stem cells. Cell proliferation 36 Suppl 1, 17-32.

Wheelock, M.J., and Johnson, K.R. (2003). Cadherin-mediated cellular signaling. CurrOpinCell Biol 15, 509-514.

Whiteman, E.L., Liu, C.J., Fearon, E.R., and Margolis, B. (2008). The transcription factor snail represses Crumbs3 expression and disrupts apico-basal polarity complexes. Oncogene 27, 3875-3879.

Whitney, E.M., Ghaleb, A.M., Chen, X., and Yang, V.W. (2006). Transcriptional profiling of the cell cycle checkpoint gene kruppel-like factor 4 reveals a global inhibitory function in macromolecular biosynthesis. Gene expression 13, 85-96.

Wicha, M.S., Liu, S., and Dontu, G. (2006). Cancer stem cells: an old idea--a paradigm shift. Cancer Res 66, 1883-1890; discussion 1895-1886.

Wirapati, P., Sotiriou, C., Kunkel, S., Farmer, P., Pradervand, S., Haibe-Kains, B., Desmedt, C., Ignatiadis, M., Sengstag, T., Schutz, F., et al. (2008). Meta-analysis of gene expression profiles in breast cancer: toward a unified understanding of breast cancer subtyping and prognosis signatures. Breast Cancer Res 10, R65.

176

Wu, J.M., Fackler, M.J., Halushka, M.K., Molavi, D.W., Taylor, M.E., Teo, W.W., Griffin, C., Fetting, J., Davidson, N.E., De Marzo, A.M., et al. (2008). Heterogeneity of breast cancer metastases: comparison of therapeutic target expression and promoter methylation between primary tumors and their multifocal metastases. ClinCancer Res 14, 1938-1946.

Xiao, H., Chung, J., Kao, H.-Y., and Yang, Y.-C. (2003). Tip60 Is a Co-repressor for STAT3, pp. 11197-11204.

Xiong, Y., Hannon, G.J., Zhang, H., Casso, D., Kobayashi, R., and Beach, D. (1993). p21 is a universal inhibitor of cyclin kinases. Nature 366, 701-704.

Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J., and Kosik, K. (2009). MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell 137, 647-658.

Yanagisawa, M., Huveldt, D., Kreinest, P., Lohse, C.M., Cheville, J.C., Parker, A.S., Copland, J.A., and Anastasiadis, P.Z. (2008). A p120 catenin isoform switch affects Rho activity, induces tumor cell invasion, and predicts metastatic disease. J Biol Chem 283, 18344-18354.

Yancy, H.F., Mason, J.A., Peters, S., Thompson, C.E., 3rd, Littleton, G.K., Jett, M., and Day, A.A. (2007). Metastatic progression and gene expression between breast cancer cell lines from African American and Caucasian women. Journal of carcinogenesis 6, 8.

Yang, J., Mani, S.A., Donaher, J.L., Ramaswamy, S., Itzykson, R.A., Come, C., Savagner, P., Gitelman, I., Richardson, A., and Weinberg, R.A. (2004). Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117, 927- 939.

Yang, J., and Weinberg, R.A. (2008). Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell 14, 818-829.

Yang, S.H., Jaffray, E., Senthinathan, B., Hay, R.T., and Sharrocks, A.D. (2003). SUMO and transcriptional repression: dynamic interactions between the MAP kinase and SUMO pathways. Cell cycle (Georgetown, Tex 2, 528-530.

Yang, X.H., Flores, L.M., Li, Q., Zhou, P., Xu, F., Krop, I.E., and Hemler, M.E. (2010). Disruption of Laminin-Integrin-CD151-Focal Adhesion Kinase Axis Sensitizes Breast Cancer Cells to ErbB2 Antagonists. Cancer Research 70, 2256-2263.

177

Yang, X.R., Sherman, M.E., Rimm, D.L., Lissowska, J., Brinton, L.A., Peplonska, B., Hewitt, S.M., Anderson, W.F., Szeszenia-DÄ…browska, N., Bardin-Mikolajczak, A., et al. (2007). Differences in Risk Factors for Breast Cancer Molecular Subtypes in a Population-Based Study. Cancer Epidemiology Biomarkers & Prevention 16, 439-443.

Yang, Y., Goldstein, B.G., Chao, H.H., and Katz, J.P. (2005a). KLF4 and KLF5 regulate proliferation, apoptosis and invasion in esophageal cancer cells. Cancer biology & therapy 4, 1216-1221.

Yang, Z., Rayala, S., Nguyen, D., Vadlamudi, R.K., Chen, S., and Kumar, R. (2005b). Pak1 phosphorylation of snail, a master regulator of epithelial-to-mesenchyme transition, modulates snail's subcellular localization and functions. Cancer Res 65, 3179-3184.

Yasunaga, J., Taniguchi, Y., Nosaka, K., Yoshida, M., Satou, Y., Sakai, T., Mitsuya, H., and Matsuoka, M. (2004). Identification of aberrantly methylated genes in association with adult T-cell leukemia. Cancer Research 64, 6002-6009.

Yates, C.C., Shepard, C.R., Stolz, D.B., and Wells, A. (2007). Co-culturing human prostate carcinoma cells with hepatocytes leads to increased expression of E-cadherin. Br J Cancer 96, 1246-1252.

Yee, L.D., Williams, N., Wen, P., Young, D.C., Lester, J., Johnson, M.V., Farrar, W.B., Walker, M.J., Povoski, S.P., Suster, S., et al. (2007). Pilot study of rosiglitazone therapy in women with breast cancer: effects of short-term therapy on tumor tissue and serum markers. Clin Cancer Res 13, 246-252.

Yet, S.F., McA'Nulty, M.M., Folta, S.C., Yen, H.W., Yoshizumi, M., Hsieh, C.M., Layne, M.D., Chin, M.T., Wang, H., Perrella, M.A., et al. (1998). Human EZF, a Kruppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains. J Biol Chem 273, 1026-1031.

Yoon, H.S., Chen, X., and Yang, V.W. (2003). Kruppel-like Factor 4 Mediates p53- dependent G1/S Cell Cycle Arrest in Response to DNA Damage. Journal of Biological Chemistry 278, 2101-2105.

Yoon, H.S., Ghaleb, A.M., Nandan, M.O., Hisamuddin, I.M., Dalton, W.B., and Yang, V.W. (2005). Kruppel-like factor 4 prevents centrosome amplification following gamma- irradiation-induced DNA damage. Oncogene 24, 4017-4025.

178

Yoon, H.S., and Yang, V.W. (2004). Requirement of Kruppel-like Factor 4 in Preventing Entry into Mitosis following DNA Damage. Journal of Biological Chemistry 279, 5035- 5041.

Yori, J.L., Johnson, E., Zhou, G., Jain, M.K., and Keri, R.A. (2010a). Kruppel-like factor 4 inhibits epithelial-to-mesenchymal transition through regulation of E-cadherin gene expression. The Journal of biological chemistry 285, 16854-16863.

Yori, J.L., Seachrist, D.D., Johnson, E., Lozada, K.L., Abdul-Karim, F.W., Chodosh, L.A., Schiemann, W.P., and Keri, R.A. (2010b). Kruppel-like factor 4 inhibits tumorigenic progression and metastasis in a mouse model of breast cancer. Cancer Research (submitted).

Yoshida, T., Gan, Q., Franke, A.S., Ho, R., Zhang, J., Chen, Y.E., Hayashi, M., Majesky, M.W., Somlyo, A.V., and Owens, G.K. (2010). Smooth and cardiac muscle-selective knock-out of Kruppel-like factor 4 causes postnatal death and growth retardation. The Journal of biological chemistry 285, 21175-21184.

Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920.

Yu, M., Smolen, G.A., Zhang, J., Wittner, B., Schott, B.J., Brachtel, E., Ramaswamy, S., Maheswaran, S., and Haber, D.A. (2009). A developmentally regulated inducer of EMT, LBX1, contributes to breast cancer progression. Genes Dev 23, 1737-1742.

Zhang, H., and Stavnezer, E. (2009). Ski regulates muscle terminal differentiation by transcriptional activation of Myog in a complex with Six1 and Eya3. J Biol Chem 284, 2867-2879.

Zhang, W., Geiman, D.E., Shields, J.M., Dang, D.T., Mahatan, C.S., Kaestner, K.H., Biggs, J.R., Kraft, A.S., and Yang, V.W. (2000). The Gut-enriched Kruppel-like Factor (Kruppel-like Factor 4) Mediates the Transactivating Effect of p53 on the p21WAF1/Cip1 Promoter. Journal of Biological Chemistry 275, 18391-18398.

Zhang, W., Shields, J.M., Sogawa, K., Fujii-Kuriyama, Y., and Yang, V.W. (1998). The gut-enriched Kruppel-like factor suppresses the activity of the CYP1A1 promoter in an Sp1-dependent fashion. The Journal of biological chemistry 273, 17917-17925.

179

Zhang, X.-L., Zhang, D., Michel, F.J., Blum, J.L., Simmen, F.A., and Simmen, R.C.M. (2003). Selective Interactions of KrÃ¼ppel-like Factor 9/Basic Transcription Element- binding Protein with Progesterone Receptor Isoforms A and B Determine Transcriptional Activity of Progesterone-responsive Genes in Endometrial Epithelial Cells, pp. 21474- 21482.

Zhao, J., Bian, Z.C., Yee, K., Chen, B.P., Chien, S., and Guan, J.L. (2003). Identification of transcription factor KLF8 as a downstream target of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression. Molecular cell 11, 1503-1515.

Zhao, W., Hisamuddin, I.M., Nandan, M.O., Babbin, B.A., Lamb, N.E., and Yang, V.W. (2004). Identification of Kruppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer. Oncogene 23, 395-402.

Zheng, B., Han, M., Bernier, M., Zhang, X.H., Meng, F., Miao, S.B., He, M., Zhao, X.M., and Wen, J.K. (2009a). Kruppel-like factor 4 inhibits proliferation by platelet- derived growth factor receptor beta-mediated, not by retinoic acid receptor alpha- mediated, phosphatidylinositol 3-kinase and ERK signaling in vascular smooth muscle cells. The Journal of biological chemistry 284, 22773-22785.

Zheng, H., Pritchard, D.M., Yang, X., Bennett, E., Liu, G., Liu, C., and Ai, W. (2009b). KLF4 gene expression is inhibited by the notch signaling pathway that controls goblet cell differentiation in mouse gastrointestinal tract. Am J Physiol Gastrointest Liver Physiol 296, G490-498.

Zhou, B.P., Deng, J., Xia, W., Xu, J., Li, Y.M., Gunduz, M., and Hung, M.-C. (2004). Dual regulation of Snail by GSK-3[beta]-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol 6, 931-940.

Zhou, B.P., Liao, Y., Xia, W., Spohn, B., Lee, M.-H., and Hung, M.-C. (2001). Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER- 2/neu-overexpressing cells. Nat Cell Biol 3, 245-252.

Zhou, Q., Hong, Y., Zhan, Q., Shen, Y., and Liu, Z. (2009). Role for Kruppel-like factor 4 in determining the outcome of p53 response to DNA damage. Cancer Res 69, 8284- 8292.

Zhou, Y., Hofstetter, W.L., He, Y., Hu, W., Pataer, A., Wang, L., Wang, J., Zhou, Y., Yu, L., Fang, B., et al. (2010). KLF4 inhibition of lung cancer cell invasion by suppression of SPARC expression. Cancer biology & therapy 9.

180

Zhuang, Z., Lininger, R.A., Man, Y.G., Albuquerque, A., Merino, M.J., and Tavassoli, F.A. (1997). Identical clonality of both components of mammary carcinosarcoma with differential loss of heterozygosity. Mod Pathol 10, 354-362.

181