C/EBPδ EXPRESSION AND FUNCTION IN PROSTATE CANCER BIOLOGY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

The Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

by

Daniel C. Sanford

* * * * *

The Ohio State University 2006

Dissertation Committee:

Dr. James DeWille, Adviser

Dr. Patrick Green Approved by

Dr. Michael Ostrowski ______Adviser Dr. Robert Hamlin Graduate Program in Molecular, Cellular and Developmental Biology

ABSTRACT

CCAAT/Enhancer binding proteins (C/EBPs) are a family of transcription factors that play key roles in growth, differentiation and apoptosis. Previous studies demonstrated that C/EBPδ, one of the C/EBP family members, plays an important role in regulating growth arrest in mammary epithelial cells.

Similarities in the activation of growth arrest pathways between mammary and prostate epithelial cells have been described; however, the function of C/EBPδ

in prostate cancer cell biology has not been extensively investigated. The focus

of this dissertation was the induction and function of C/EBPδ in prostate cancer

cell biology. The purpose of this dissertation was three-fold. First, investigate the induction of C/EBPδ in response to prostate cancer cell growth arrest and determine the signaling pathways required for C/EBPδ induction. Secondly,

investigate the potential function of the C/EBPδ growth arrest signaling pathway

in prostate cancer progression and metastasis. Finally, identify potential

C/EBPδ downstream effector genes to further elucidate the function of C/EBPδ

in prostate cancer cell biology.

ii The first part of this project investigated the expression of C/EBPδ and

the upstream signaling events required for C/EBPδ induction under growth

arrest conditions in prostate cancer cell lines. Previous studies have

demonstrated that interleukin 6 (IL-6) induce growth arrest in the LNCaP

prostate cancer cell line. We hypothesized that C/EBPδ expression would be

induced under these conditions. Consistent with previous results, we

demonstrated that IL-6 induced LNCaP cell growth arrest, even in the presence

of complete growth media. Northern blot analysis demonstrated that C/EBPδ

mRNA expression was significantly induced with the addition of IL-6 but not with

serum and growth factor withdrawal. Further analysis showed that signal

transducer and activator of transcription 3 (STAT3) was also activated

(phosphorylated) with the addition of IL-6 but not with serum and growth factor

withdrawal, suggesting that STAT3 played an important role in the induction of

C/EBPδ in response to IL-6. Reporter gene studies identified a STAT3 binding

site within the C/EBPδ promoter that is required for IL-6 induced C/EBPδ

expression. Subsequent examination of additional prostate cancer cell lines

demonstrated that cells harboring defects in this pathway were resistant to the

growth suppressive effects of IL-6. We then demonstrated that STAT3

activation and the induction of C/EBPδ gene expression was specific to G0/G1 growth arrest using cell cycle blocking studies. In addition, we showed that

C/EBPδ expression alone suppressed the growth of prostate cancer cells. This work identified a STAT3/C/EBPδ growth arrest pathway which was activated in

iii prostate cancer cells in response to IL-6. In addition, we demonstrated that

defects in this growth arrest pathway render prostate cancer cells resistant to

IL-6 mediated growth arrest. Finally, C/EBPδ overexpression significantly

inhibited prostate cancer cell growth which suggested that C/EBPδ may function

as a novel growth suppressor gene in prostate cancer cells.

The second focus of this dissertation was the investigation of the

potential role of the IL-6/STAT3/C/EBPδ growth arrest pathway in prostate

cancer progression and metastasis. Previous studies demonstrated that the

primary site of prostate cancer metastasis is bone and the growth of prostate

metastases in bone is significantly reduced, allowing cancer cells to evade

chemotherapy. We hypothesized that the IL-6/STAT3/C/EBPδ growth arrest pathway may be activated in prostate cancer cells which have metastasized to bone. We first demonstrated that osteoblast conditioned media (OCM) significantly inhibited LNCaP cell growth. Northern and western blot analyses demonstrated that OCM induced both STAT3 activation and C/EBPδ gene expression in LNCaP cells. Cytokine array analysis demonstrated that OCM contained high levels of IL-6, approximately 6.5ng/ml when quantitated by

ELISA. Antibody blocking studies specifically targeting IL-6 signaling inhibited

OCM induced STAT3 activation, C/EBPδ expression and growth arrest in

LNCaP cells. Furthermore, siRNA mediated gene knockdown experiments reduced activated STAT3 and C/EBPδ protein levels resulting in a decrease in

OCM induced growth arrest. Finally, we demonstrated that prolonged exposure

iv to IL-6 eliminated IL-6 mediated growth arrest. These findings suggest that the

IL-6/STAT3/C/EBPδ growth arrest signaling pathway may play an important role in prostate cancer progression.

The final portion of this dissertation investigated the downstream effector genes regulated in response to C/EBPδ induction. Microarray and ChIP on Chip assays identified several candidate C/EBPδ downstream effector genes which have been shown to function in growth arrest, including beta 1C integrin,

FOXO3A, ING4, BTG2 and MCM7. Interestingly, both assays also identified downstream genes with functions in cell survival, including TRX2, Mcl-1 and beta 1 arrestin. Subsequent analysis of the potential pro-survival function of

C/EBPδ demonstrated that doxorubicin regulated C/EBPδ protein expression in a STAT3 independent manner. SiRNA-mediated C/EBPδ knockdown reduced bcl-xl expression levels and increased doxorubicin-induced apoptosis in LNCaP cells. Further studies suggested that the pro-survival function of C/EBPδ may not be restricted to LNCaP cells but may also exist in mammary epithelial cells.

Overall, in addition to identifying candidate C/EBPδ downstream target genes with functions in growth arrest, these studies identified a novel pro-survival function for C/EBPδ in prostate cancer biology.

In summary, this dissertation identified the activation of STAT3 and induction of C/EBPδ as important mediators of IL-6 induced growth arrest in prostate cancer cells. Defects in this pathway render cells insensitive to IL-6 mediated growth arrest. This dissertation also demonstrated that the activation

v of this growth arrest pathway may play an important role in the metastasis of prostate cancer cells to bone as well as the progression of the disease. Finally, to our knowledge, our studies are the first to investigate the function of C/EBPδ on a genome-wide scale. We identified several C/EBPδ downstream target genes involved in growth regulation and also identified a novel pro-survival function for C/EBPδ in prostate cancer biology.

vi

Dedicated to my parents and my wife Jamie for all their support.

vii ACKNOWLEDGMENTS

This body of work could not have been completed without the help of many individuals over the years. I would first like to thank my advisor, Dr. Jim DeWille. I cannot express how important your mentorship has been. You have allowed me to explore the various avenues of my research and provided a strong foundation for my scientific career. I am also grateful for our discussions about life, computers, sports, eBay and everything else that ever came up. Hopefully some of my computer knowledge rubbed off and will make your battles with the computer a little easier. I would also like to thank my committee members, Dr. Patrick Green, Dr. Michael Ostrowski, Dr. Michael Robinson and Dr. Robert Hamlin for their valuable suggestions throughout my graduate career. I would also like to thank both my current (Dr. Dahai Tang, Yingjie Zhang, Junling Si, Shanggen Zhou, Bin Li) and past (Larry Dearth, Gloria Sivko, Andrew Gigliotti, Julie Hutt) lab members for providing a fun and productive work environment. A special thanks to the friends I have met along the way including Matt and Tiffiney Hartman, Stacey Hull, Patrick Baker, Matt and Kristen Anderson, Yvette Nout, Adrienne Lee, Ellen Nixon, Matt Buccellato, Tom and Mary Carsillo, Drew Dangle, Jeff Voorhees and Amy Wanken. I will miss the basketball games with Dr. Tang and the softball games in the summer.

viii I would like to thank my parents. Mom and Dad you have been there through it all and I cannot express how thankful I am for everything you have done over the years. Finally I would like to thank my wife Jamie. You are my best friend and I cannot imagine how I would have done this without you. Thanks for keeping me focused on the important things and for always being there.

ix VITA

July 22, 1977 ...... Born – Hornell, New York

1999 ...... B.A. Biology, Elmira College Elmira, New York

1999 - present ...... Graduate Teaching and Research Associate, The Ohio State University Columbus, Ohio

PUBLICATIONS

1. Sanford DC, and JW DeWille. 2005. C/EBPδ is a downstream mediator of IL-6 induced growth inhibition of prostate cancer cells. The Prostate. 63:143-54.

2. Sivko, G. S., D. C. Sanford, L. D. Dearth, D. Tang, and J. W. DeWille. 2004. CCAAT/Enhancer binding protein delta (C/EBPdelta) regulation and expression in human mammary epithelial cells: II. Analysis of activating signal transduction pathways, transcriptional, post- transcriptional, and post-translational control. J Cell Biochem 93:844-56.

FIELDS OF STUDY

Major Field: Molecular, Cellular and Developmental Biology

x TABLE OF CONTENTS

Page Abstract ...... ii

Dedication ...... vii

Acknowledgments ...... viii

Vita ...... x

List of Tables ...... xiii

List of Figures...... xiv

Abbreviations...... xvi

Chapters:

1. Introduction ...... 1

Statement of Purpose ...... 1 Prostate Cancer ...... 2 Prostate Cancer Metastases and Bone...... 4 The IL-6 Family of Cytokines ...... 5 Interleukin 6 ...... 6 IL-6 and Prostate Cancer ...... 12 The JAK-STAT Pathway ...... 14 Signal Transducers and Activators of Transcription (STATs)...... 17 Function of STAT Proteins ...... 18 STAT3...... 19 STAT3 and Prostate Cancer ...... 20 CCAAT/Enhancer Binding Proteins (C/EBPs)...... 23 The Structure of the C/EBP Family Members ...... 24 Function of C/EBP Proteins ...... 26 C/EBPδ and G0 Growth Arrest ...... 30 C/EBPδ and the Prostate ...... 33

xi 2. C/EBPδ is a Downstream Mediator of IL-6 Induced Growth Inhibition of Prostate Cancer Cells...... 35

Abstract...... 35 Introduction ...... 36 Materials and Methods...... 39 Results ...... 43 Discussion...... 59

3. Osteoblasts inhibit prostate cancer cell growth via activation of the IL-6/STAT3 growth arrest pathway...... 66

Abstract...... 66 Introduction ...... 67 Materials and Methods...... 71 Results ...... 73 Discussion...... 83

4. Identification of C/EBPδ downstream effector genes: A novel pro-survival function for C/EBPδ...... 89

Abstract...... 89 Introduction ...... 90 Materials and Methods...... 93 Results ...... 97 Discussion...... 110

5. Perspectives and Future Work...... 117

The IL-6/STAT3/C/EBPδ growth arrest pathway and prostate cancer ...... 117 Prostate cancer metastasis to bone...... 119 The function of C/EBPδ in prostate cancer growth arrest...... 120 Doxorubicin induction of C/EBPδ protein levels ...... 121 The novel function of C/EBPδ in survival ...... 122

Bibliography...... 123

xii LIST OF TABLES

Table Page

1.1 Abnormal IL-6 production and its association with cancers ...... 10

4.1 Microarray identified target genes regulated by C/EBPδ overexpression...... 98

xiii LIST OF FIGURES

Figure Page

1.1 Representation of the well characterized activities of IL-6...... 7

1.2 Overview of the JAK/STAT signaling pathway...... 16

1.3 Diagram of the STAT domain structure...... 17

1.4 Structure of the C/EBP family members...... 24

1.5 Model for C/EBPβ bound to DNA...... 25

2.1 IL-6 decreases [3H]Thymidine incorporation in the LNCaP but not the PC-3 human prostate epithelial cell line...... 45

2.2 Induction of C/EBPδ mRNA following IL-6 treatment...... 46

2.3 Phosphorylation of JAK2, STAT3 and induction of C/EBPδ protein following IL-6 treatment of LNCaP cells...... 47

2.4 Analysis of STAT3 activation and C/EBPδ expression following IL-6 addition in various prostate cancer cell lines...... 50

2.5 Identification of an IL-6 responsive region within the human C/EBPδ gene promoter ...... 52

2.6 Analysis of the STAT3 and Sp1 sites...... 54

2.7 Cell cycle blocking studies ...... 56

2.8 C/EBPδ expression inhibits the growth of prostate cancer cell lines...... 58

3.1 Osteoblast conditioned media induces growth arrest, STAT3 activation and C/EBPδ expression in LNCaP cells ...... 75

xiv 3.2 OCM induced STAT3 activation, C/EBPδ gene expression and growth arrest is the result of secreted IL-6...... 77

3.3 siRNA mediated knockdown of STAT3 reduces OCM mediated LNCaP growth arrest ...... 79

3.4 Prolonged exposure to IL-6 eliminates IL-6 mediated growth arrest of LNCaP cells in response to exogenous IL-6...... 82

4.1 Microarray validation by RT-PCR ...... 99

4.2 ChIP on Chip analysis to identify C/EBPδ bound gene promoters...... 101

4.3 IL-6 reduces doxorubicin (DOX) induced apoptosis...... 103

4.4 Doxorubicin increases C/EBPδ protein levels in the absence of phosphorylated STAT3 ...... 104

4.5 Doxorubicin does not affect C/EBPδ mRNA levels in LNCaP cells...... 105

4.6 SiRNA mediated C/EBPδ knockdown reduces Bcl-xl protein levels and increases cleaved caspase-3 levels in doxorubicin treated LNCaP cells ...... 107

4.7 Doxorubicin reduces C/EBPδ protein levels in mammary epithelial cells but C/EBPδ overexpression renders cells resistant to doxorubicin induced apoptosis...... 109

5.1 Summary of dissertation studies...... 118

xv ABBREVIATIONS

ADM activation domain modules

APR acute phase response

APRE acute phase response elements

AR androgen receptor

ARE androgen responsive elements

ATCC american tissue culture collections

ATF activating transcription factor

BAD Bcl2- antagonist of cell death

Bcl-2 B-cell leukemia /lymphoma 2

Bcl-xl B-cell leukemia/lymphoma like protein-long isoform

β−gal β-galactosidase

Bp base pair

BSF2 B-cell stimulatory factor 2

BTG2 B-cell translocation gene family member 2

bZIP leucine zipper motif

C/EBP CCAAT enhancer binding protein cdk2 cyclin dependent Kinase 2 cdk4 cyclin dependent kinase 4

xvi CGM complete growth media

ChIP chromatin immunoprecipitation

CHOP C/EBP homologous protein

CLC cardiotrophin-like cytokine

CML chronic myelogenous leukemia

CNTF ciliary neurotrophic factor

CNTFRα ciliary neurotrophic factor receptor α

CP cyclophilin

CREB cyclic AMP response element binding protein

CT-1 cardiotrophin-1

DMEM dulbecco’s modified eagle medium

DNA deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate

Dox doxorubicin

FBS fetal bovine serum

GADD153 growth arrest and DNA damage inducible gene 153 gadd45γ growth arrest and DNA damage inducible gene 45 γ

GAM growth arrest media

GAPDH glyceraldehyde-3-phosphate dehydrogenase gas1 growth arrest specific gene 1

GNA11 guanine nucleotide binding protein alpha 11 gp130 glycoprotein 130

HFOB human fetal osteoblasts

xvii HGF hybridoma growth factor

HPV human papiloma virus

HSF hepatocyte stimulatory factor

IFN interferon

IFNB2 interferon beta 2

IGF-I insulin like growth factor 1

IGF-II insulin like growth factor 2

IL interleukin

IL-6BP interleukin 6 binding protein

IL-6R interleukin 6 receptor

IL-6Rα interleukin 6 receptor α

ING4 inhibitor of growth family member 4

JAK janus kinase

LAP liver enriched activating protein

LIF leukemia inhibitory factor

LIFR leukemia inhibitory factor receptor

LIP liver enriched inhibiting protein

MAPK mitogen-activated protein kinases

Mcl-1 myeloid cell leukemia sequence 1

MCM7 minichromosome maintenance deficient 7

MDR1 multi-drug resistant gene 1

MEC mammary epithelial cell mRNA messenger ribonucleic acid

xviii NE neuroendocrine

NFIL-6 nuclear factor IL-6

NFIL-6β nuclear factor IL-6 β

NSE neuron specific enolase

OCM osteoblast conditioned media

OSM oncostatin M

OSMR oncostatin M receptor

PBS phosphate buffered saline

PCR polymerase chain reaction

PI-3 phosphoinositide 3

PSA prostate specific antigen pSTAT phosphorylated signal transducer and activator of transcription

PTEN phosphatase and tensin homolog

PVDF polyvinylidene fluoride

Rb retinoblastoma

RBBP-8 retinoblastoma binding protein 8

RNA ribonucleic acid

RT-PCR reverse transcription – polymerase chain reaction

SDS sodium dodecyl sulfate

SFM serum free media sgp130 soluble glycoprotein 130

SH2 src homology 2 sIL-6R soluble interleukin 6 receptor xix siRNA short interfering ribonucleic acid

STAT signal transducer and activator of transcription

STAT-C signal transducer and activator of transcription – constitutive active form

TGF-β transforming growth factor β

TNF-α tumor necrosis factor α

TRX2 thioredoxin 2

VEGF vascular endothelial growth factor

VHL von hippel-lindau

xx CHAPTER 1

INTRODUCTION

Statement of Purpose

Prostate cancer is the most common cancer among men and the second leading cause of cancer-related mortality in the United States (100). In 2005, the

American Cancer Society estimated that there would be 234,300 new cases of prostate cancer and approximately 30,000 men would die of the disease (132).

Prostate cancer, like most cancers is a heterogeneous disease with regards to hormone sensitivity, growth regulation and known genetic alterations. These alterations can be the result of environmental or inherited factors or a combination of both. In general, early stage prostate cancer is androgen sensitive, responding to androgen ablation therapy (94). Many cases of prostate cancer, however, progress to a hormone insensitive state that is unresponsive to conventional treatments and is associated with poor prognosis (142). The genetic alterations that facilitate this progression to malignancy are poorly understood.

Cancer cells differ from normal cells in many ways, including: amplification of proto-oncogenes and the production of growth factors resulting in increased proliferation, loss of differentiation, immortalization, evasion of host immune response and apoptosis, increased invasiveness, loss of contact

1 inhibition and/or the ability to override anti-proliferative signals, loss of tumor suppressor gene expression/function and the ability to sustain angiogenesis

(105). A major focus of cancer biology is the elucidation of the mechanisms by which tumor suppressor and proto-oncogenes are regulated and how they control cell proliferation. The central focus of this dissertation is the characterization of CCAAT/enhancer binding protein delta (C/EBPδ) expression and function in G0 growth arrest in prostate cancer cell biology.

Prostate Cancer

Normal prostate epithelial cell growth and survival is dependent on androgen stimulation. Androgens regulate total prostate cell numbers by concurrently stimulating cell proliferation and inhibiting cell death (126, 127). Like normal prostate epithelial cells, early stage prostate cancer cell growth is androgen sensitive (dependent) (64). In these early stages, the most common treatment for prostate cancer is androgen ablation therapy. Androgen ablation therapy initiates cancer regression as cell proliferation rates decrease and cell death rates increase. Unfortunately, androgen ablation therapies are not an effective treatment for metastatic prostate cancer because a percentage of these cancer cells have become androgen independent, no longer requiring androgens for growth and survival (94). Presently, there are few effective treatment options for patients with androgen-independent prostate cancer. A major focus of current prostate cancer research is the identification of genetic changes that occur as subpopulations of malignant prostate epithelial cells begin to grow independent of androgens.

2 While prostate cancer is the most frequently diagnosed cancer in US

males, hereditary prostate cancer accounts for 10% or less of all patients with the

disease (30). This suggests that a majority of prostate cancers arise from

progressive genetic alterations. Out of the many established classical oncogenes

and tumor suppressor genes, only mutations in p53 and PTEN appear to

contribute substantially to prostate cancer. Previous studies have demonstrated

that p53 mutations are found in less than 10% of organ-confined prostate

cancers but can be as high as 50% at metastatic sites (14, 200). There is,

however, no general consensus on the prognostic importance of p53 mutations in prostate cancer (21, 310). Defects in PTEN have been identified in 5% of primary prostate tumors and between 30-60% in prostate metastases implicating an important role for the Akt intracellular signaling pathway in prostate cancer progression (28, 275). Similar mutational results have been observed in studies

focused on androgen signaling. A number of reports have found fewer androgen

receptor mutations in primary prostate cancers compared to metastatic prostate

cancers (147, 176, 285, 286, 289). Metastatic prostate cancers may harbor

mutations in the androgen receptor at frequencies as high as 50%. Taken

together, these results (as well as the ineffectiveness of androgen ablation

therapy on metastatic prostate cancers) have focused prostate cancer research

on the development of prostate cancer metastases.

3 Prostate Cancer Metastases and Bone

The most common site of prostate cancer metastasis is bone. Bone

metastases are present in ~90% of patients dying from prostate cancer (23, 229).

Most patients with advanced prostate cancer will experience complications from

bone metastases that are incurable (142, 191, 229, 336). Despite its prevalence,

the mechanisms involved in the development of prostate cancer metastases in

bone remain incompletely understood. The process of metastasis is complex and

involves a number of interactions between the host and tumor cells. Prostate

cancer cells that have metastasized to bone can be influenced by factors derived

from bone marrow, bone matrix products released during osteoclast absorption or osteoblasts secreted factors. Prostate cancer metastases generally form osteoblastic lesions in bone, in contrast to bone metastases from kidney, breast and lung cancers which are more often osteolytic in nature (35, 229).

Interestingly, prostate cancer lethality is attributable to an increased

resistance to apoptosis rather than enhanced proliferation rates (12). Previous studies have demonstrated that human prostate cancer cells typically have a low proliferative growth fraction (12). Primary and metastatic prostate cancer cells exhibit an exceptionally slow growth rate with <10% of prostate cancer cells proliferating per day. This accounts for the low response observed with commonly used chemotherapeutic agents, which target cells with high proliferation rates (12, 251). Despite these observations the mechanisms whereby osteoblasts regulate prostate cancer cell growth have not been elucidated. Previous studies have demonstrated that osteoblasts secrete a

4 number of factors including TGF-β (17, 125), Insulin-like growth factors I and II

(IGF-I and IGF-II) (83, 214), IL-6 (130, 156, 230), IL-1β (37) and tumor necrosis factor α (TNF-α) (58). IL-6, a member of the IL-6 family of cytokines, is one of the documented osteoblast-derived factors that can profoundly influence prostate cancer cell proliferation (156, 163, 194, 230, 240).

The IL-6 Family of Cytokines

The interleukin-6 (IL-6) family of cytokines is pleiotropic, acting on many different cell types. They often show redundancy and can affect the action of other cytokines in additive, synergistic or antagonistic manners. IL-6 family cytokines activate target genes involved in differentiation, survival, apoptosis and proliferation (109, 110, 113, 195). The IL-6 family of cytokines is comprised of IL-

6, IL-11, LIF (leukemia inhibitory factor), OSM (oncostatin M), CNTF (ciliary neurotrophic factor), CT-1 (cardiotrophin-1), CLC (cardiotrophin-like cytokine)

(277) and two newly characterized family members, IL-27 and IL-31 (67, 219).

Unlike hormones, which are stored as preformed molecules, cytokines are rapidly synthesized and secreted at very small concentrations by cells following stimulation. Generally their expression is transient and can be regulated at both the transcriptional and translational levels (104). Cytokines act on target cells via interactions with specific cell surface receptors and are capable of acting in either an autocrine or paracrine manner. The IL-6 family cytokine receptors are all type

І membrane proteins (extracellular N-terminus, one transmembrane domain) with the exception of the CNTF receptor which is also linked to the plasma membrane by a glycosylphosphatidylinositol anchor (110). Receptors involved in the

5 recognition of the IL-6 family of cytokines can be further subdivided into the non-

signaling α -receptors (IL-6Rα, IL-11Rα and CNTFRα) and the signal transducing receptors (gp130, LIFR and OSMR). The gp130 subunit is ubiquitously expressed, while expression of the other receptor subunits is tightly regulated.

Thus, the expression of specific receptor subunits determines the responsiveness of a particular cell type to IL-6 family cytokine stimulation (109,

110). In recent years, IL-6 has emerged as an important, but poorly understood factor in normal prostate biology and in the pathogenesis of prostate cancer (163,

194, 240). For this reason, the remainder of this literature review will focus specifically on IL-6.

Interleukin 6

IL-6, also known as Interferon Beta 2 (IFNB2), B-Cell Differentiation

Factor, B-Cell Stimulatory Factor 2 (BSF2), Hepatocyte Stimulatory Factor

(HSF) and Hybridoma Growth Factor (HGF) is the translation product of a 1.3 kb mRNA derived from the IL-6 gene located on chromosome 7 (15). Functionally,

IL-6 binds to the IL-6 receptor (IL-6Rα subunit) on the surface of target cells. This

IL-6 and IL-6Rα complex then associates with gp130, inducing its dimerization and the initiation of signaling (236, 277). Upon stimulation, gp130-associated

kinases JAK1, JAK2 and Tyk2 become activated and phosphorylate the

cytoplasmic tail of gp130, providing docking sites for the signal transducers and

activators of transcription (STAT) molecules. Interestingly, a naturally occurring

soluble form of the IL-6R (sIL-6R), generated by either limited proteolysis of the

membrane protein or translation from an alternatively spliced mRNA, has been

6 identified (173, 190, 238). Through a process called trans-signaling (190, 218,

238), the sIL-6R together with IL-6 can stimulate cells that only express gp130

(175, 237, 276). This process allows IL-6 signaling to stimulate more diverse

target cell populations. This can be counteracted by a soluble form of gp130

(sgp130), which prevents signaling through membrane bound gp130 by binding

the IL-6/sIL-6R complex (186, 199).

IL-6 is a pleiotropic cytokine with varied systemic functions (Figure 1.1)

(11). IL-6 is secreted in diverse cell types including fibroblasts, endothelial cells,

keratinocytes, monocyctes/macrophages, T cells, B cells, osteoblasts and in a

variety of tumor cell lines (304).

Figure 1.1. Representation of the well characterized activities of IL-6. Borrowed from (295).

7 Knockout mouse studies have demonstrated that targeted disruption of

the IL-6 gene results in defective hepatocyte regeneration and liver failure (53).

In addition, the absence of STAT3 production was observed, suggesting that

STAT3 induction during liver regeneration is strictly mediated by IL-6 (53). IL-6 is not constitutively produced under normal circumstances, but is readily produced in response to several stimuli including inflammation, infection and in response to other cytokines (32, 145, 203, 252, 303, 335). IL-6 is primarily involved in the regulation of the immune and inflammatory responses as well as inducing the terminal differentiation of B cells. It has a central role in the acute phase response, acting on hepatocytes to increase the synthesis of acute-phase proteins (231). Previous studies have also demonstrated that IL-6 contributes to the body’s defenses by increasing body temperature and stimulating the release of adrenocorticotropic hormone (177). The role of IL-6 in pro-inflammatory and anti-inflammatory activities suggests it may have an important function in regulating physiological responses to disease.

IL-6 has been implicated in a number of human diseases. Kawano et al. presented evidence that constitutive expression of IL-6 or its receptor may be responsible for the generation of myelomas (141). In Paget’s disease of the bone, the number and size of osteoclasts are greatly increased compared to normal bone tissue. Studies demonstrated that a portion of this can be attributed to elevated IL-6 levels (235). A nucleotide variant in the promoter of the IL-6 gene has been associated with decreased bone mineral density suggesting IL-6 may play a role in the development of osteoporosis (212). The Kaposi sarcoma

8 associated herpes virus encodes a functional homolog of IL-6 (vIL-6), which,

when expressed in infected cells, induces angiogenesis and hematopoiesis (41).

In contrast to IL-6, which binds to gp130 only after it forms a complex with IL-

6Rα, vIL6 directly activates gp130. Abnormal IL-6 production has also been reported in a variety of cancers (Table 1.1, borrowed with modification from

(295)). In most of these studies, elevated levels of IL-6 are related to disease severity and outcome. In recent years, there has been increased interest in IL-6 expression and function in prostate cancer.

9

Continued

Table 1.1. Abnormal IL-6 production and its association with cancers. Borrowed with adaptation from (295)

10 Table 1.1 continued

11 IL-6 and Prostate Cancer

Clinical studies indicate that plasma IL-6 levels are elevated in prostate

cancer patients with advanced disease, and some human prostate cancer cell lines secrete IL-6 and also express the IL-6 receptor (93, 194, 298). IL-6 levels correlate with prostate cancer tumor burden as well as with serum prostate specific antigen (PSA) levels and clinically evident metastases (1, 69). As previously mentioned, the most effective treatment for prostate cancer is androgen ablation therapy (111). While a majority of prostate tumors regress following androgen ablation, a percentage of tumor cells remain which are androgen refractory (insensitive) and no longer require androgens for growth.

Interestingly, among prostate cancer cell lines, only androgen refractory cells produce detectable levels of IL-6. This data suggests a correlation between IL-6 expression and the development of androgen insensitive prostate cancer cells.

Functionally, androgen binds to the androgen receptor (AR) which in turn triggers the interaction of AR with specific androgen responsive elements (AREs) located in the promoters of androgen regulated genes (127). This induces the activation or repression of genes regulating prostate epithelial cell development, differentiation and proliferation. In prostate cancer, the acquisition of androgen independent growth is required for tumor cell survival. Previous studies have demonstrated that androgen-independent growth can be promoted by activation of the AR through AR gene mutation and/or amplification, co-activators and cross-talk between the protein kinase and AR pathways (52, 57, 168, 327).

Accumulating evidence suggests that an additional mechanism for the

12 development of androgen independent tumors is non-steroidal activation of the

AR by cytokines and growth factors (56, 80, 93, 165, 227, 300, 301, 325).

Recent reports indicate that IL-6 is one of the cytokines that can activate the AR

in a ligand-independent manner and increase the expression of several ARE

driven reporters, including PSA (40, 114, 300). This data suggests that IL-6 stimulation of the AR pathway may provide a mechanism for prostate cancer cells to continue to proliferate in the absence of androgens.

A number of other pathways have been identified in prostate cancer cells which can be activated by IL-6. In LNCaP cells, IL-6 can regulate growth and neuroendocrine (NE) differentiation as observed by morphological changes and expression of neuron-specific enolase (NSE) (188). Under these conditions, IL-6

activates the Etk/Bmx pathway, resulting in LNCaP cell NE differentiation (226).

IL-6 has also been shown to function as an anti-apoptotic signal in human

prostate cancer cell lines by activating PI-3 kinase (42). Despite reports indicating that IL-6 induces differentiation in LNCaP cells, the role of IL-6 in prostate cancer cell growth is controversial. IL-6 has been shown to induce both growth arrest (43, 115, 188, 264, 314) and growth stimulation (153, 171) in the

LNCaP prostate cancer cell line. These differences may be due to differences in the duration of IL-6 treatment, variations in prostate cancer cell line passage number, or other unspecified factors.

The LNCaP cell line is unique in that it is the only commonly used prostate cancer cell line whose growth remains androgen sensitive (responsive). The two other most commonly used prostate cancer cell lines (PC-3 and Du145) are both

13 androgen insensitive. It is of interest that both the Du145 and PC-3 cell lines

secrete IL-6, and reports have demonstrated that their growth is minimally stimulated in response to exogenous IL-6 treatment (43, 207). These results

suggest that IL-6 may initially function as a growth inhibitor in early stage

(androgen sensitive) prostate cancer cell lines. However, as cells progress to an

androgen insensitive state, they secrete IL-6 and are no longer growth inhibited

by exogenous IL-6. Interestingly, Hobisch and coworkers found that long term

exposure of LNCaP cells to IL-6 abolished IL-6 mediated growth arrest and led to

the development of LNCaP cells which secrete IL-6 (115). Taken together, these results suggest that the effects of IL-6 on prostate cancer cells are varied and may be dependent on the length of exposure. Despite the differences in the

responses observed with IL-6 treatment of prostate cancer cells, the activation of the JAK/STAT pathway is consistent.

The JAK-STAT Pathway

As previously mentioned, binding of IL-6 to the IL-6Rα triggers dimerization of gp130, which in turn leads to the recruitment and activation of non-receptor protein tyrosine kinases (192). Like many other cytokines, IL-6 induces the rapid activation of the Janus kinase (JAK) family of tyrosine kinases.

The JAK family consists of four members – JAK1, JAK2, JAK3 and Tyk2. JAK1,

JAK2 and Tyk2 are ubiquitously expressed while JAK3 expression appears to be limited to cells of haematopoietic lineage and does not interact with the IL-6/IL-

6Rα complex (7). Of the family members, JAK1 plays an important role in IL-6 signaling, as cells lacking JAK1 have severely impaired IL-6 signaling (103, 233).

14 Amino acid sequence analysis of the JAK family revealed seven highly conserved domains designated JH1-JH7. The hallmark of the JAK family is the

presence of both a kinase (JH1) and pseudokinase (JH2) domain in series (320).

The JH1 domain consists of the active kinase domain and an autophosphorylation site. Phosphorylation at this site is required for full kinase activity. Interestingly, several critical residues are missing in the JH2 domain rendering it catalytically inactive. However, the JH2 domain appears to function in regulating the kinase activity of the JH1 domain. Taken together, these two domains comprise almost half of the molecule while the remaining domains

(which form the N-terminus) play critical roles in regulating the interaction between JAKs and their cognate cellular receptors.

In an unstimulated cell, JAKs are bound to the membrane proximal region of cytokine receptors. This interaction occurs through a highly conserved motif designated box-1 which is present in cytokine receptors. Box-1 is one of three conserved motifs present in the cytoplasmic region of the receptor with the other two motifs designated box-2 and box-3 (Figure 1.2) (144). Box-2 appears to contribute to JAK binding presumably by increasing its affinity, while box-3 plays an important role in transcription factor binding. Upon stimulation, receptor dimerization results in the phosphorylation of JAKs at specific tyrosine residues located within the kinase domain, which in turn leads to JAK activation. Activated

JAKs then phosphorylate residues within box-3 of the receptor chain, which then

15 serve as docking sites for various transcription factors. The major family of

transcription factors activated in response to IL-6 mediated JAK activation is the

signal transducers and activators of transcription (STAT) family of proteins.

Figure 1.2. Overview of the JAK/STAT signaling pathway. Borrowed from (50)

16 Signal Transducers and Activators of Transcription (STATs).

The STAT family of transcription factors consists of seven members, all of

which are latent in the cytoplasm until their activation by signaling molecules

which include cytokines and growth factors. STAT proteins contain a conserved

N-terminal oligerimization domain, a DNA binding domain, a src homology 2

(SH2) domain and a transactivation domain (Figure 1.3) (160, 209). Upon

binding of extracellular signaling molecules, the JAK-STAT pathway is activated and STAT proteins are recruited to the activated receptor complex. The SH2 domain mediates this recruitment and requires phosphorylation at specific residues located within the receptor for binding (108, 112, 266). The interaction of the SH2 domain with the receptor tyrosine kinase (JAK) results in the phosphorylation (activation) of the STAT protein (Figure 1.2). Activated STAT proteins then form homo- or heterodimers and translocate to the nucleus (160,

209). In the nucleus they bind specific DNA consensus sites in gene promoters to induce gene transcription (61).

Figure 1.3. Diagram of the STAT domain structure. pY and pS represent phosphorylation sites within the transactivation domain and vary between STAT family members. Borrowed from (46)

17 Function of STAT Proteins

As mentioned above, there are seven members in the STAT family of transcription factors, STAT1, 2, 3, 4, 5a, 5b and 6. The first members of the

STAT family to be discovered were STAT1 and STAT2 as a result of research on interferon (IFN) signaling pathways (81, 246, 254). IFNγ induced growth inhibition of cultured cells requires functional STAT1 (19). Additional studies have indicated that STAT1 is required for growth inhibition by other cytokines, including IL-4, suggesting that STAT1 may act as a tumor suppressor gene (33, 140). STAT2 plays an important role in the formation of the interferon stimulated gene factor-3

(ISGF-3) complex induced by IFNα (215). STAT3 was originally identified as an acute phase response (APR) factor, activated in response to IL-6 treatment (3).

Among the STAT family of proteins, only mice deficient in STAT3 are embryonic lethal (281). In recent years STAT3 has been linked to transformation and constitutive STAT3 activation has been observed in many types of cancer (16,

20). STAT4 is activated by IL-12, which plays an important function in T helper cell differentiation (288). STAT5a is responsible for the development of the mammary gland and lactation (169). STAT5b is also involved in the mammary gland development but also functions in regulating gene expression in the liver in response to growth hormones (61, 299). STAT6 is critical for Th2 differentiation and functions in the adaptive immune system (118). Of the seven STAT family members, many cancer researchers have focused their attention on STAT3

18 because it is the major STAT family member activated by IL-6 (which is elevated

in many human diseases) and is also constitutively activated in various cancers.

For these reasons, the remainder of this discussion will focus on STAT3.

STAT3

STAT3 is expressed in most tissues and its expression can be observed

early in the post-implantation period (71). Embryos deficient in STAT3 exhibit

severe defects in development which results in early (E6.5 - E7.5) fetal death

(281). As a result of this lethality, tissue-specific STAT3 deletional studies have

been employed to investigate the function of STAT3. As a result of these studies,

it has become apparent that the function of STAT3 is not conserved between

different cell types but instead appears to be very cell type specific. In epidermal cells, STAT3 is essential for skin remodeling (during wound healing and the hair cycle) and also plays an important role in cell migration (243). Previous studies have shown that IL-6 induces proliferation (and also suppresses apoptosis) in T cells, but STAT3 deficient T cells are defective in IL-6 induced proliferation and have elevated apoptosis levels (280). In contrast, apoptosis in terminally differentiated hematopoietic cells requires STAT3 activation (184, 208). STAT3 also plays a critical role in IL-10 induced anti-inflammatory response as macrophages deficient in STAT3 demonstrate a constitutively activated phenotype and are more sensitive to lipopolysaccharide (279). In the mammary gland, STAT3 functions in a pro-apoptotic role, as STAT3 null mammary glands demonstrate delayed programmed cell death during involution (34). One explanation for the diverse biological functions of STAT3 is the activation of

19 additional signaling pathways (for example the MAPK pathway). To complicate matters, while STAT3 functions as a growth inhibitor in many tissues, constitutive

STAT3 activation has been linked to both viral and oncogene mediated transformation.

The first evidence suggesting a role for STAT3 in transformation was the observation that activation of the src oncogenic tyrosine kinase resulted in

STAT3 activation (328). Further analyses revealed that a dominant negative form of STAT3 (STAT3β) could suppress src mediated transformation (18, 297).

In addition, STAT3 activation has been observed in a number of cells transformed by viruses including the Epstein-Barr virus and HTLV-1 (59, 183,

317). Interestingly, no STAT3 mutations have been identified which induce constitutive activation or cellular transformation. This suggests that defects in the activation of STAT3 signaling and not STAT3 itself are responsible for its observed transformation functions. In addition to viral and oncogene mediated transformation, STAT3 activation has been observed in a variety of cancers including breast, prostate, ovarian, head and neck, brain, lung, multiple melanoma and leukemias (16, 85, 120, 166, 187, 244, 263). For the purpose of this literature review, we will focus on the role of STAT3 in prostate cancer.

STAT3 and Prostate Cancer

The importance of STAT3 in prostate cancer biology was first described in early 2000 (264). Like IL-6, the role of STAT3 in prostate cancer is controversial.

Previous studies have demonstrated that constitutive STAT3 activation occurs frequently in prostate tumors (187). In addition, STAT3 activation is also elevated

20 in androgen insensitive prostate cancer cells compared to androgen sensitive cells (66, 187). The same observations have been made in the Du145, PC-3 and

LNCaP prostate cancer cells lines. The Du145 prostate cancer cell line is an androgen insensitive prostate cancer cell line which expresses constitutively activated STAT3 (171). This constitutive activation of STAT3 is most likely the result of IL-6 secreted by Du145 cells acting in an autocrine manner (207).

Recent reports indicate that siRNA mediated STAT3 knockdown inhibits Du145 cell growth and induces apoptosis (155). Another commonly used androgen insensitive prostate cancer cell line is the PC-3 cell line, which also secretes IL-6

(264). Interestingly, IL-6 may function as an autocrine growth stimulator in PC-3 cells (43, 207, 255). IL-6 mediated growth stimulation of PC-3 cells appears to be the result of defective STAT3 expression in PC-3 cells (264). Chung and coworkers transfected wild type STAT3 into PC-3 cells and observed that the transfected STAT3 was constitutively activated due to the secretion of endogenous IL-6 (265). In addition, the growth of PC-3 cells expressing constitutively activated STAT3 was severely impaired compared to normal PC-3 cell growth (265). These results suggest that IL-6 may stimulate growth in prostate cancer cells harboring defects in the STAT3 signaling pathway. The

LNCaP cell line does not secrete detectable levels of IL-6 but does express the

IL-6Rα. A number of studies have reported STAT3 activation in LNCaP cells treated with exogenous IL-6 (55, 114, 115, 153, 171, 264, 265, 268). Previous studies have observed both growth inhibition (55, 115, 264, 265) and growth stimulation (153, 171) in response to STAT3 activation in the LNCaP cell line. It

21 is unclear how such contradictory results could be obtained. One possible

explanation is the activation of alternative intracellular signaling pathways as a

result of cell passage number or duration of cytokine treatment. Work by

Coqueret and coworkers has demonstrated that the activation of growth inhibitory

vs. growth promoting genes in response to STAT3 activation can be altered by

activation of the AKT pathway (10). Previous studies have also shown that

LNCaP cells generated by prolonged exposure to IL-6 demonstrate alterations in

the IL-6 signaling pathway which includes up regulation of members of the MAPK signaling pathway (268). To further investigate the role of STAT3 activation in prostate cancer cells, we will focus our attention on the STAT3 downstream effector genes which have been identified in prostate cancer.

Despite numerous reports investigating the role of activated STAT3 in prostate cancer cells, the identification of STAT3 downstream effector genes is limited. Activated STAT3 has been shown to directly interact with the androgen receptor, allowing it to regulate androgen receptor mediated gene expression

(40, 178), suggesting that STAT3 plays an important role in the development of

androgen independent prostate cancer cells. Steiner et. al. demonstrated that

activated STAT3 stimulates the expression of vascular endothelial growth factor

(VEGF), a potent regulator of angiogenesis (98, 267). Recent reports suggest

that activated STAT3 may function in an anti-apoptotic role in prostate cancer.

Activated STAT3 has been linked to the regulation of numerous anti-apoptotic

proteins including bcl-2, bcl-xl, Mcl-1, survivin and phosphorylated bad (84, 154,

198, 225). These results suggest that STAT3 plays an important role in prostate

22 cancer cell survival in addition to its function in growth arrest. Unfortunately, no

growth arrest specific downstream effector genes regulated by activated STAT3

have been identified in prostate cancer cells.

CCAAT/Enhancer Binding Proteins (C/EBPs)

C/EBPs are a highly conserved family of basic leucine zipper type (bZIP)

DNA binding proteins (228). Most C/EBPs are encoded by intronless genes and

exhibit a high degree of homology in the basic and leucine zipper regions (228).

Six C/EBP family members have been identified: C/EBPα (CEBPA), C/EBPβ

(CEBPB, CRP2, NF-IL6, LAP, AGP/EBP, IL-6BP,NF-M), C/EBPγ (CEBPG),

C/EBPδ (CEBPD, CRP3, NF-IL6β, CELF), C/EBPε (CEBPE) and C/EBPζ

(C/EBP Homologous Protein 10, CHOP10, GADD153) (228). C/EBPs function in

cell-type specific growth and differentiation (228). At the mechanistic level,

C/EBPs bind to DNA as homo- and heterodimers with other C/EBP family

members or with other leucine zipper containing proteins such as fos, jun and

ATF/CREB through a basic C-terminal region (119, 157, 228, 307, 308). C/EBP

family members primarily function as transcriptional activators, activating the

expression of downstream effector genes that function in cell growth,

differentiation, the acute phase response, liver regeneration and metabolism

(228).

23 The Structure of the C/EBP Family Members

All C/EBPs consist of three structural domains: a DNA binding domain, a leucine zipper domain and an N-terminal activation domain (Figure 1.4) (228).

The leucine zipper domain consists of a heptad repeat of four or five leucine residues which assume an α helical configuration (121, 150, 309).

Figure 1.4. Structure of the C/EBP family members. The activation domains are in green, the leucine zipper domain in yellow and the basic DNA binding domain in red. Negative regulatory domains are in blue. Borrowed from (228)

24 The binding of C/EBPs to DNA requires dimerization, which leads to the

formation of an inverted Y shaped structure (Figure 1.5). The DNA binding

domain of each protein makes up the “arms” of the Y and binds to one half of a

palindromic recognition sequence in the major groove of the DNA (Figure 1.5).

The activation domain mediates the transcriptional activation, repression and

auto regulatory functions of the C/EBP family members. Work by Osada et. al.

identified the optimal C/EBP binding site to be a symmetrical repeat

RTTGCGYAAY, where R is A or G and Y is C or T, however substantial variations are tolerated (210).

Figure 1.5. Model for C/EBPβ bound to DNA. Borrowed from (228).

25 Unlike the leucine zipper domain, C/EBPs share little sequence

homology within their activation domains accounting for the tissue specific

functions of each family member. The only conserved regions within the

activation domain are three small sub-regions designated activation domain modules (ADM). These regions are required for direct interaction with the basal

transcriptional machinery to activate gene transcription. C/EBPγ is the only

C/EBP family member that lacks an activation domain and subsequently acts as a dominant negative dimerization partner (49).

While the C/EBP family consists of six members, alternative isoforms of

C/EBP α, β, and ε have been identified. In the case of C/EBPα and β, the use of alternative translation initiation codons or proteolysis accounts for the existence of the different isoforms (65, 164, 211, 318). There are two isoforms for C/EBPα

(42 and 30 kDa) and three isoforms for C/EBPβ (38, 35 and 20 kDa) (Figure 1.4).

C/EBPε has four known isoforms (32, 30, 27 and 14 kDa) which are generated via differential splicing and the use of alternate promoters (Figure 1.4) (158, 322).

Like C/EBPγ, the 20 kDa isoform of C/EBPβ (also known as LIP) and the 14 kDa isoform of C/EBPε lack an activation domain and function as dominant negative

C/EBP dimerization partners.

Function of C/EBP Proteins

C/EBPα was the first member of the C/EBP family to be identified in 1988

in the laboratory of Dr. Steve McKnight (149). The expression of C/EBPα is highest in lung, intestine, liver and adipose tissue (157, 228). C/EBPα is essential

26 for liver and lung function and plays a critical role in adipocyte and granulocyte

differentiation. Overexpression of C/EBPα in various cell types results in growth

arrest, indicating that C/EBPα is a potent inhibitor of proliferation (302, 315).

Further investigation into the mechanisms of C/EBPα mediated growth arrest has demonstrated that C/EBPα interacts directly with a number of key cell cycle regulatory proteins (39, 290, 291, 313). C/EBPα directly binds to p21, stabilizing

the protein and facilitating growth arrest and differentiation (293). Interactions

between C/EBPα and RB-E2F complexes have been proposed to play important

roles in cell cycle progression (291, 292). In addition, C/EBPα also interacts with

free E2F to suppress cellular proliferation (223, 260). Finally, C/EBPα has been shown to bind cdk2 and cdk4, inhibiting their activities (107, 313).

C/EBPβ expression is induced in response to inflammatory stimuli including LPS, IL-6, IL-1 and TNF-α in hepatocytes, macrophages and intestinal epithelial cells (2, 89, 222). C/EBPβ plays an important role in terminal differentiation, macrophage activation and the acute phase response in the liver

(222, 247, 283). Unlike C/EBPα, C/EBPβ appears to play an important role in promoting proliferation (26, 99). C/EBPβ has been linked to the proto-oncogene ras as C/EBPβ deficiency results in reduced tumorgenesis in v-Ha-ras transgenic mice, and ras has been shown to stimulate C/EBPβ transactivation (338).

Mammary epithelial cells derived from C/EBPβ knockout mice demonstrate

defects in proliferation that results in impaired ductal morphogenesis and

lactation (232, 248). Recently, a pro-survival function for C/EBPβ has been

27 identified. In macrophage cells, C/EBPβ plays an important role in oncogenic

transformation by regulating the expression of IGF-І, a survival factor (319).

C/EBPβ also plays a critical role in the survival of keratinocytes in response to carcinogen treatment (270). Interestingly, C/EBPβ has also been shown to

activate the human MDR1 gene (38) which plays an important role in cancer cell

drug resistance (6, 38, 95, 256).

C/EBPε expression is limited primarily to lymphoid and myeloid cells.

Overexpression of C/EBPε results in differentiation of promyelocytic leukemia

cells (296). C/EBPε has also been shown to repress the transcription of Myc (an

E2F target gene), suggesting it may function like C/EBPα, via interactions with

E2F and Rb (87).

As mentioned earlier, C/EBPγ lacks an activation domain and functions primarily as a dominant negative dimerization partner (49). C/EBPγ is expressed

in all tissues and appears to play an important role in natural killer cell

maturation, as C/EBPγ knockout mice have impaired natural killer cell activity and decreased interferon-γ production (139, 234).

C/EBPζ was originally named growth arrest and DNA damage inducible gene 153 (gadd153), as it was identified in response to DNA damage (78).

C/EBPζ plays an important role in growth arrest and apoptosis in response to endoplasmic reticulum stress (213, 339). Interestingly, most myxoid

28 liposarcomas contain an oncogenic variant of the C/EBPζ gene generated by

fusion of the TLS gene N-terminus to the C/EBPζ C-terminus (54). Unlike

C/EBPζ, this oncogenic fusion protein fails to induce growth arrest (9).

C/EBPδ expression is rapidly induced in the liver, spleen, kidney, heart and brain upon induction of the acute phase response by LPS (4). During adipocyte differentiation, both C/EBPδ and C/EBPβ are required to induce

C/EBPα expression (60). In the mammary gland, C/EBPδ expression is dramatically induced during the initial “reversible” phase of involution (90, 91).

Adult female C/EBPδ knockout mice exhibit excessive mammary ductal branching and an increase in total mammary epithelium compared to wild type mice (92). C/EBPδ (as well as α and β) is widely expressed in the nervous system. In hippocampal neurons, both the expression and DNA binding activities of C/EBPδ (and β) are enhanced by cAMP and Ca2+ (330). This suggests that

C/EBPδ plays an important role in the long term memory process, as

hippocampal neurons play an important role in long-term memory and cAMP and

Ca2+ are critical inducers of the memory process. In addition, work by Rogers and coworkers has demonstrated that C/EBPδ expression may be elevated in the cortex of Alzheimer’s disease patients (161). Interestingly, C/EBPδ deficient mice have a selectively enhanced contextual fear response, suggesting that C/EBPδ

may also play an important role in learning (269).

29 C/EBPδ and G0 Growth Arrest

The cell cycle is divided into four phases G1, S, G2 and M. During the S

and M phases, a cell executes two basic events- the generation of a single copy

of its genetic material, and the partitioning of all cellular components into identical

daughter cells (202). The G1 and G2 phases represent “gaps” in the cycle when

cells prepare for successful completion of the S and M phases. After each round

of division every proliferating cell must make the important decision whether to undergo another round of cellular division or exit the cell cycle. When cells exit the cell cycle, they enter a non-dividing, quiescent state known as G0 (5).

Just as cycling cells must decide whether to remain in or exit the cell cycle, G0 cells must decide whether to remain in G0 or re-enter the cell cycle.

Most cells in an adult organism reside in the G0 state, however some retain the

capacity to re-enter the cell cycle, the exception being those cells which are

terminally differentiated (306). Despite its importance, little is known about the

regulation of G0 growth arrest.

Cells which have entered G0 demonstrate dramatic decreases in both

biochemical and cellular activity (134, 135). Both gene transcription and

translation decrease upon G0 arrest and G0 cells have significantly fewer

ribosomes compared to cycling cells (135, 159). While general cellular functions

decrease during G0 growth arrest, a subset of genes have been identified which are activated or up-regulated during G0 including von Hippel-Lindau (VHL), p130

and growth arrest specific gene 1 (gas1) (44, 217, 261, 262). Alterations in G0

30 growth arrest genes have been identified in many common cancers. Individuals

harboring hereditary mutations of the VHL tumor suppressor gene demonstrate

increased incidence of vascular tumors and renal cell carcinomas (151, 217).

Work from our laboratory has established an important role for C/EBPδ

during G0 growth arrest in mammary epithelial cells (MEC). Initial studies demonstrated that C/EBPδ expression was dramatically induced in several mouse MECs in response to G0 growth arrest (206). The induction of C/EBPδ

appears to be specific to MEC G0 growth arrest, since the levels of C/EBPδ did

not increase in other cell lines tested or when MECs were arrested in other

stages of the cell cycle (206). Further analyses demonstrated that both nuclear

C/EBPδ protein levels and DNA binding activity increased during G0 growth

arrest and diminished upon cell cycle re-entry (204-206). Subsequent studies

using C/EBPδ antisense cell lines revealed that cells with diminished C/EBPδ

protein levels were resistant to serum and growth factor induced G0 growth arrest. Conversely, G0 growth arrest was accelerated in cells overexpressing

C/EBPδ protein, further suggesting C/EBPδ plays an important role in G0 growth

arrest (122, 206).

The transcriptional regulation and structure of the mouse C/EBPδ

promoter has been characterized by our lab as well as others (29, 123).

Promoter analysis identified binding sites for both STAT3 and Sp1 within the

proximal mouse C/EBPδ promoter. Work by Johnson and coworkers demonstrated that both the STAT3 and Sp1 binding sites are required for

31 induction of C/EBPδ mRNA (29). Analysis of protein lysates from G0 growth

arrested mouse MECs identified significant increases in activated

(phosphorylated) STAT3 levels (123). In addition, further studies have

demonstrated that dominant negative STAT3 decreased C/EBPδ promoter

activity while STAT3 overexpression increased C/EBPδ promoter activity. Recent

work from our lab has also shown that activation of STAT3 by oncostatin M

induced C/EPBδ expression and G0 growth arrest in MECs (122, 259). Taken

together, these results suggested that C/EBPδ gene expression is regulated by a

STAT3 dependent pathway during G0 growth arrest of MECs. It is important to

note that C/EBPδ may also be subject to autoregulation as C/EBPδ promoter

activity is increased in C/EBPδ overexpressing cells, and autoregulation of

C/EBPδ has been observed in other cell lines (205, 321).

C/EBPδ has also been identified as an important growth regulator in other

cell lines. In M1 myeloid leukemia cells, C/EBPδ plays an important role in the

regulation of growth arrest and DNA damage inducible gene 45γ (gadd45γ)

during growth arrest and differentiation (137). Gadd45γ is a member of the gadd

family of proteins which are induced in response to a variety of DNA damages

and cellular stresses (77, 78, 116). In addition, a recent report by Koeffler and

coworkers demonstrates that C/EBPδ expression induced growth arrest and

myeloid differentiation in BCR-ABL positive leukemic cells (88). BCR-ABL is an

oncogenic fusion protein which causes chronic myelogenous leukemia (CML). In

CML patients, leukemic cells loose the ability to differentiate into mature

32 granulocytes. Ectopic expression of C/EBPδ resulted in G0/G1 growth arrest and

eventually differentiation. Their results suggest that C/EBPδ may hold an

important therapeutic potential in CML patients. Taken together, these results

suggest that C/EBPδ plays an important role in growth arrest in a variety of cell

types.

C/EBPδ and the Prostate

Interestingly, the initial characterization of the human C/EBPδ gene structure and chromosomal location was performed in LNCaP prostate cancer cells (45). Trapman and colleagues were attempting to identify C/EBP-like transcription factors which might be expressed in the prostate. They identified a genomic clone which contained the entire human C/EBPδ gene. It is of interest that no other C/EBP family member was identified in their screen for C/EBP-like transcripts, suggesting that C/EBPδ may be the most abundant C/EBP transcript in human prostate cells. Since their initial observations, there have been only a few publications focused on C/EBPδ in the prostate. Androgen removal (which results in initiation of growth arrest and programmed cell death) increases

C/EBPδ gene expression in the rat ventral prostate (323). In addition, C/EBPδ gene expression is reduced in advanced stage androgen independent human prostate xenografts (323). In human clinical studies, C/EBPδ gene expression is significantly reduced in advanced prostate cancer metastases (152). C/EBPδ was identified among the 100 genes exhibiting the greatest degree of differential expression between primary and advanced metastatic prostate cancers (152).

33 Recently Koeffler and coworkers demonstrated that C/EBPδ is required for 1,25- dihydroxyvitamin D3 mediated prostate cancer cell growth inhibition (124). Taken together, these studies suggest that C/EBPδ may be an important, but poorly understood regulator of prostate cancer cell growth.

34 CHAPTER 2

C/EBPδ IS A DOWNSTREAM MEDIATOR OF IL-6 INDUCED GROWTH INHIBITION OF PROSTATE CANCER CELLS

Abstract

Although a number of reports have investigated the effects of IL-6 family

cytokines on prostate cell growth, there is limited information available identifying

IL-6 inducible downstream effector genes and their function in growth control.

Previous studies have demonstrated that IL-6 treatment results in the activation

of STAT3 in prostate cancer cells. The goal of this study was to investigate the

influence of IL-6 treatment and activation of the JAK/STAT signal transduction

pathway on C/EBPδ gene expression and growth inhibition of human prostate cancer cells. Expression of C/EBPδ and STAT3 activation were assayed using northern and western blotting techniques. Proliferation was assessed by [3H]

Thymidine incorporation, flow cytometry and colony formation analyses. The analysis of the transcriptional regulation of C/EBPδ was performed using luciferase-reporter constructs. In this report we demonstrate that IL-6 treatment induces STAT3 activation (pSTAT3); pSTAT3 binds to the human C/EBPδ gene promoter and induces its expression. We also demonstrate that C/EBPδ over- expression is capable of suppressing prostate cancer cell growth. These results

35 demonstrate that C/EBPδ gene expression is increased in IL-6 treated LNCaP

cells. Increased C/EBPδ gene expression plays an important role in IL-6/STAT3

mediated growth arrest of LNCaP prostate cancer cells. Ongoing studies are

investigating the mechanism by which C/EBPδ controls prostate cancer cell growth and the potential role of C/EBPδ in the survival and chemo resistance of prostate cancer metastasis.

Introduction

Prostate cancer is the most common form of cancer in US men with an

estimated 189,000 new cases diagnosed in 2002 (133). Prostate cancer is

second to lung cancer as a cause of cancer mortality in US males, accounting for

about 30,000 deaths in 2002 (133). The incidence of prostate cancer is rising

worldwide, highlighting the need to better understand the underlying causes and

to develop more effective treatment strategies (101). Prostate cancer typically

follows a slow growing; unpredictable clinical course characterized by

progression from an initial hormone sensitive state to a hormone insensitive,

chemotherapy resistant state that is refractory to treatment (94). Identification of

biological and pharmacological factors that control prostate epithelial cell growth,

including the identification of factors that activate growth inhibitory pathways, is

critically important to provide a better understanding of the development,

prevention and treatment of prostate cancer.

IL-6 is a pleiotropic cytokine that has emerged as a potentially important,

but poorly understood factor in normal prostate biology and in the pathogenesis of prostate cancer (43, 115, 171, 188, 194, 207, 264). At the functional level IL-6

36 binds to the 80 kDa binding subunit of the IL-6 receptor and initiates a signal

transduction cascade that involves gp130, members of the Janus kinase family of

non-receptor protein tyrosine kinases and activated (phosphorylated) STAT3 (61,

104). Activated STAT3 translocates to the nucleus and binds to acute phase

response elements (APRE) in gene promoters (29, 61, 104). Although STAT3

has been identified as a major intracellular mediator of IL-6 in prostate cells, IL-6

treatment of LNCaP prostate cancer cells has also been shown to activate the

MAP kinase pathway and the Etk/Bmx pathway (163, 226).

We previously reported that C/EBPδ, a member of the CCAAT/Enhancer

binding protein (C/EBP) family of transcription factors, plays a key role in

mammary epithelial cell G0 growth arrest (62, 63, 91, 92, 122, 123, 204-206).

C/EBPδ gene expression is highly induced in response to serum and growth factor withdrawal, contact inhibition and growth inhibitory cytokines (122, 123,

204, 206). Our previous reports also indicated that C/EBPδ mRNA and protein are highly unstable, similar to proteins that play essential roles in the regulation of cell growth and cell fate determining pathways (62, 63). In addition, we have also demonstrated that C/EBPδ gene expression is regulated at the level of transcription, highlighting the importance of signal transduction pathways and transcription factors that function in the transcriptional control of C/EBPδ gene expression (205). C/EBPs are a highly conserved family of leucine zipper type

(bZIP) DNA binding proteins (228). C/EBPs function in cell growth, differentiation and death pathways (228). Most C/EBPs are encoded by intronless genes and exhibit a high degree of homology in the basic and leucine zipper regions (228). 37 Six C/EBP family members have been identified including C/EBPα, C/EBPβ (also

called CRP2, NF-IL6, LAP, AGP/EBP, IL-6BP, or NF-M), C/EBPγ, C/EBPδ

(CRP3, NF-IL6β, CELF), C/EBPε and C/EBP Homologous Protein10 (CHOP10,

GADD153) (228).

The function of C/EBPδ in prostate development, normal adult prostate

biology and prostate cancer has not been extensively investigated. The limited

evidence available supports a growth suppressor role for C/EBPδ in prostate

epithelial cell growth control. In human prostate cancer patients, comparative

gene expression studies demonstrate that C/EBPδ gene expression is significantly reduced in prostate cancer metastases compared to the primary tumor (152). Experimental studies in the rat have shown that androgen removal, which is associated with reduced growth and regression of the prostate, increases C/EBPδ gene expression in the rat ventral prostate (323). Also,

C/EBPδ gene expression is reduced in androgen independent, proliferative

human prostate xenografts (323).

Although a number of reports have investigated the effects of cytokines on

prostate cell growth, there is limited information available identifying IL-6

inducible downstream effector genes and their function in growth control. The

goal of this study was to investigate the influence of IL-6 treatment and activation

of the JAK/STAT signal transduction pathway on C/EBPδ gene expression and

growth inhibition of human prostate cancer cell lines. The results indicate that IL-

6 treatment induces activation (phosphorylation) of STAT3, increases C/EBPδ

38 gene expression and inhibits the growth of LNCaP prostate cancer cells. PC-3 cells, a prostate cancer cell line that lacks the IL-6 inducible JAK/STAT pathway, do not exhibit increased C/EBPδ gene expression or growth inhibition in response to IL-6. We have also identified a STAT3 binding site that is required for IL-6 mediated induction of C/EBPδ gene expression and a Sp1 site that is required for basal expression of the human C/EBPδ gene promoter. Finally, we demonstrate that overexpression of C/EBPδ is sufficient to suppress prostate cancer cell growth indicating that activation of C/EBPδ may be a viable new strategic approach in the prevention, treatment or control of prostate cancer.

Materials and Methods

Cell Culture- LNCaP, PC-3, 22RV1 and Du145 (ATCC) prostate cell lines were cultured in complete growth medium (CGM) consisting of DMEM/F12 (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and

100µg/ml streptomycin (Invitrogen). PZ-HPV-7 (ATCC) prostate cells were cultured in Keratinocyte-SFM supplemented with epidermal growth factor and bovine pituitary extract (Invitrogen).

Growth Arrest Experiments- 70% confluent cells were cultured in serum-free media (growth arrest medium (GAM)). For IL-6 induced growth arrest experiments cells were cultured in CGM or GAM supplemented with 50ng/ml recombinant human IL-6 (Peprotech). For drug studies LNCaP cells cultured in complete growth media were treated with either Nocodazole (400ng/ml) or

Hydroxyurea (2mM). At the indicated times, cells were harvested for Northern or

Western blot analysis. 39 [3H] Thymidine experiments- Cells were plated at 70% confluence in 12-well plates. The cells were then switched to the experimental media and pulsed for 2

hours with [3H] thymidine (1µCi/ml) before the indicated isolation time point. Cells

were harvested by precipitation with cold 10% trichloroacetic acid, solubilized in

0.2 N NaOH/1% SDS, and counted by liquid scintillation counting. Results

presented are representative of six wells per time point.

Flow cytometry experiments- For flow cytometry, cells were washed with 1X cold PBS and then fixed with 70% ethanol. The cells were then stained with

Propidium Iodide (Boehinger Manheim).

Northern Blot Analysis- Total RNA was isolated at the indicated times using

RNAzol B (Tel Test). Northern blots were performed with 30µg of total RNA as described (206). Filters were probed with the following 32P-labeled cDNAs:

C/EBPδ and cyclophilin (CP) (loading control).

Western Blot Analysis- Whole cell protein lysates were prepared from tissue-

cultured cells. Cells were initially washed with cold PBS, scraped into

microcentrifuge tubes and centrifuged at 14,000 x g to pellet the cells. The

supernatant was then removed and the cells were resuspended in whole cell

lysis buffer (20mM Tris, pH 8.0, 137mM NaCl, 10% glycerol, 1% Nonidet P-40,

0.1%SDS, 0.5% sodium deoxycholate, and 2mM EDTA). Protein kinase and

phosphatase activities were inhibited with the addition of Complete tablets

(Roche Molecular Biochemicals), 1mM NaF, 1mM NaVO3, 1mM NaMoO4, and

10nM okadaic acid to the protein isolation solution. Proteins were quantified using the BCA microprotein assay kit (Pierce). Proteins were separated by SDS

40 polyacrylamide gel electrophoresis and transferred to PVDF membranes by

electroblotting. Evenness of loading was verified by examination of gels stained with Coomassie Blue. Western blots were probed with primary antibodies to

STAT3, phospho-STAT3 (Tyr705) (New England BioLabs), C/EBPδ (Santa Cruz

Biotechnology) and β-actin. Horseradish peroxidase-conjugated antibodies (New

England BioLabs) were used to detect primary antibodies, and the signal was developed using ECL system (Amersham Pharmacia Biotech). Results are representative of experiments performed 2-5 times.

Human C/EBPδ Promoter-Reporter Constructs- A 1.7kb fragment of the human C/EBPδ promoter (generous gift of Dr. Trapman, Erasmus University,

Rotterdam, The Netherlands) was cloned into the pGL3 basic luciferase vector.

Deletion constructs were created by PCR with end points at -1700, -800, -393, -

233, -75 and -43 respectively relative to the transcription start site. A -393bp

promoter fragment with a mutated STAT3 binding site (STAT3 mutant TTCCCAG

→ GGCCTAG) was created by site directed mutagenesis. A -393bp promoter

mutant construct which contained a mutated Sp1 binding site (Sp1 mutant

GGGCGG → GGGCAA) was also created by site directed mutagenesis. A third -

393bp promoter mutant construct was created which contained both the STAT3

and Sp1 (Double Mutant) mutated binding sites.

Transient Transfection assays- Transfections were performed with 60-80% confluent cells in 60mm culture dishes. The Lipofectamine Plus transfection kit

(Invitrogen) was used as a vehicle to co-transfect 2 µg of each C/EBPδ promoter construct along with 100ng of SV40-βgal (control for transfection efficiency, 41 Clontech). Three hours after transfection the cells were rinsed with PBS and the

media changed to complete growth media or complete growth media containing

50ng/ml IL-6. Cells were harvested 24 hours later using the Luciferase Assay Kit

(Promega). Lysates were also assayed for β-galactosidase activity using the

Luminescent β-galactosidase detection kit II (Clontech). Luminescence was measured using a Hewlett-Packard Lumicount micro plate luminometer. C/EBPδ

promoter activities were normalized to β-galactosidase activity and the fold

induction in promoter activity following IL-6 treatment was calculated. Results

shown are the average fold increase from 3-6 independent experiments with 3

replicates per fragment and treatment per experiment.

Clonogenic Assay – The full length human C/EBPδ gene was cloned into the pcDNA3 mammalian expression vector. Three human prostate cancer cell lines

(LNCaP, PC-3 and 22RV1) were grown to approximately 70% confluence in

100mm dishes and transfected with 4µg of the C/EBPδ-pcDNA3 construct.

Positive control cells were transfected with empty pcDNA3 vector; negative control cells were non-transfected. Twenty four hours after transfection, the cells were shifted to media containing 400µg/ml G418 (selection media). After 14 days the cells were rinsed with PBS and stained with crystal violet in 20% methanol.

42 Results

IL-6 treatment reduces the growth rate of LNCaP human prostate

epithelial cells. IL-6 treatment of prostate cancer cells has been associated with

both growth inhibition and growth promotion (43, 115, 171, 188, 207, 264).

These differences in experimental outcomes may be due to differences in the

duration of IL-6 treatment, variations in prostate cancer cell line passage number,

or other unspecified factors. We initially investigated the effects of short term

(24 hours) treatment of LNCaP cells with increasing levels of IL-6 (0, 10, 50, 100

and 200ng/ml). Increasing the concentration of IL-6 decreased [3H] thymidine

incorporation in LNCaP cells, indicating that short term IL-6 treatment decreases

LNCaP proliferation (Fig 2.1 A). The growth inhibitory effect of IL-6 treatment on

LNCaP cells was next compared to growth inhibition in response to standard

serum and growth factor withdrawal conditions (“growth arrest media”,

GAM))(Fig. 2.1 B). Compared to exponentially growing LNCaP cells cultured in

complete growth media (CGM), [3H] thymidine incorporation was significantly

reduced (~60%) in LNCaP cells cultured in CGM plus IL-6 (Fig. 2.1 B). [3H] thymidine incorporation was similar between CGM + IL-6 treated and serum and growth factor withdrawn cultures, indicating that both treatments resulted in significant LNCaP cell growth inhibition. Combining IL-6 treatment and serum and growth factor withdrawal (IL-6 GAM) produced an additive effect on growth inhibition as demonstrated by the further reduction in [3H] thymidine incorporation

in IL-6 GAM treated cultures compared to GAM alone (Fig. 2.1 B). Consistent

with the [3H] thymidine incorporation experiments, flow cytometry demonstrated

43 that the percentage of LNCaP cells in S phase decreased approximately 50%

when cells were cultured in CGM plus IL-6, compared to CGM (Fig 2.1 C). In

contrast to LNCaP cells, IL-6 treatment did not reduce PC-3 cell proliferation. [3H] thymidine incorporation in PC-3 cells cultured in CGM, or CGM plus IL-6 was similar at all time points measured (0, 24, 48 and 72 hours), indicating that PC-3 cells are unresponsive to growth inhibition by IL-6 (Fig. 2.1 D). It is important to note, however, that PC-3 cells are sensitive to growth inhibition, as [3H] thymidine

incorporation decreased 40% when PC-3 cells were cultured under serum growth

factor withdrawal conditions (GAM) (Fig. 2.1 D). [3H] thymidine incorporation in

PC-3 cells cultured in GAM plus IL-6 did not differ from PC-3 cells cultured in

GAM, further demonstrating that PC-3 are refractory to IL-6 growth inhibition (Fig

2.1 D).

44

Figure 2.1. IL-6 decreases [3H]Thymidine incorporation in the LNCaP but not the PC-3 human prostate epithelial cell line. A: [3H]Thymidine incorporation of LNCaP cells cultured in complete growth media (CGM) supplemented with increasing concentrations of IL-6 for 24hrs. B: [3H]Thymidine incorporation of LNCaP cells in complete growth media, complete growth media with 50ng/ml IL- 6, growth arrest media (GAM), and growth arrest media with 50ng/ml IL-6 (*p<0.0001) C: Flow cytometry analysis indicating the percentage of LNCaP cells in S phase when cultured in complete growth media (CGM), complete growth media with 50ng/ml IL-6 (CGM+), growth arrest media (GAM), and growth arrest media with 50ng/ml IL-6 (GAM+). D: [3H]Thymidine incorporation of PC-3 cells in complete growth media, complete growth media with 50ng/ml IL-6, growth arrest media, and growth arrest media with 50ng/ml IL-6.

45 IL-6 treatment increases LNCaP C/EBPδ mRNA levels. We and others have reported that IL-6 family cytokine treatment induces C/EBPδ gene expression (29, 122). C/EBPδ mRNA levels were assessed in LNCaP human prostate cancer cells cultured in complete growth media (CGM) and complete growth media supplemented with growth inhibitory levels of IL-6 (50 ng/ml).

C/EBPδ mRNA expression increased in LNCaP cells cultured in CGM + IL-6 compared to cells cultured in CGM alone (Fig. 2.2). IL-6 treated LNCaP cells exhibited marked induction of C/EBPδ mRNA levels as early as 1 hour post treatment. C/EBPδ mRNA levels remained elevated at 24 hours. C/EBPδ mRNA levels remain elevated in LNCaP cells treated with IL-6 for up to 72 hours (data not shown).

Figure 2.2. Induction of C/EBPδ mRNA following IL-6 treatment. Near confluent plates were switched from complete growth media (CGM) to complete growth media, growth arrest media (GAM) and complete growth media with 50ng/ml IL- 6. RNA was isolated at 0, 1 and 24 hours and analyzed for C/EBPδ expression. CP (cyclophylin) was used as a loading control.

46 IL-6 treatment activates LNCaP JAK2 and STAT3 and increases

C/EBPδ protein levels. Previous reports have shown that IL-6 treatment of

LNCaP cells activates the JAK/STAT signal transduction pathway (171, 264). In

this study, we extend these results, demonstrating both specific activation of

JAK2 and STAT3 and induction of C/EBPδ mRNA (Fig 2.2) and protein levels

(Fig. 2.3). C/EBPδ protein levels are detectable as early as 2 hour after IL-6

treatment and remain elevated after 24 hours of IL-6 treatment. Growing LNCaP

cells cultured in CGM do not exhibit JAK/STAT activation or induction of C/EBPδ

protein levels. IL-6 treatment did not alter the levels of C/EBPβ, p44/42, STAT1,

phospho-STAT1, or p38 (data not shown).

Figure 2.3. Phosphorylation of Jak2, STAT3 and induction of C/EBPδ protein following IL-6 treatment of LNCaP cells. Near confluent plates were cultured in complete growth media (CGM) alone or complete growth media containing 50ng/ml IL-6. Whole cell protein was isolated at 0, 2 and 24 hour time points for western blots. Westerns were probed with primary antibodies to phosphorylated- JAK2 (pJAK2), STAT3, phosphorylated-STAT3 (pSTAT3) and C/EBPδ. β-actin was used as a loading control. 47 IL-6 treatment results in increased pSTAT3 levels, increased C/EBPδ gene expression and growth inhibition in LNCaP cells. IL-6 treatment induces variable activation of the pSTAT3/C/EBPδ growth inhibitory pathway and defective growth inhibition in PC-3, Du145, HPV and 22RV1 human prostate cells. Previous reports from our lab and others have demonstrated that IL-6 family cytokine-induced activation of the pSTAT3/C/EBPδ pathway induces growth inhibition (29, 122, 188, 264). We next investigated the influence of IL-6 cytokine treatment on activation of the pSTAT3/C/EBPδ growth control pathway and growth inhibition in 5 human prostate derived cell lines: LNCaP, PC-3, Du145, HPV and 22RV1. IL-6 treatment significantly induced C/EBPδ mRNA levels in LNCaP cells (Fig. 2.4 A).

In contrast, C/EBPδ mRNA levels were undetectable in both untreated and IL-6 treated PC-3 cells. Du145 cells exhibited constitutively elevated C/EBPδ mRNA levels. HPV cells exhibited detectable levels of C/EBPδ mRNA under untreated

(growing) conditions; C/EBPδ mRNA levels were induced in IL-6 treated HPV cells. C/EBPδ mRNA levels were minimally detectable in untreated and IL-6 treated 22RV1 cells. Phosphorylated (activated) STAT3 (pSTAT3) was undetectable in non-treated LNCaP, PC-3, HPV or 22RV1 cells. Du145 cells exhibited constitutively elevated pSTAT3 levels. C/EBPδ protein levels generally reflected C/EBPδ mRNA levels (Fig. 2.4 A) and also paralleled pSTAT3 levels

(Fig. 2.4 B). C/EBPδ protein levels were significantly induced in IL-6 treated

LNCaP cells. C/EBPδ protein levels were also induced in IL-6 treated HPV cells

48 compared to untreated controls. C/EBPδ protein levels were undetectable in IL-6 treated PC-3 cells and minimally detectable in 22RV1 cells. Du145 cells exhibited constitutively elevated C/EBPδ protein levels. STAT3 protein levels were present at relatively constant levels in all five cell lines regardless of treatment (Fig. 2.4 B). IL-6 treatment significantly reduced [3H] Thymidine incorporation into LNCaP cells (Fig. 2.4 C). In contrast, none of the other prostate derived cell lines exhibited a significant decrease in [3H] Thymidine incorporation in response to IL-6 treatment.

49

Figure 2.4. Analysis of STAT3 activation and C/EBPδ expression following IL-6 addition in various prostate cancer cell lines. A: Northern blot analysis of C/EBPδ expression levels in a variety of prostate cancer cell cultured in either complete growth media alone or complete growth media containing 50ng/ml IL-6. RNA was isolated at 24 hours post IL-6 addition. CP (cyclophylin) was used as a loading control. B: Prostate cancer cells were cultured in complete growth media alone or complete growth media containing 50ng/ml IL-6. Whole cell protein was isolated at 24 hours post IL-6 addition. Western blots were probed with antibodies to STAT3, phosphorylated-STAT3 (pSTAT3) and C/EBPδ. β-actin was used as a loading control. C: [3H]Thymidine incorporation assays. Cells were cultured in complete growth media or complete growth media containing 50ng/ml IL-6 for 24 hours.

50 The C/EBPδ gene promoter IL-6 response element is localized between -393 and -233 base pairs (bp) upstream of the transcription start site. In order to identify the IL-6 response element within the human C/EBPδ gene promoter we cloned a 1.7kb C/EBPδ promoter fragment into the pGL3 luciferase reporter construct. Deletion constructs were produced with end points at -800, -393, -233, -75, -43bp, respectively, relative to the transcription start site

(Fig. 2.5 A). Each of the five C/EBPδ gene promoter constructs with endpoints between -1700 and -75 bp exhibited similar basal transcriptional activity (Fig. 2.5

B). A dramatic decrease in basal transcriptional activity was observed when the region between -75 and -43 bp was deleted (Fig. 2.5 B). IL-6 treatment significantly increased the relative promoter activity of each of the five C/EBPδ gene promoter constructs with endpoints between -1700 and -393 bp. The highest level of IL-6 activation was observed in the -393 C/EBPδ gene promoter construct; IL-6 treatment did not significantly activate the -233, -75 or -43 C/EBPδ gene promoter constructs, suggesting the loss of an IL-6 inducible response element with the deletion from -393 to -293 (Fig. 2.5 C).

51

Figure 2.5. Identification of an IL-6 responsive region within the human C/EBPδ gene promoter. A: Luciferase reporter constructs containing the indicated 5’ promoter deletions were cotransfected with SV40β-gal into LNCaP cells. The luciferase data were normalized to β-galactosidase values to control for differences in transfection efficiencies. The values represent averages +/- standard deviations of 5 independent experiments carried out in triplicate. B: Normalized luciferase expression from each construct is shown relative to the value for -393 construct. C: Fold induction represents luciferase activity after IL-6 treatment relative to the basal levels.

52 Mutation of the STAT3 binding site in the C/EBPδ gene promoter eliminates IL-6 inducibility in LNCaP cells. Analysis of the deletion constructs localized the IL-6 inducible response element between -393 and -233 bp upstream of the transcription start site. This region contains a putative STAT3 binding site at -282. To provide further evidence that the STAT3 binding site was critical to IL-6 induction of the C/EBPδ gene promoter a -393bp C/EBPδ promoter reporter construct was prepared in which the STAT3 binding site was mutated

(Fig. 2.6 A). This mutation did not affect the basal transcription levels of the -393

C/EBPδ gene promoter fragment (Fig. 2.6 B). However, when the mutant STAT3 promoter fragment was treated with IL-6 the fold induction decreased from 3.5

(wild type promoter) to 1.3 with the STAT3 mutation (Fig. 2.6 C).

The region between -75 and -43 bp, which was required for basal transcription of the C/EBPδ gene promoter, contains a consensus Sp1 binding site. To evaluate the functional role of this Sp1 site we mutated the Sp1 site in the -393 and -393stat3 promoter fragments (Fig. 2.6 A). Mutation of the Sp1 site

within the -393 bp promoter fragment significantly decreased the basal

transcription levels (Fig. 2.6 B), but did not alter the fold induction observed with

the addition of IL-6 (Fig. 2.6 C). When both the Sp1 and STAT3 binding sites

were mutated in the -393bp (double mutant) promoter fragment both the basal

transcription level and the fold induction with IL-6 were dramatically reduced (Fig.

2.6 B, C).

53

Figure 2.6. Analysis of the STAT3 and Sp1 sites. A: Diagram of mutations. Mutations were introduced in the (-393) promoter construct at either the STAT3 or Sp1 site, or at both sites. B: Normalized luciferase expression from each construct are shown relative to the value for the wild type (-393) promoter. C: Fold induction represents luciferase activity after IL-6 treatment relative to the basal levels.

54 STAT3 activation and induction of C/EBPδ gene expression is

specific to G0/G1 growth arrest. To better understand the role of C/EBPδ in cell

cycle control we investigated the induction of C/EBPδ gene expression in

response to treatments that blocked the cell cycle at G0 /G1, G1 /S and G2/M.

LNCaP cells were cultured under two conditions that induce a general G0 /G1

growth arrest: serum and growth factor withdrawal (Growth arrest Media, GAM)

and CGM + IL-6. LNCaP cells were also cultured in CGM + hydroxyurea (G1 /S block, (75)) or CGM + Nocodazole (G2/M block, (250)). Exponentially growing

LNCaP cells cultured in CGM alone were used as a negative control. Only IL-6

treatment exhibited significant induction of C/EBPδ mRNA (Fig. 2.7 A). Similarly,

only IL-6 treatment (G0 /G1 block) significantly increased pSTAT3 levels and

C/EBPδ protein levels (Fig. 2.7 B). There was no evidence of pSTAT3 induction, increased C/EBPδ mRNA or increased C/EBPδ protein levels in GAM (G0 /G1), hydroxyurea (G1 /S) or nocodazole (G2/M) treated LNCaP cells (Fig. 2.7 A, B).

These results suggest that IL-6 induces a G0 /G1 growth arrest state that may be

differentiated from serum and growth factor withdrawal-induced G0 /G1 growth arrest and that treatments that induce blocks in other cell cycle stages, i.e. G1 /S and G2/M do not induce C/EBPδ gene expression. Cell cycle stage-specific

blocks were verified by flow cytometry (data not shown).

55

Figure 2.7. Cell cycle blocking studies. A: Northern blot analysis of LNCaP cells cultured in complete growth media (CGM), growth arrest media (GAM), CGM + 50ng/ml IL-6, CGM + 2mM hydroxyurea or CGM + 400ng/ml nocodazole. RNA was isolated at 24 hours and analyzed for C/EBPδ expression. CP (cyclophylin) was used as a loading control. B: Western blot analysis of LNCaP cells cultured in complete growth media (CGM), growth arrest media (GAM), CGM + 50ng/ml IL-6, CGM + 2mM hydroxyurea or CGM + 400ng/ml nocodazole. Whole cell protein was isolated at 24 hours. Western blots were probed with antibodies to STAT3, phosphorylated-STAT3 (pSTAT3) and C/EBPδ. β-actin was used as a loading control

56 C/EBPδ expression suppresses growth of prostate cancer cell lines.

Published reports from our lab and others have associated C/EBPδ gene

expression with growth inhibition in mammary and lung epithelial cells (31, 90,

91, 122, 123, 204-206, 274). We used the clonogenic assay to assess the ability

of C/EBPδ to suppress the growth of prostate cancer cell lines. A C/EBPδ-

pcDNA3 expression construct was transfected into the LNCaP, PC-3 and 22RV-1

human prostate cancer cell lines. As a positive control, cells were also

transfected with empty vector (pcDNA3). Transfected cells along with one set of

untransfected control cells were cultured in selection media (G418) for 12 days

and then stained to determine the number of surviving colonies. C/EBPδ

expression dramatically reduced the number and size (<10 cells) of surviving

colonies from all three prostate cell lines compared to pcDNA3 vector transfected

(positive) controls (Fig. 2.8). In contrast, untransfected cells (negative controls) died within the 12 days of culture in selection media (Fig. 2.8). These results demonstrate that C/EBPδ expression is sufficient to inhibit the growth of human

prostate cancer cell lines.

57

Figure 2.8. C/EBPδ expression inhibits the growth of prostate cancer cell lines. The LNCaP, PC-3 and 22RV1 human prostate cells lines were transfected with 4ug of pcDNA3 (positive control) or pcDNA3 containing the full length human C/EBPδ gene sequence. 24 hours after transfection cells were switched to complete growth media containing 400µg/ml of G418 and maintained in this media for 14 days. Untransfected cells from each line were also cultured in G418 media as a negative control. After 12 days of G418 treatment cells were fixed and stained with 0.5% crystal violet in 20% methanol.

58 Discussion

The goal of the present study was to investigate the influence of IL-6 on intracellular signaling, downstream gene activation and growth inhibition in prostate cancer cell lines. The identification of C/EBPδ as an IL-6 inducible downstream effector gene extends previous investigations which have also observed IL-6 induced growth inhibition of LNCaP cells (43, 264). In addition, these results are consistent with previous reports from our lab demonstrating that

IL-6 family cytokine treatment of mammary epithelial cells activates the

JAK/STAT signal transduction pathway, increases C/EBPδ gene expression and results in growth inhibition (122). Not all reports, however, have observed that

IL-6 treatment inhibits LNCaP cell growth (153, 171, 207). Divergent or even contradictory cytokine-mediated effects may be the result of events occurring in either the extracellular and intracellular environment of the target cell (129). In the extracellular environment, differences in duration of the cytokine treatment can influence the biological response of the target cell (115). For example, in this report we show that short term (24 hours) IL-6 treatment of low passage

LNCaP cells is associated with growth inhibition (Fig. 2.1). In contrast, chronic, long term IL-6 treatment results in the emergence of LNCaP cells that are refractory to IL-6 induced growth inhibition, and may exhibit IL-6 induced growth

(115). The apparent explanation for these contradictory IL-6 inducible growth effects is an alteration in the activation of IL-6 induced intracellular signaling pathways (55). Intracellular events, including variations in the expression or activation of intracellular signal transduction pathway components, or activation

59 of parallel signal transduction pathways, can influence the cellular response to cytokine treatment (55). For example, IL-6 treatment activates multiple intracellular signal transduction pathways that individually, and in combination, may induce variable biological effects. IL-6 treatment of LNCaP cells upregulates androgen receptor gene expression via a complex mechanism that involves activation of the MAP kinase pathway (163). Although we did not observe significant activation of the MAP kinase pathway following short term IL-

6 treatment of LNCaP cells (data not shown), long term IL-6 treatment has been shown to increase LNCaP MAP kinase activity and has been associated with increased in vivo growth of transplanted LNCaP cells (163). In addition, IL-6 treatment of LNCaP cells activates the Etk/Bmx pathway, resulting in LNCaP cell neuroendocrine differentiation (226). We also observed IL-6 induced increased neuroendocrine differentiation of LNCaP cells as evidenced by alterations in cell morphology (increased cellular elongation and appearance of cellular projections) and increased expression of neuron-specific enolase, a neuroendocrine differentiation marker (data not shown).

Although IL-6 activates multiple intracellular signal transduction pathways

STAT3 activation is a central mediator of IL-6 in target cells (264). In previous studies with non-transformed mammary epithelial cells and in the present study with LNCaP prostate cancer cells we have consistently observed that IL-6 family cytokine treatment results in STAT3 activation and growth inhibition (122).

However, clinical studies have linked constitutive activation of STAT3 with increased growth in a variety of cancer cells, including prostate cancer cells (66).

60 In addition, ectopic expression of STAT3-C, a constitutively active, recombinant

form of STAT3, increases cell transformation (20). In preliminary experiments we

found that ectopically expressed STAT3-C increased growth (colony formation)

of HC11 non-transformed mouse mammary epithelial cells (data not shown).

This suggests that STAT3-C activates a different array of cellular genes than

STAT3 that is activated by physiologically regulated intracellular signaling

pathways. That is, STAT3-C would appear to activate a predominantly growth

promoting gene array, whereas, IL-6 induced (regulated) STAT3 activation would

result in the activation of a predominantly growth inhibitory gene array.

Activation of STAT3 by Src or other cellular kinases may also influence the array

of downstream genes activated and may result in differential biological

responses. A comparison of the gene array profiles expressed following IL-6

mediated (physiological) STAT3 activation vs. ectopically expressed STAT3-C

may provide a better understanding of the different biological responses induced

by these alternate forms of activated STAT3.

Recent reports by Coqueret and coworkers provide new insights into the

molecular mechanism of STAT3 mediated transcriptional activation (10). They

show that STAT3-mediated transcriptional activation of growth promoting genes

waf1 (i.e., c-myc, pim-1 and cyclin D1) vs. growth inhibitory genes (i.e., p21 ) is altered by constitutive activation of the PI-3 kinase/AKT pathway (10). These findings suggest that cells exhibiting a constitutively activated PI-3 kinase/AKT pathway may express a different array of genes in response to activated STAT3 compared to cells in which the PI-3 kinase/AKT pathway activity is inactive.

61 Interestingly, the phosphatase and tensin homologue (PTEN) gene is deleted in

LNCaP cells resulting in constitutive PI-3 kinase/Akt activation (10). Although the

PI-3K/Akt pathway is constitutively activated in LNCaP cells the basal level of activation can be further upregulated by androgen withdrawal (10, 268).

In this study the influence of IL-6 treatment on STAT3 activation,

C/EBPδ gene expression and growth inhibition was investigated in 5 prostate- derived cell lines with varying results. The PC-3 and 22RV1 prostate cancer cell lines demonstrated little to no induction of C/EBPδ or STAT3 activation with IL-6 addition (Fig. 2.4). The absence of STAT3 activation in PC-3 cells with IL-6 addition is consistent with previous reports which have indicated PC-3 cells have a defect in the STAT3 activation pathway (264). In addition, these cell lines demonstrated no decrease in [3H] thymidine incorporation levels (Fig. 2.4 C). The absence of STAT3 activation in these cells most likely accounts for the lack of

C/EBPδ expression observed with IL-6 addition and further demonstrates that activated STAT3 is required for C/EBPδ gene expression. Constitutive STAT3 activation has been observed in various types of tumors including breast, head and neck and prostate (96, 97, 316). Consistent with previous reports we observed constitutively activated STAT3 in the Du145 cell line (171) and also demonstrate the constitutive expression of the STAT3 downstream effector gene,

C/EBPδ. These results suggest a downstream functional loss in the

STAT3/C/EBPδ growth inhibitory pathway in these IL-6 non-responsive cell lines.

Interestingly, the PZ-HPV-7 human prostate cell line (normal prostate cells transformed with HPV-18 DNA) demonstrated activation of STAT3 and 62 expression of C/EBPδ with IL-6 treatment. However, unlike LNCaP cells, the

HPV infected cells showed no reduction in [3H] thymidine incorporation levels with IL-6 addition (Fig. 2.4 C). Human papillomavirus E6 and E7 proteins play important roles in cell transformation. The E6 protein binds to p53 and enhances p53 degradation (245). E7 binds to hypophosphorylated Rb preventing Rb/E2F complex formation and promoting cell cycle progression (311). Previous reports have demonstrated C/EBP family members, including C/EBPδ, interact with Rb and p53 (36, 47, 51, 87). These results suggest the possibility that C/EBPδ growth arrest function is mediated by an interaction with p53 or RB. Current studies in our lab are focusing on the C/EBPδ protein-protein interactions with

Rb, E2F1 and other key cell cycle regulatory proteins.

C/EBPδ gene promoter analysis studies demonstrated that the STAT3 and

Sp1 binding sites play key roles in the transcriptional activation of the human

C/EBPδ gene. Although these results are consistent with those from the mouse

C/EBPδ gene promoter, the primary C/EBPδ gene promoter sequences between the two species (mouse and human) exhibit significant structural differences (29,

123). The mouse C/EBPδ gene promoter contains a Sp1 site (-120 to -115) immediately upstream to the STAT3 site (-110 to -102) (29). The close proximity of the two essential transcription factor binding sites was first described by

Johnson and coworkers and it suggests that the two transcription factors may interact in the IL-6 inducible transcriptional activation of the mouse C/EBPδ gene promoter (29). In addition, a second Sp1 site (-56 to -51) also plays a key role in

63 IL-6 induction of the mouse C/EBPδ gene promoter that is independent of the

upstream Sp1 site (29). In contrast to the mouse C/EBPδ gene promoter, the

human C/EBPδ gene promoter lacks an upstream Sp1 site 5’ to the functional

STAT3 binding site (-282 to -274). A downstream functional Sp1 site located at -

58 to -53 is required for basal activity, but IL-6 induction of the human C/EBPδ

gene promoter does not require an intact Sp1 site. Current studies are

investigating the differential role of chromatin remodeling and the assembly of

transcriptional co-activators on the human and mouse C/EBPδ gene promoters.

Although we previously observed that serum and growth factor withdrawal

induces C/EBPδ gene expression and growth arrest in mammary epithelial cells,

we did not observe serum and growth factor withdrawal induction of C/EBPδ in

the LNCaP prostate cancer cell line. The absence of activated STAT3 and

C/EBPδ gene expression with the removal of serum and growth factors suggests

that LNCaP cells are arresting in the G1 rather G0 phase of the cell cycle, or a

STAT3/C/EBPδ independent G0 growth arrest pathway exists in LNCaP cells.

Current studies are focusing on the discrimination between G0 and G1 phase arrest in LNCaP cells. We also demonstrate with the use of cell cycle blockers

(nocodazole, hydroxyurea) that STAT3 activation and C/EBPδ expression are not induced under other cell cycle arrest conditions.

Recent in vitro studies indicate that osteoblast conditioned media significantly enhances the percentage of prostate cancer cells in G0/G1 (220).

This microenvironment-induced growth inhibition is clinically relevant as it

64 increases prostate cancer cell survival and resistance to apoptosis in response to chemotherapeutic drugs (220). It is well-recognized that the enhanced survival and chemo resistance of prostate cancer cell bone metastases is a significant

cause of morbidity and mortality in prostate cancer patients, but the molecular

mechanism underlying these phenomena has not been identified. In initial

experiments we have observed that IL-6 is the predominant cytokine secreted by cultured osteoblasts. In addition, preliminary results indicate that LNCaP cells cultured in osteoblast conditioned media exhibit increased STAT3 activation

(pSTAT3), increased C/EBPδ gene and a decreased growth rate. These results suggest that C/EBPδ may play a major role in the molecular mechanism underlying the enhanced survival and chemo resistance of metastatic prostate

cancer cells in the bone microenvironment.

In conclusion, this study demonstrates that C/EBPδ gene expression is

increased in IL-6 treated LNCaP human prostate cancer cells. Increased

C/EBPδ gene expression plays an important role in IL-6/STAT3 mediated growth

arrest of LNCaP prostate cancer cells. Ongoing studies are investigating the

mechanism by which C/EBPδ controls prostate cancer cell growth and the

potential role of C/EBPδ in the survival and chemo resistance of prostate cancer

metastasis.

65 CHAPTER 3

OSTEOBLASTS INHIBIT PROSTATE CANCER CELL GROWTH VIA ACTIVATION OF THE IL-6/STAT3 GROWTH ARREST PATHWAY.

Abstract

Most patients with advanced prostate cancer will experience complications from bone metastases that are incurable. Previous studies have reported that the proliferative growth fraction of human prostate cancers is <10%, resulting in increased resistance to chemotherapy and mortality in prostate cancer patients.

A novel in vitro osteoblast/LNCaP coculture system has been described by

Isaacs and coworkers which mimics this clinical phenotype. In this system,

LNCaP cells are cocultured with osteoblasts, resulting in decreased LNCaP growth and enhanced resistance to chemotherapeutic agents. This coculture system provides a unique opportunity to examine the interactions that occur between osteoblasts and prostate cancer cell in the bone microenvironment.

The goal of this study was to investigate the mechanisms whereby osteoblasts mediate LNCaP growth inhibition by identifying the factors secreted by osteoblasts and their effect on intracellular signaling and gene activation in the

LNCaP prostate cancer cell line. We first demonstrate that osteoblast conditioned media (OCM) inhibits LNCaP cell growth and induces STAT3 activation and

66 C/EBPδ gene expression in LNCaP cells. We then demonstrate that OCM

contains high levels of IL-6, and using blocking antibodies and siRNA gene

knockdown studies identify an important role for the IL-6/STAT3 pathway in OCM

mediated growth arrest. Finally, we show that prolonged exposure of prostate

cancer cells to IL-6 renders cells resistant to IL-6 mediated growth arrest.

Interestingly, the growth of these IL-6 resistant prostate cancer cells may be stimulated in response to exogenous IL-6 addition. These findings suggest that

the IL-6/STAT3 growth arrest pathway plays an important role in prostate cancer

metastasis to bone.

Introduction

Prostate cancer is the most common form of cancer in US men with an

estimated 234,300 new cases diagnosed in 2005 (132). Prostate cancer is

second to lung cancer as a cause of cancer mortality in US males, accounting for

about ~30,000 deaths in 2005 (132). In general, prostate cancers are relatively

slow growing and their lethality is generally attributable to an increased

resistance to apoptosis rather than aggressive proliferation (12). This probably

accounts for the low response observed with commonly used chemotherapeutic

agents, which target cells in S phase. While prostate cancer is a heterogeneous

disease that is characterized by a prolonged and unpredictable clinical course, a

common observation is the appearance of peripheral metastases which are

associated with the untreatable, terminal phase of the disease (23, 48, 167, 229).

In prostate cancer patients the most common site of metastasis is bone. Bone

67 metastases are present in ~90% of patients dying from prostate cancer and most

patients with advanced prostate cancer will experience complications from bone

metastases that are incurable (23, 229).

To investigate the influence of the bone microenvironment on prostate

cancer cell growth, Isaacs and coworkers developed a novel osteoblast/prostate

cancer coculture system (220). Using this system they demonstrated that LNCaP

cell growth was dramatically reduced when cocultured with osteoblasts, and that

this reduced growth was associated with resistance to chemotherapeutic agents

(220). Their results suggested that osteoblasts secreted factors which inhibited prostate cancer cell growth; however the mechanism of osteoblast-mediated prostate cancer cell growth inhibition was not identified. A better understanding of both the osteoblast secreted factors and the mechanisms whereby they reduce prostate cancer cell growth rates may lead to the development of new therapeutic approaches which could specifically target metastases in the bone microenvironment.

The bone is a dynamic organ composed of two major cell types regulating bone remodeling, osteoblasts and osteoclasts (70, 287). Interactions between osteoblasts and/or osteoclasts with invading metastatic cancer cells have been described in a number of studies. Interestingly, prostate cancer metastases generally form osteoblastic lesions in bone compared to other cancers which typically form osteolytic metastases, suggesting that osteoblasts may play an important role in the metastasis of prostate cancer cells to bone (35, 170). A number of factors secreted by osteoblasts have been identified which play critical

68 roles in the bone microenvironment including TGF-β, Insulin-like growth factors I and II (IGF-I and IGF-II), IL-6, IL-1β and tumor necrosis factor α (TNF-α) (17, 37,

83, 125, 130, 214). While the primary functions of these factors in the bone microenvironment are well characterized, their importance in prostate cancer metastasis to bone is incompletely understood. In recent years, a number of reports have described an important, but poorly understood role for IL-6 in prostate cancer growth regulation (156, 163, 194, 230, 240).

IL-6 is a pleiotropic cytokine that induces a wide spectrum of responses in various cell types. Clinical studies indicate that plasma IL-6 levels increase in prostate cancer patients as the disease progresses (1, 69, 194). Increased IL-6 levels also correlate with the development of hormone-refractory prostate cancers (69). In addition, IL-6 has been shown to activate androgen receptor mediated gene expression and most androgen insensitive prostate cancer cell lines secrete IL-6 (40, 55, 114, 207). Previous work from our lab and others demonstrated that IL-6 inhibits LNCaP prostate cancer cell growth (43, 115, 188,

241, 265). Subsequent analyses showed that STAT3 activation was required for

IL-6 mediated LNCaP cell growth inhibition (241, 264, 265). Taken together, these results suggest that the IL-6/STAT3 pathway functions as an important regulator of prostate cancer cell growth.

Work in our lab has identified C/EBPδ as a STAT3 downstream effector gene (122, 123, 241, 259). C/EBPδ is a member of the CCAAT/enhancer binding protein family of leucine zipper type DNA binding proteins (228). C/EBPs function in a variety of cellular mechanisms including cell growth, differentiation and death 69 (136, 228). The function of C/EBPδ in prostate cancer biology has not been extensively investigated. Previous work in the rat ventral prostate has demonstrated that androgen removal, which is associated with reduced growth, increases C/EBPδ gene expression (323). In addition, androgen independent human prostate xenografts have reduced C/EBPδ gene expression (323).

Previous work from our lab identified C/EBPδ as a downstream mediator of IL-6 induced growth inhibition in prostate cancer cells (241). We also demonstrated that C/EBPδ overexpression suppressed prostate cancer cell growth (241).

In the present study, we investigated the effect of osteoblast conditioned media (OCM) on prostate cancer cell growth. We demonstrate that OCM inhibits

LNCaP cell growth and induces the activation of STAT3 and subsequent induction of C/EBPδ. Using cytokine array technology, we show that OCM contains elevated levels of IL-6, IL-7 and GM-CSF. IL-6 and gp130 blocking antibodies suppressed OCM mediated STAT3 activation, C/EBPδ expression and growth arrest. SiRNA mediated knockdown of STAT3 suggested that STAT3 plays a role in OCM mediated LNCaP growth inhibition. Finally, we demonstrate that prolonged exposure to IL-6 abolishes IL-6 induced prostate cancer cell growth inhibition and may confer a growth advantage. Taken together, our results suggest that the IL-6/STAT3 pathway plays an important role in the low proliferation rates observed when prostate cancer cells metastasize to bone.

70 Materials and Methods

Cell Culture- LNCaP and human fetal osteoblasts (hFOB) cell lines were

purchased from the American Type Culture Collection. LNCaP cells were

cultured as previously described (241). The conditionally immortalized human

fetal osteoblastic cell line was maintained in a 1:1 mixture of phenol-free

DMEM/F12 containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml

streptomycin and supplemented with 300 µg/ml geneticin at 34°C, the permissive

temperature for the expression of the large T antigen. To generate osteoblast

conditioned media (OCM), hFOBs were plated at approximately 70% confluence

and maintained at 39°C for 48 hours in DMEM/F12 containing 10% FBS and

pen/strep without the addition of geneticin (G418). After 48 hours, the OCM was

removed from the hFOBs and centrifuged briefly to remove any debris. OCM was

then used to treat LNCaP cells at 37°C.

Crystal Violet Staining- Following treatments, cells were washed with PBS and

stained with 0.5% crystal violet in 20% methanol for 5 minutes.

[3H] Thymidine Incorporation- LNCaP cells were cultured in poly-D-lysine

coated dishes (BD Biosciences). [3H]Thymidine incorporation studies were

carried out as previously described (241, 259).

Northern Blot Analysis- Total RNA isolations and northern blot analyses were

performed as previously described (241, 259). Northern blots were probed with

C/EBPδ and cyclophylin (CP, loading control) 32P-labeled cDNAs.

71 Western Blot Analysis- Whole cell protein lysates were prepared and western blots performed as previously described (241, 259). Polyclonal rabbit C/EBPδ

antibody was obtained from Active Motif. C/EBPβ antibody was obtained from

Santa Cruz Biotechnology, Inc. Phosphorylated STAT3-Tyr705 (pSTAT3),

STAT3, p21, cleaved caspase-3, β-actin, bcl-xl and cyclin D1 antibodies were obtained from Cell Signaling Technology, Inc.

Cytokine Array Study- The human cytokine array 3.1 (RayBiotech Inc.) was used to identify cytokines present in OCM. OCM was prepared as described above and analyzed by cytokine array analysis following the manufacturer’s directions.

IL-6 Quantitation by ELISA- A human IL-6 quantiGlo ELISA Kit (R&D Systems) was used to quantitate IL-6 levels in OCM following the manufacturer’s instructions. OCM was prepared as described above.

GP130 and IL-6 Blocking Studies- Human gp130, IL-6 and mouse IgG

(negative control) antibodies (R&D Systems) were used to disrupt the IL-6 signaling pathway. Blocking antibodies were added (15ng/ml) to OCM and incubated for 10 minutes. LNCaP cells were then cultured in OCM containing blocking antibodies for the indicated times.

SiRNA Analysis- STAT3 and scrambled (Junk) siRNA smartpools were purchased from Dharmacon for gene specific knock down studies.

Nucleofection™ technology (Amaxa Biosystems, Germany) was utilized to introduce siRNAs into LNCaP cells. SiRNAs were nucleofected at a concentration of 200 nM. LNCaP cells (2 X 106) were nucleofected with the

72 appropriate siRNA and seeded on 6cm poly-D-lysine coated cell dishes (BD

Biosciences). After 24 hours, nucleofected LNCaP cells were then treated with

either complete growth media (CGM) or OCM for an additional 24 hours.

Generation of IL-6 Overexpressing Cells- The full length human IL-6 gene was

cloned into the pcDNA3 mammalian expression vector. LNCaP cells were grown

to approximately 70% confluence and nucleofected with 2µg of the pcDNA3-IL6

construct. As a control an additional plate of LNCaP cells were nucleofected with

empty pcDNA3 vector, negative control cells were non-nucleofected. Stable cells

were selected with G418 (300µg per ml).

Results

Osteoblast conditioned media (OCM) inhibits LNCaP cell growth and induces STAT3 activation (pSTAT3) and C/EBPδ expression. Previous work

by Isaacs and coworkers demonstrated that coculturing LNCaP cells with human fetal osteoblasts enhanced the number of LNCaP cells in G0/G1 (220). We first

wanted to investigate the ability of OCM to inhibit LNCaP cell growth. LNCaP cell

growth was inhibited when cells were cultured in OCM compared to CGM (Fig.

3.1 A). Consistent with this result, OCM significantly reduced [3H] thymidine

incorporation levels in LNCaP cells, further suggesting that OCM inhibited

LNCaP cell growth (Fig. 3.1 B). Previous work in our lab had identified C/EBPδ

as a novel gene induced in mammary and prostate epithelial cells during G0 growth arrest (122, 204-206, 241, 258, 259). We investigated the ability of OCM to induce C/EBPδ expression and found that C/EBPδ mRNA expression increased in LNCaP cells cultured in OCM compared to cells cultured in CGM 73 (Fig. 3.1 C). We next investigated the influence of OCM on the activation of

STAT3, as previous work in our lab demonstrated that STAT3 activation was required for the induction of C/EBPδ mRNA (123, 258). pSTAT3 was induced in

LNCaP cells cultured in OCM but not in cells maintained in CGM (Fig. 3.1 D).

C/EBPδ protein levels were also induced in LNCaP cells in response to OCM.

STAT3 levels were not affected by OCM treatment. We then examined the effects of pSTAT3 (in response to OCM) on other previously identified STAT3 target genes (bcl-xl, c-Myc, cyclin D1 and p21 (16)). C-Myc levels were reduced

in response to pSTAT3 while bcl-xl levels were not affected by pSTAT3. The

levels of p21 decreased slightly following 4 days of OCM treatment (Fig. 3.1 D,

lane 4). Cyclin D1 levels were also slightly reduced following 4 day OCM

treatment. To demonstrate that the effects of OCM on LNCaP cells were the

result of growth inhibition and not induction of cell death, we analyzed protein

levels of activated (cleaved) caspase-3. Cleaved caspase-3 levels were slightly

induced following treatment of LNCaP cells with OCM for 4 days, suggesting that

OCM primarily induced LNCaP cell growth arrest rather than cell death.

74

Figure 3.1. Osteoblast conditioned media induces growth arrest, STAT3 activation and C/EBPδ expression in LNCaP cells. A: Crystal violet staining of LNCaP cells cultured in complete growth media (CGM) or osteoblast conditioned media (OCM) for 24 hours. B: [3H]Thymidine incorporation assays. Cells were cultured in CGM or OCM for 24 and 48 hours (* = p value < 0.001). C: Northern blot analysis showing that C/EBPδ mRNA is induced in LNCaP cells following OCM treatment. D: Western blot analysis of LNCaP cells treated with CGM or OCM for 2 and 4 days (2D, 4D). Western blots were probed with antibodies to phosphorylated-STAT3-Tyr-705 (pSTAT3), STAT3, C/EBPδ, c-myc, bcl-xl, p21, cyclin D1, cleaved caspase 3 and β−actin.

75 OCM contains IL-6 and blocking IL-6 signaling inhibits OCM

mediated STAT3 activation, C/EBPδ expression and growth arrest.

Osteoblasts secrete a variety of factors in the bone microenvironment. We

investigated the expression of various cytokines in OCM using a cytokine antibody array. We found detectable amounts of IL-6, IL-8 and GM-CSF (Fig. 3.2

A). The levels of IL-6 in OCM were quantitated by ELISA and determined to be approximately 6,812ng/ml. To investigate the significance of IL-6 in OCM

mediated LNCaP cell growth inhibition, blocking studies were performed using

antibodies to either IL-6 or gp130 (IL-6 receptor signaling component). The

addition of either IL-6 or gp130 blocking antibodies to OCM significantly inhibited

pSTAT3 and C/EBPδ expression (Fig. 3.2 B, C). These antibodies had no effect

on STAT3 or β−actin levels. Mouse IgG was used as a negative control. In

addition, we investigated the effect of blocking IL-6 signaling on OCM mediated

LNCaP cell growth arrest. [3H]Thymidine incorporation levels increased

significantly in LNCaP cells cultured in OCM containing either IL-6 or gp130

blocking antibodies compared to cells cultured in OCM alone (Fig. 3.2 D).

76

Figure 3.2. OCM induced STAT3 activation, C/EBPδ gene expression and growth arrest is the result of secreted IL-6. A: Cytokine array analysis of OCM. Cytokine arrays were treated with either OCM or CGM. B: Northern blot of C/EBPδ mRNA expression in LNCaP cells treated with OCM in the presence or absence of IL-6 and gp130 blocking antibodies. C: Western blot analysis of LNCaP cells treated with OCM in the presence of IL-6 and gp130 blocking antibodies (15ng/ml). Mouse IgG was used as a negative control. D: [3H]Thymidine incorporation assays. LNCaP cells were treated with OCM in the absence or presence of IL-6 and gp130 blocking antibodies. Mouse IgG was used as a negative control.

77 SiRNA mediated knockdown of STAT3 inhibits OCM mediated growth

arrest in LNCaP cells. Previous studies in our lab and others have demonstrated that IL-6 mediated growth arrest of LNCaP cells requires STAT3 activation (43, 241, 264). We investigated the importance of STAT3 activation in

OCM mediated LNCaP cell growth arrest using siRNA mediated knockdown technology. STAT3 siRNA constructs significantly reduced STAT3 and pSTAT3 levels in the presence of OCM (Fig. 3.3 A). C/EBPδ protein levels were also reduced in STAT3 siRNA nucleofected cells treated with OCM (Fig. 3.3 A, lane

3). As a control, C/EBPβ levels were assessed to demonstrate STAT3 specifically regulates C/EBPδ expression. C/EBPβ protein levels were unaffected by siRNA treatment. A non-sense scrambled (junk) siRNA was used as a negative control and did not alter STAT3, pSTAT3, C/EBPβ or C/EBPδ protein levels (Fig. 3.3 A, lane 2). We next investigated the effects of siRNA mediated

STAT3 knockdown on OCM induced LNCaP cell growth inhibition. SiRNA mediated knockdown of STAT3 in LNCaP cells increased [3H] thymidine

incorporation rates in the presence on OCM (Fig. 3.3 B). [3H]Thymidine

incorporation rates were not affected by junk nucleofected siRNAs.

78

Figure 3.3. siRNA mediated knockdown of STAT3 reduces OCM mediated LNCaP growth arrest. A: Western blot analysis of OCM treated LNCaP cells in the presence or absence of scrambled (Junk) and STAT3 siRNAs. Following nucleofection, cells were treated with OCM for 24 hours and whole cell lysates collected. Western blots were probed with antibodies to phosphorylated-STAT3- Tyr-705 (pSTAT3), STAT3, C/EBPδ, C/EBPβ and β−actin. B: [3H]Thymidine incorporation assays. Following siRNA nucleofection (200nM) cells were plated in 24 well poly-D-lysine coated plates for 24 hours and then cultured in OCM for an additional 24 or 48 hours.

79 Prolonged exposure to IL-6 desensitizes LNCaP cells to IL-6

mediated growth arrest. The role of IL-6 in prostate cancer biology is

controversial with reports of both stimulation and inhibition of LNCaP cell growth

(43, 153, 171, 241, 264, 265). Previous studies have demonstrated that LNCaP

cells may lose their ability to growth arrest in response to IL-6 treatment if they

are exposed to IL-6 for prolonged periods of time (115). We nucleofected LNCaP

cells with an IL-6 overexpression construct and stably selected for IL-6

overexpressing clones. Initially, overexpression of IL-6 inhibited prostate cancer cell growth (Fig. 3.4 A). We performed western blot analyses on stably selected cells to determine the effect of prolonged IL-6 exposure on STAT3 activation. IL-

6 treatment of pcDNA3 vector nucleofected cells resulted in phosphorylation of

STAT3, induction of C/EBPδ and a reduction of both c-Myc and cyclin D1 levels

as expected (Fig. 3.4 B lanes 1-2). Three different IL-6 overexpressing clones

(clones 1, 2 and 23) demonstrated constitutive STAT3 activation and C/EBPδ

expression (although at lower levels than IL-6 treated pcDNA3 vector

nucleofected cells) in the presence or absence of exogenous IL-6 (Fig. 3.4 B

lanes 3-8). Cyclin D1 levels were not reduced with exogenous IL-6 treatment in

the clones stably expressing IL-6. Exogenous IL-6 treatment of IL-6 clone 23,

however, increased cyclin D1 (Fig. 3.4 B, lanes 7-8). C-Myc levels also increased

in clone 23 in response to exogenous IL-6 treatment. The levels of c-Myc in IL-6

clone 1 were not affected by exogenous IL-6 addition (Fig. 3.4 B, lanes 3-4),

while IL-6 clone 2 showed a reduction in c-Myc levels with additional IL-6

treatment (Fig. 3.4 B,lanes 5-6). We next investigated the effect of prolonged IL-6

80 exposure on IL-6 mediated LNCaP cell growth arrest using [3H]thymidine

incorporation studies. LNCaP cells stably expressing IL-6 were not growth

arrested with exogenous IL-6 treatment, while pcDNA3 vector selected cells remained sensitive to IL-6 treatment and growth arrested (Fig. 3.4 C).

81

Figure 3.4. Prolonged exposure to IL-6 eliminates IL-6 mediated growth arrest of LNCaP cells in response to exogenous IL-6. A: Crystal violet staining of pcDNA3 and pcDNA3-IL-6 nucleofected cells. Cells were nucleofected with 2µg DNA and cultured in complete growth media for 48 hours. B: Western blot analysis of pcDNA3 vector nucleofected cells compared with three different stably nucleofected IL-6 overexpression clones (IL-6 clones 1, 2 and 23). Cells were cultured in the presence or absence of exogenous IL-6 (50 ng/ml) for 24 hours and whole cell lysates prepared. Western blots were probed with antibodies to phosphorylated-STAT3-Tyr-705 (pSTAT3), STAT3, C/EBPδ, cyclin D1, c-Myc and β-actin. C: [3H]Thymidine incorporation assays. pcDNA3 vector and IL-6 overexpressing clones were cultured in CGM in the presence or absence of exogenous IL-6 (50 ng/ml). 82 Discussion

In general, the phenotype of bone metastases is determined by the balance between the activities of osteoblasts and osteoclasts. In prostate cancer, bone metastases are frequently osteoblastic (35, 170). Interestingly, prostate cancer growth in the bone micro-environment is significantly reduced

(proliferative growth fraction is <10%)(12). Previous work has demonstrated that this in vivo phenomenon can be reconstituted in vitro using an osteoblast/LNCaP coculture system (220).

The goal of the present study was to investigate the factors secreted by osteoblasts and their effect on intracellular signaling, downstream gene activation and growth inhibition in the LNCaP prostate cancer cell line. The LNCaP cell line was chosen because it is representative of prostate cancers which are typically lethal to patients. LNCaP cells express the androgen receptor, secrete PSA and do not require androgens for growth (117). In addition, LNCaP cells typically demonstrate an osteoblastic phenotype in bone while other prostate cancer cell lines (PC-3 and Du145) tend to be osteolytic (117, 138, 271). The human fetal osteoblastic cell line (hFOB) was chosen to generate osteoblast conditioned media (OCM) as previous studies had demonstrated that coculture of these cells with LNCaP cells mimicked the growth inhibition observed in vivo when prostate cancer cells metastasize to bone (220). In addition, this cell line has been shown to be a good model system for osteoblast biology, as the cells are conditionally immortalized but not transformed (106). hFOBs also have minimal chromosomal

83 abnormalities and exhibit many of the properties of differentiated osteoblasts

compared to other commonly used bone cell lines (273). For these reasons,

OCM was generated from hFOBs to treat LNCaP prostate cancer cells.

We first demonstrated that OCM inhibits LNCaP cell growth (Fig. 3.1 A,

B). This is consistent with previous work by Isaacs and coworkers in which the osteoblast/LNCaP coculture system was developed to enhance G0/G1 checkpoint

control in prostate cancer cells (220). However, other groups have observed

growth in LNCaP cells treated with conditioned media from other osteoblast cell

lines (13, 172). This may be the result of events occurring in either the

extracellular or intracellular environment of the target cell. The source of the

conditioned media may also be responsible for these divergent results as human

osteoblast-like (HOBIT) cells and primary osteoblast cultures were used to

generate the conditioned media used in these studies. Interestingly, in both

reports, IL-6 signaling was required for the conditioned media induced growth of

prostate cancer cells.

Previous work in our lab identified C/EBPδ, a novel growth arrest gene,

which is up regulated during mammary and prostate epithelial cell G0 growth arrest conditions (122, 123, 204-206, 241, 258, 259). We examined the induction of C/EBPδ mRNA with OCM treatment and found that C/EBPδ mRNA levels are highly induced in LNCaP cells in response to OCM (Fig. 3.1 C). Work from our lab on the transcriptional regulation of C/EBPδ has demonstrated that activated

STAT3 is required for the induction of C/EBPδ expression (123, 241, 258, 259).

Western blot analysis confirmed that STAT3 activation occurs in LNCaP cells in 84 response to OCM treatment (Fig. 3.1 D). We also determined that OCM contains

IL-6, IL-8 and GM-CSF (Fig. 3.2 A). Previous work in our lab demonstrated that

IL-6 family cytokine treatment of mammary and prostate epithelial cells activated the STAT3 signal transduction pathway, increased C/EBPδ expression and inhibited cell growth (122, 241, 259). Here we show the same growth arrest pathway is activated following prostate cancer treatment with OCM (Fig. 3.1).

While a number of cytokines and other secreted factors are present in OCM, we demonstrated that IL-6 plays an essential role in OCM mediated growth arrest.

Eliminating IL-6 signaling with the use of blocking antibodies inhibited OCM mediated STAT3 activation, C/EBPδ expression and growth arrest in LNCaP cells (Fig. 3.2 C, D). In addition, although IL-6 activates multiple intracellular signaling pathways, we demonstrate that STAT3 activation plays an important role in LNCaP cell growth inhibition in response to OCM with the use of STAT3 specific siRNA constructs (Fig. 3.3).

The identification of the IL-6/STAT3 growth arrest pathway and its potential role in the prostate cancer cell / bone microenvironment is significant.

The effect of IL-6 on LNCaP prostate cancer cell growth is controversial, with some investigators reporting growth stimulation and others growth arrest (43, 55,

153, 171, 241, 264). It is possible that IL-6 initially acts as a growth inhibitor in prostate cancer cells. However, following prolonged exposure the inhibitory effect is lost and cells may acquire a growth advantage in the presence of IL-6. In order to address this, we nucleofected LNCaP cells with an IL-6 overexpression construct and stably selected for IL-6 expressing clones. Initially, LNCaP cell

85 growth was significantly inhibited by IL-6 (Fig. 3.4 A). This result was in

agreement with previous studies from our lab as well as work from a number of

other laboratories (43, 241, 264). Prolonged exposure to IL-6 rendered LNCaP

cells insensitive to exogenous IL-6 addition and eliminated IL-6 mediated growth

arrest (Fig. 3.4 C). In addition, the ability of these IL-6 resistant cells to induce

STAT3 activation with exogenous IL-6 addition was also lost (Fig. 3.4 B). This is

in agreement with previous work (115). Interestingly, previous studies have

demonstrated that LNCaP cells generated after long term exposure to IL-6 may

acquire a growth advantage and may become dependent on IL-6 for their growth

(115). This could explain the growth inhibition observed when activated STAT3

levels are reduced in constitutively STAT3 activated cell lines (84). IL-6 has been

shown to inhibit the growth of prostate cancer xenografts in mice using the

LNCaP cell line (314). It is of interest that this study also demonstrated that

Du145 prostate cancer cell tumor growth was unaffected by IL-6 treatment.

Previous work from our lab showed that Du145 cells express constitutive STAT3

activation and their growth is not inhibited with the addition of exogenous IL-6

(241). These results suggest that IL-6 inhibits the growth of prostate cancer cells

with a functional STAT3 signaling pathway. However, prolonged exposure to IL-6

or defects in the STAT3 signaling pathway, render prostate cancer cells

insensitive to IL-6 mediated growth arrest. These findings have also been observed in vivo. Previous studies have demonstrated that inoculation of LNCaP cells in bone failed to develop tumors (0/9) compared to Du145 (9/9) and PC-3

(7/9) cells (76). The PC-3 prostate cancer cell line harbors a defect in the STAT3

86 signaling pathway (43). It is possible that LNCaP cells were unable to develop

tumors in this study as a result of the IL-6 present in the bone microenvironment.

Du145 and PC-3 cells, however, which harbor defects in the STAT3 signaling

pathway, were not inhibited by IL-6 and formed tumors in the bone

microenvironment. Additional studies have demonstrated that IL-6 expressing

(resistant) LNCaP cells have accelerated in vivo growth rates and fail to induce

activation of STAT3 (268). Taken together, these results suggest that IL-6

induces growth arrest in prostate cancer cells via the STAT3 signaling pathway.

Defects in this pathway disrupt IL-6 mediated growth arrest.

Previous studies also suggest that IL-6 may function as a survival agent in

prostate cancer cells. IL-6 has been shown to protect LNCaP cells from

apoptosis through activation of the STAT3 pathway (154, 155). It is conceivable

that in addition to inducing prostate cancer cell growth arrest, activated STAT3

may up regulate the expression of pro-survival genes. The growth arrest and

survival functions of IL-6 mediated STAT3 activation may be independent of one

another. Thus prolonged exposure to IL-6 may render prostate cancer cells

insensitive to IL-6 mediated growth arrest; however the expression of pro-survival genes may persist with constitutive STAT3 activation.

We demonstrate that the same survival genes may be upregulated when prostate cancer cells metastasize to bone. Osteoblasts secrete IL-6 into the bone microenvironment. Osteoblast secreted IL-6 activates the IL-6/STAT3 signaling pathway in metastatic cells. STAT3 activation induces a growth arrest response

which decreases the number of prostate cancer cells entering the cell cycle

87 (lowers the growth fraction). This lowering of the growth fraction protects the cells from chemotherapeutic agents used to treat metastatic cancers which typically target rapidly dividing cells. In addition, STAT3 activation by osteoblast secreted IL-6 may also up regulate the expression of pro-survival genes in prostate cancer cells allowing them to survive in this unique microenvironment.

In conclusion, this study demonstrated that the IL-6/STAT3 growth arrest pathway plays an important role in OCM mediated LNCaP cells growth arrest.

STAT3 activation is induced when LNCaP cells are treated with osteoblast

conditioned media. This STAT3 activation is the result of osteoblast derived IL-6.

Disrupting the IL-6/STAT3 growth arrest pathway with blocking antibodies or

siRNAs decreased OCM induced STAT3 activation, C/EBPδ expression and

growth arrest. In addition, prolonged exposure to IL-6 eliminates IL-6 mediated

growth arrest and may be an important event in prostate cancer progression.

88 CHAPTER 4

IDENIFICATION OF C/EBPδ DOWNSTREAM EFFECTOR GENES: A NOVEL PRO-SURVIVAL FUNCTION FOR C/EBPδ.

Abstract

Previous work from our laboratory demonstrated that CCAAT/enhancer

binding protein δ (C/EBPδ) plays a key role in G0 growth arrest in prostate and mammary epithelial cells. In this report, we investigated the downstream effector genes induced in response to C/EBPδ expression in LNCaP prostate cancer cells. We first identified potential C/EBPδ downstream target genes using cDNA microarray analysis. To further identify downstream target genes specifically regulated by C/EBPδ, we performed ChIP on Chip experiments. Interestingly, in addition to identifying genes involved in growth arrest, both the microarray and

ChIP on Chip experiments identified various downstream target genes which have been shown to function in survival. Doxorubicin treatment of LNCaP cells increased C/EBPδ protein levels but not C/EBPδ mRNA levels. The observed increase in C/EBPδ was independent of STAT3 activation suggesting a unique mechanism of C/EBPδ regulation in response to doxorubicin. SiRNA mediated

C/EBPδ knockdown increased doxorubicin induced apoptosis in LNCaP cells as assessed by caspase 3. Finally, we demonstrated that C/EBPδ may

89 also function in a pro-survival role in mammary epithelial cells as C/EBPδ

overexpression protected MCF-12A cells from doxorubicin induced apoptosis.

These results suggest that C/EBPδ may play a novel role in survival.

Introduction

C/EBPδ is a member of the CCAAT/enhancer binding protein family of

transcription factors (228). Six C/EBP family members have been identified

including C/EBPα, C/EBPβ, C/EBPδ, C/EBPε, C/EBPγ and C/EBPζ (228).

C/EBPs consist of three major structural domains including a DNA binding domain, a leucine zipper domain and an activation domain (228). They regulate transcription by forming homo or heterodimers with other C/EBP family members

or other leucine zipper proteins (121, 228). In addition, C/EBPs have been shown

to regulate cell cycle progression by binding to key regulatory proteins including

p21, Rb, p107, E2F, cdk2 and cdk4 (36, 89, 107, 216, 290-293, 313). C/EBPs

are expressed in a tissue specific manner and function in a wide range of cellular

activities including proliferation, differentiation and apoptosis (228).

Previous reports from our laboratory identified an important role for

C/EBPδ in the initiation and maintenance of G0 growth arrest in mammary

epithelial cells (MECs) (122, 123, 204-206, 258, 259). We first demonstrated that

C/EBPδ mRNA, protein and DNA binding activity significantly increase during

mouse MEC G0 growth arrest (204-206). Further analyses demonstrated that

mouse MECs expressing C/EBPδ antisense RNA were resistant to G0 growth arrest upon serum and growth factor withdrawal while C/EBPδ overexpressing

MECs demonstrated accelerated G0 growth arrest under the same conditions 90 (206). Subsequent studies focused on the transcriptional pathways regulating the

expression of C/EBPδ and demonstrated that STAT3 activation was required for

the induction of C/EBPδ during G0 growth arrest of MECs in response to serum

and growth factor withdrawal or IL-6 family cytokine treatment (122, 123, 258,

259). We also demonstrated that the C/EBPδ mRNA and protein are highly

unstable (62, 63), paralleling other important proteins that play essential roles in

growth regulation (102). The importance of C/EBPδ in mammary epithelial cell

growth regulation has also been demonstrated in vivo. C/EBPδ knockout mice display increased mammary ductal branching and total mammary gland volume compared to wild type mice (92). Taken together, these results suggest that

C/EBPδ plays an important role in the growth regulation of MECs.

The importance of C/EBPδ in growth arrest is not limited to MECs. C/EBPδ plays an important role in growth arrest and differentiation in M1 myeloid leukemia cells (137). In addition, C/EBPδ expression in BCR-ABL positive leukemic cells induced growth arrest and differentiation (88). A recent report from our lab has demonstrated that C/EBPδ also functions as an important regulator of prostate cancer cell growth arrest (241). We identified C/EBPδ as an important downstream effector gene induced in response to activation of the IL-6/STAT3 growth arrest pathway and also demonstrated that prostate cancer cell lines harboring defects in this pathway were resistant to IL-6 induced growth arrest.

Finally, we showed that C/EBPδ overexpression was sufficient to suppress prostate cancer cell growth (241). Work from other labs also suggested that

91 C/EBPδ may be an important regulator of prostate cancer cell growth. Clinical

studies identified C/EBPδ as one of 100 genes with the greatest degree of differential expression between primary and advanced metastatic prostate cancers, demonstrating that C/EBPδ expression levels were significantly reduced in advanced prostate cancer metastases (152). Yang and coworkers investigated the effects of androgens on C/EBPδ expression and found that androgen removal increased C/EBPδ gene expression in the rat ventral prostate (323).

Interestingly, they also demonstrated that C/EBPδ gene expression was down-

regulated in advanced stage, androgen independent human prostate tumors. In

addition, a recent report demonstrated that C/EBPδ is essential for the significant

growth inhibition of prostate cancer cells in response to 1,25(OH)2D3 treatment

(124). Collectively, these results suggest that C/EBPδ is an important regulator of

cell growth. However, the function of C/EBPδ has not been elucidated.

The goal of this study was to investigate the downstream effector genes

upregulated in response to C/EBPδ gene expression in prostate cancer cells.

With the use of cDNA microarray and ChIP on Chip analysis, we identified a

number of potential C/EBPδ downstream target genes. In addition to identifying

genes involved in growth arrest, both analyses identified downstream target

genes with important pro-survival roles. Further investigation into the potential

pro-survival function of C/EBPδ demonstrated that doxorubicin induced C/EBPδ

protein levels independent of STAT3 activation. SiRNA mediated knockdown of

C/EBPδ sensitized LNCaP cells to doxorubicin induced apoptosis as assessed

92 by cleaved caspase-3. Finally, we demonstrated that the pro-survival function of

C/EBPδ is not restricted to prostate cancer cells, as MCF-12A mammary

epithelial cells overexpressing C/EBPδ are resistant to doxorubicin induced

apoptosis.

Materials and Methods

Cell Culture- LNCaP prostate cancer cells were cultured in complete growth

medium (CGM) consisting of DMEM/F12 (Invitrogen) supplemented with 10%

fetal bovine serum (FBS), 100 units/ml penicillin, 100µg/ml streptomycin and 500 ng/ml fungizone (Invitrogen). MCF-10A and MCF-12A cells were cultured in

DMEM/F12 media supplemented with 20ng/ml recombinant human EGF, 100 ng/ml cholera toxin, 10µg/ml bovine insulin, 500ng/ml hydrocortisone, 5% horse

serum, 100units/ml penicillin, 100µg/ml streptomycin and 500ng/ml fungizone.

DNA Microarray Analysis- LNCaP cells were transfected (Lipofectamine Plus,

Invitrogen) with either 4µg of pcDNA3 (vector alone) or a pcDNA3-C/EBPδ

construct. After 24 hours in CGM, total RNA was isolated and prepared for

Affymetrix GeneChip analysis using RNABee (TelTest Inc.). GeneChip analysis

was performed on the Human Genome U133 Plus 2.0 Array followed by data

analysis using the Affymetrix GeneChip® DNA Analysis Software.

RT-PCR- Total RNA was isolated using RNABee from pcDNA3-vector and

pcDNA-C/EBPδ transfected LNCaP cells. One µg of each sample was treated

with DNase І (amplification grade) and reversed transcribed with an oligo dT

primer using the Superscript™ First-Strand Synthesis system (Life

Technologies). One µl of RT aliquots were PCR amplified with the following 93 primer pairs; GNA11 (sense 5’-AGA GCA AAG CCC TGT TCC-3’, anti-sense 5’-

ATG ATC TTG TCG CTG TCG-3’), Beta 1 Arrestin (sense 5’-GTG AAG AAG

ATC AAG ATC TC-3’, anti-sense 5’-CGT GTC TTC GTG CTT GCG-3’), Beta 1C

Integrin (sense 5’-TCT GTC GCC CAG CCT GGA GTG-3’, anti-sense 5’-TTT

CCC TCA TAC TTC GGA TTG-3’), C/EBPδ (sense 5’-AGC GCC TAC ATC GAC

TCC ATG G-3’, anti-sense 5’-CAA GCT CAC CAC GGT CTG TGC-3’) and

GAPDH (sense 5’-CTC ACT GGC ATG GCC TTC CG-3’ , anti-sense 5’-ACC

ACC CTG TTG CTG TAG CC-3’). PCR amplified samples were run on 1.5%

agarose gels stained with ethidium bromide and photographed on a Kodak

EDAS290 gel documentation system.

Nucleofection- Nucleofector Technology™ (Amaxa Biosystems) was used to

deliver both plasmid DNA and siRNA constructs into the LNCaP and MCF-12A

cell lines. An optimized nucleofection protocol was established in LNCaP cells

which used cell line nucleofector kit V and nucleofection program O-17. Following

nucleofection, LNCaP cells were plated on poly-D-lysine coated cell culture

dishes (BD Biosciences). For MCF-12A cells, the optimized protocol required cell

line nucleofector kit V and the nucleofection program P-20. Following

nucleofection of either cell line, cells were permitted 24 hours to recover before

additional treatment. The nucleofection of DNA into either cell line required 2µg

of plasmid DNA. C/EBPδ and Junk (scrambled, negative control) siRNA

smartpools were purchased from Dharmacon for gene specific knock down

studies. SiRNAs were nucleofected at a concentration of 200 nM.

94 “ChIP on Chip” assay- LNCaP cells were nucleofected with either a C/EBPδ-v5

tagged expression construct or vector alone and cultured in CGM for 24 hours.

The following day, nucleofected cells were treated with IL-6 (50 ng/ml) for 24

hours. ChIP assays were performed using the Chromatin Immunoprecipitation

(ChIP) Assay Kit, (Upstate Biochemicals). Briefly, cells were cross-linked with 1%

formaldehyde, harvested, and sonicated (with optimization) to generate chromatin/DNA fragments <1 kb. Immunoprecipitations were performed using an anti-v5 antibody (Invitrogen) to pull down the C/EBPδ-v5/DNA complexes. As a negative control, vector nucleofected cells were also immunoprecipitated with anti-v5 antibody. The recovered chromatin/DNA crosslinks were reversed and digested with Proteinase K followed by phenol/chloroform extraction. Released

DNA fragments from the vector control and C/EBPδ-v5 immunoprecipitations were amplified by PCR with either standard dNTPs or allylamine-dUTP

(incorporated into the DNA product for dye labeling). The C/EBPδ and vector

bound PCR amplified DNA products were individually labeled with either Alexa

Fluor 555 or Alexa Fluor 647 (improved labeling/detection compared with Cy5 or

Cy3) using the BioPrime DNA labeling system (Invitrogen). The labeled probes

were co-hybridized on a CpG Island Microarray containing ~12K CpG island DNA

fragments (available from the University of Toronto). Reciprocal dye labeling

was performed to eliminate false positives resulting from dye-array interactions.

Microarray hybridization and washing was performed at the OSU Microarray Unit.

Following washing, the microarray was scanned with the

95 GenePIx 400A scanner and the data analyzed using GenePix 3.0 software (OSU

MicroArray Unit). CpG island tags generating a differential Alexa Fluor signal of

>2 were designated as C/EBPδ-bound DNA fragments.

Western Blot Analysis- Whole cell protein lysates were prepared and western blots performed as previously described (241, 259). Polyclonal rabbit C/EBPδ

antibody was obtained from Active Motif. Cleaved caspase-3, β-actin,

phosphorylated STAT3-Tyr705 (pSTAT3), STAT3, p21, p53, bcl-xl and survivin

antibodies were obtained from Cell Signaling Technology, Inc.

IL-6 and Doxorubicin Experiments- For IL-6 and doxorubicin studies, LNCaP

cells were cultured in CGM supplemented with either 50 ng/ml of IL-6

(Peprotech) or 2µM doxorubicin (Sigma) for 24 hours. For experiments requiring

treatment with both IL-6 and doxorubicin, LNCaP cells were first cultured in IL-6

(50 ng/ml) for 24 hours followed by addition of doxorubicin (2µM final) for the

indicated times. MCF-10A and MCF-12A cells were cultured in CGM to

confluence and doxorubicin added at final concentrations of 1 or 2 µM for 24

hours. MCF-12A cells which were nucleofected with either vector or C/EBPδ- overexpression constructs were treated with 1µM doxorubicin for 24 hours following completion of the nucleofection protocol.

Northern Blot Analysis- Total RNA was isolated and northern blots performed with 30µg of total RNA as previously described (206). Filters were probed with the following 32P-labeled cDNAs: C/EBPδ and cyclophylin (CP) (loading control).

96 Results

Identification of C/EBPδ downstream target genes by microarray analysis. Previous work from our lab reported that C/EBPδ plays a key role in mammary and prostate epithelial cell G0 growth arrest (122, 123, 204-206, 241,

258, 259). In addition, reports from other labs have also identified C/EBPδ as an important regulator of prostate cancer cell growth arrest (124, 323). Despite these observations, the function of C/EBPδ in prostate cancer biology has not been elucidated. Since C/EBPδ is a transcription factor, one of its primary functions is regulating the expression of downstream effector genes. To identify

C/EBPδ downstream effector genes, we transfected a C/EBPδ-overexpression construct into LNCaP cells and performed microarray analysis. Table 4.1 lists genes that were induced in response to C/EBPδ overexpression and have also been demonstrated to function in cell growth regulation. In addition, two genes were identified whose expression was significantly reduced in response to overexpressed C/EBPδ. The existence of potential C/EBP binding sites in the proximal promoters of these genes was investigated using the MatInspector program available at http://www.genomatix.de. This program identified C/EBP binding sites in 8 out of 10 proximal gene promoters further suggesting that their expression may be regulated by C/EBPδ.

97

Potential C/EBP Fold induction with binding site Demonstrated to GENE NAME C/EBPδ present in Function in overexpression promoter Beta 1C inhibits prostate cancer 4.7 yes Integrin cell proliferation (189) DNA damage, Gadd45α induction. FOXO3A 4.5 no Growth regulation (22, 82, 174, 294, 326) guanine cell growth, expression nucleotide 3.1 yes reduced in breast binding protein cancer samples (8) alpha 11 differentiation, cell Mcl-1 2.9 no survival (148, 182) Interacts with APC MAPRE3 2.8 yes (193, 272) involved in activation of Beta 1 Arrestin 2.6 yes PI3K, anti-apoptotic activity (224) mitochondrial up-regulated during tumor 2.5 yes differentiation and suppressor quiescence (249) gene 1 may function as Kruppel-type transcriptional zinc finger 2.1 yes repressor, tumor protein suppressor (146) osteoclast TNFSF11 -6.7 yes differentiation (278) Origin recognition role in chromosomal -6.9 yes complex replication (128) subunit

Table 4.1. Microarray identified target genes regulated by C/EBPδ overexpression. Microarray analysis was performed on the Human Genome U133 Plus 2.0 Array followed by data analysis using the Affymetrix GeneChip® DNA Analysis Software. Potential C/EBP binding sites were identified with the MatInspector program available at http://www.genomatix.de.

98 Validation of microarray data using RT-PCR. To verify the gene expression profiles obtained by microarray analysis, we performed RT-PCR on a subset of genes identified in the microarray study. The expression of GNA11, beta 1 arrestin and beta 1C integrin were induced in response to C/EBPδ overexpression in LNCaP prostate cancer cells (Fig. 4.1). RT-PCR was also performed using C/EBPδ specific primers to confirm overexpression. GAPDH was used as a control.

Figure 4.1. Microarray validation by RT-PCR. LNCaP cells were transfected with either pcDNA3 empty vector or pcDNA3-C/EBPδ followed by RNA isolation and RT-PCR analysis.

99 C/EPBδ targets identified by “ChIP on Chip” analysis. To further

identify potential C/EBPδ downstream targets genes we utilized the novel ChIP on Chip assay. This assay identifies target gene promoters which C/EBPδ can directly interact with. Immunoprecipitated C/EBPδ and vector bound DNAs were amplified by PCR in the presence or absence of allylamine-dUTP (Fig. 4.2 A).

C/EBPδ bound DNAs were labeled with Alexa Fluor 555 (red) dye and vector bound DNAs with Alexa Fluor 647 (green) dye and co-hybridized on the CpG island microarray (Fig. 4.2 B). The reciprocal dye labeling experiment was also performed and only genes identified under both conditions were considered

C/EBPδ bound promoters (Fig. 4.2 C). We identified several C/EBPδ bound promoters including: inhibitor of growth family member 4 (ING4), retinoblastoma binding protein 8 (RBBP-8), BTG family member 2 (BTG2), thioredoxin 2 (TRX2) and minichromosome maintenance deficient 7 (MCM7).

100

Figure 4.2. ChIP on Chip analysis to identify C/EBPδ bound gene promoters. A: LNCaP cells were nucleofected with either a C/EBPδ-v5 tagged expression construct or vector alone. Sonication conditions for LNCaP cells were optimized to generate DNA fragments between 300 and 1200 base pairs. Lanes 1 and 3 represent PCR amplification with standard dNTP’s while lanes 2 and 4 represent PCR amplification with allylamine-dUTP (aa-dUTP) which is incorporated into the DNA product for dye labeling. B: PCR amplified DNA (from A) was labeled with the appropriate Alexa Fluor Reactive Dye. Negative control (vector nucleofected) DNA was labeled with the Alexa 647 (green) dye while the positive (C/EBPδ nucleofected) DNA was labeled with the Alexa 555 (red) dye. C: The reciprocal dye labeling was also performed to eliminate false positives resulting from dye- array interactions. The white arrows in B and C designate gene promoters that were bound by C/EBPδ under both dye labeling conditions.

101 IL-6 protects LNCaP cells from doxorubicin induced apoptosis. While

both the microarray and ChIP on Chip assays identified a number of candidate

C/EBPδ downstream target genes, no overlap was observed between the two

assays. However, both identified downstream target genes which have been

shown to function as pro-survival genes. Microarray analysis identified Mcl-1 and

beta 1 arrestin while the ChIP on Chip assay identified TRX2 and BTG2.

Previous studies have described a pro-survival function for each of these genes

(72, 182, 201, 224, 284, 312). These results suggest that C/EBPδ may play an important role in prostate cancer cell survival. In addition, pro-survival functions have been described for both C/EBPα and C/EBPβ in a variety of cell types (24,

25, 131, 270, 319, 324, 338). Previous work from our lab and others demonstrated that IL-6 induced C/EBPδ gene expression and growth arrest in

LNCaP cells (241). In addition, work from other labs has shown that IL-6 may also function to protect LNCaP cells from apoptosis (154, 225). Hence, we wanted to investigate the potential pro-survival function of IL-6 in LNCaP cells.

IL-6 decreased cleaved caspase-3 levels in doxorubicin treated LNCaP cells which were pre-treated with IL-6 compared to non IL-6 treated cells (Fig. 4.3).

102

Figure 4.3. IL-6 reduces doxorubicin (DOX) induced apoptosis. LNCaP cells were cultured in Complete Growth media (CGM) or CGM + IL-6 (50ng/ml) for 24 hours followed by treatment with DOX (2µM) for 24 hours. Cell lysates were isolated and western blot analysis performed to detect cleaved (activated) caspase-3 as an indicator of apoptosis. β-actin was used as a loading control.

Doxorubicin increases C/EBPδ protein levels in the absence of

activated STAT3. Previous work in our lab characterized the STAT3/C/EBPδ

pathway and demonstrated that it plays an important role in the growth arrest of

prostate and mammary epithelial cells in response to IL-6 family cytokines (122,

241, 258, 259). In this study, we investigated the effects of doxorubicin on the

STAT3/C/EBPδ pathway in growth arrested (IL-6 treated) and growing (CGM)

LNCaP cells. Activated (phosphorylated) STAT3 (pSTAT3) levels and C/EBPδ

protein levels increased with IL-6 addition as previously described (241) (Fig. 4.4,

lane 2). The addition of doxorubicin to IL-6 treated LNCaP cells slightly

increased C/EBPδ protein levels but did not affect pSTAT3 levels (Fig. 4.4, lanes

103 4, 6, 8). Interestingly, doxorubicin treatment of non IL-6 treated LNCaP cells significantly increased C/EBPδ protein levels in the absence of pSTAT3 (Fig. 4.4, lanes 3, 5, 7).

Figure 4.4. Doxorubicin increases C/EBPδ protein levels in the absence of phosphorylated STAT3. LNCaP cells were cultured in CGM in the absence (lanes 1, 3, 5, 7) or presence (lanes 2, 4, 6, 8) of IL-6 (50ng/ml) for 24 hours. Doxorubicin (Dox) was then added (2µM) for the indicated times (lanes 3-8). Whole cell lysates were prepared and probed with primary antibodies to phosphorylated-STAT3 (pSTAT3) and C/EBPδ.

Doxorubicin induces C/EBPδ protein levels but not C/EBPδ mRNA

levels. Previous work from our lab demonstrated that STAT3 activation is

required for the induction of C/EBPδ mRNA (122, 123, 241, 258, 259). However,

our initial results in this study suggested that doxorubicin increased C/EBPδ

protein levels independent of STAT3 activation. We next investigated the effect

of doxorubicin on C/EBPδ mRNA levels. Unlike C/EBPδ protein levels,

doxorubicin had no effect on C/EBPδ mRNA expression (Fig. 4.5 A). We

104 confirmed our initial findings by demonstrating that doxorubicin increases

C/EBPδ protein levels (Fig. 4.5 B). In addition, doxorubicin induced both p21 and cleaved caspase-3 levels but did not effect STAT3, pSTAT3 or β-actin protein

levels. IL-6 induced pSTAT3 and C/EBPδ protein levels but did not affect STAT3,

cleaved caspase-3 or β-actin protein levels (Fig. 4.5 B). p21 levels were slightly increased in response to IL-6 addition.

Figure 4.5. Doxorubicin does not affect C/EBPδ mRNA levels in LNCaP cells. A: Northern blot analysis of LNCaP cells cultured in complete growth media (CGM) alone or CGM containing either IL-6 (50ng/ml) or doxorubicin (Dox, 2µM) for 24 hours. RNA was isolated and analyzed for C/EBPδ expression. CP was used as a loading control. B: Western blot analysis of LNCaP cells cultured in complete growth media (CGM) alone or CGM containing either IL-6 (50ng/ml) or doxorubicin (Dox, 2µM) for 24 hours. Western blots were probed with antibodies to phosphorylated-STAT3 (pSTAT3), STAT3, C/EBPδ, p21, cleaved caspase-3 and β-actin.

105 SiRNA mediated C/EBPδ knockdown increases doxorubicin induced apoptosis in LNCaP cells. To better understand the function of increased

C/EBPδ levels in response to doxorubicin, we performed siRNA mediated gene knockdown studies. C/EBPδ specific siRNAs significantly reduced C/EBPδ protein levels in the presence of doxorubicin (Fig. 4.6 A). In addition, cleaved caspase-3 protein levels increased in C/EBPδ siRNA nucleofected LNCaP cells

(Fig. 4.6 A). A scrambled (junk) siRNA was nucleofected into cells as a negative control. We next investigated the effects of doxorubicin and C/EBPδ knockdown on DNA damage and apoptosis induced cellular proteins. The expression of p53 and p21 increased in response to doxorubicin while the expression of survivin was reduced (Fig 4.6 B, lane 3). SiRNA mediated C/EBPδ knockdown reduced bcl-xl protein levels, while the levels of cleaved caspase-3 increased as previously observed (Fig. 4.6 B, lane 5). The reduction in C/EBPδ levels did not effect p53, p21 or survivin protein levels. Consistent with previous reports, doxorubicin induced p53 expression resulted in the down regulation of survivin

(Fig. 4.6 B) (185, 337).

106

Figure 4.6. SiRNA mediated C/EBPδ knockdown reduces bcl-xl protein levels and increases cleaved caspase-3 levels in doxorubicin treated LNCaP cells. A: Western blot analysis of LNCaP cells nucleofected with either scrambled (junk) or C/EBPδ siRNAs followed by doxorubicin (Dox, 2µM) treatment for 24 hours. Western blots were probed with antibodies to C/EBPδ, cleaved caspase-3 and β- actin. B: Western blot analysis of LNCaP cells cultured in complete growth media (CGM) alone or with the addition of IL-6 (50ng/ml) or doxorubicin (Dox, 2µM) for 24 hours (lanes 1-3). Lanes 4 and 5 represent LNCaP cell nucleofected with either junk or C/EBPδ specific siRNAs prior to doxorubicin treatment. Whole cell lysates were prepared and probed with antibodies to pSTAT3, p53, bcl-xl, survivin, p21, cleaved caspase-3 and β-actin.

107 Doxorubicin decreases C/EBPδ expression in MCF-10A and MCF-12A

non-transformed mammary epithelial cells: however, C/EBPδ

overexpression reduces doxorubicin induced apoptosis in MCF-12A cells.

Having demonstrated an effect of doxorubicin on C/EBPδ levels in LNCaP cells, we next wanted to investigate the effects of doxorubicin on C/EBPδ levels in other epithelial cells. The MCF-10A and MCF-12A non-transformed mammary epithelial cell lines were used to assess the effects of doxorubicin on C/EBPδ protein levels. We found that C/EBPδ proteins levels decreased in both MCF-10A and MCF12A cells with the addition of doxorubicin (Fig. 4.7 A). This decrease was dose dependent in both cell lines, as the addition of a higher dose (2µM) of doxorubicin further reduced C/EBPδ protein levels (Fig. 4.7 A, lanes 3, 6). We next investigated the ability of C/EBPδ overexpression to suppress doxorubicin induced apoptosis in MCF-12A cells. In the presence of doxorubicin, C/EBPδ

overexpressing cells had reduced levels of cleaved caspase-3 compared to

control cells (Fig. 4.7 B, lane 4), suggesting that C/EBPδ may function in a pro-

survival role. C/EBPδ and cleaved caspase-3 levels were not affected in MCF-

12A cells expressing vector alone (Fig. 4.7 B, lane 3).

108

Figure 4.7. Doxorubicin reduces C/EBPδ protein levels in mammary epithelial cells but C/EBPδ overexpression renders cells resistant to doxorubicin induced apoptosis. A: Western blot analysis of MCF-10A and MCF-12A mammary epithelial cells grown to confluence and cultured for an additional 24 hours in complete growth media (CGM) alone or in the presence of doxorubicin (Dox. 1 or 2 µM). Western blots were probed with antibodies to p53, C/EBPδ and cleaved caspase-3. B: Western blot analysis of MCF-12A cells grown to confluence and cultured for an additional 24 hours in CGM alone or in the presence of doxorubicin (2µM) (lanes 1 and 2). Lanes 3 and 4 represent MCF-12A cells nucleofected with either pcDNA3 vector or pcDNA3-C/EBPδ prior to doxorubicin treatment. Western blots were probed with antibodies to C/EBPδ and cleaved caspase-3.

109 Discussion

While a number of reports from our lab and others have identified

important functions for C/EBPδ in a variety of cell types, few downstream target genes have been identified. Work in M1 myeloid leukemia cells identified growth arrest and DNA damage inducible gene 45γ (gadd45γ) as an important C/EBPδ

downstream target gene (137). Gadd45γ is generally induced in response to DNA

damaging agents and cytokines such as IL-6 (196, 333). Jung and coworkers

demonstrated that growth arrest and differentiation in M1 cells was mediated by

C/EBPδ (as well as C/EBPβ) stimulation of gadd45γ expression (137). C/EBPδ

has also been shown to activate the peroxisome proliferator-activated receptor γ

(PPARγ) promoter in 3T3-L1 preadipocytes (73).

The goal of this study was to identify downstream effector genes regulated

in response to C/EBPδ expression. We first performed microarray analysis on

LNCaP cells overexpressing C/EBPδ and identified a number of interesting target

genes including beta 1 integrin, FOXO3A, Mcl-1 and beta 1 arrestin (Table 4.1).

Beta 1C integrin has been identified as a potent inhibitor of cell cycle progression

in a variety of cell types including prostate cancer cells (79, 180, 181). FOXO3A

is a member of the forkhead family of transcription factors which play critical roles

in cell cycle and death regulation (27). Previous studies have demonstrated that

FOXO3A plays an important role in mediating cell cycle arrest in endothelial cells

in response to homocysteine (332). In addition, induced expression of FOXO3A

has also been shown to result in G0/G1 cell cycle arrest in megakaryocytes and

NIH3T3 cells (179, 282). Mcl-1 is a well characterized, anti-apoptotic member of 110 the Bcl-2 family of proteins (182). Beta 1 arrestin has also been implicated in the inhibition of apoptosis. Povsic and colleagues demonstrated that beta 1 arrestin mediates PI-3 kinase activation resulting in activation of Akt and inhibition of apoptosis (224). Taken together, our microarray results suggest that, in addition to regulating genes involved in growth arrest, C/EBPδ may also function in the regulation of genes involved in cell survival.

To complement our microarray data we utilized the novel ChIP on Chip technique to identify gene promoters bound specifically by C/EBPδ. We identified several additional novel C/EBPδ target genes including: inhibitor of growth family member 4 (ING4), retinoblastoma binding protein 8 (RBBP-8), BTG (B cell translocation gene) family member 2 (BTG2), thioredoxin 2 (TRX2) and minichromosome maintenance deficient 7 (MCM7). ING4 is a newly characterized, candidate tumor suppressor gene which has been suggested to function as a repressor of angiogenesis and tumor growth (86, 143). Previous studies have demonstrated that ING4 can directly bind to p53 and modulate p53 function (253, 334). In addition, a recent report indicates that ING4 plays a critical role in the regulation of chromatin acetylation (68). RBBP-8 (also known as CTIP) has been implicated as a key regulator in cell cycle checkpoint control and has also been shown to directly interact with BRCA1 in a number of studies (305,

329). BTG2 is a p53 transcriptional target gene with strong anti-proliferative effects (239). In rat PC12 cells, BTG2 expression has been shown to induce growth arrest and differentiation and is also required for the survival of terminally differentiated cells (72). In addition, inactivation of BTG2 in embryonic stem cells

111 results in apoptosis in response to DNA damage because of a defect in growth arrest signaling, further suggesting that BTG2 promotes growth arrest and may

inhibit apoptosis (239). Interestingly, BTG2 has also been shown to be down-

regulated in advanced prostate cancer (74). Overexpression of BTG2 in PC-3

prostate cancer cells results in growth arrest and decreased tumorigenicity of

these cells in vivo, indicating BTG2 may be an important inhibitor of prostate

cancer cell growth (74). TRX2 has been shown to play an important role in the

regulation of mitochondria-dependent apoptosis (201, 284, 312). TRX2 knockout

mice are early embryonic lethal, a result of massive apoptosis and exencephaly

(201). A recent report by Wang et. al. suggests that TRX2 may inhibit apoptosis

by regulating bcl-xl levels (312). MCM7 is a DNA replication protein that plays a

critical role in the initiation of DNA replication (197). It is of interest that C/EBPδ

would bind a gene promoter involved in inducing proliferation. It is possible that

C/EBPδ functions as a transcriptional repressor of MCM7 as previous reports

have demonstrated that C/EBPβ can function in such a manner (242, 331).

Collectively, the microarray and ChIP on Chip analyses identified several

previously unidentified C/EBPδ downstream target genes which have been

shown to function in growth regulation. The identification of these target genes

extends previous investigations which have demonstrated an important function

for C/EBPδ in growth arrest by providing insights into the mechanisms of C/EBPδ

function during G0 growth arrest.

112 It was of interest that both the microarray and ChIP on Chip experiments

identified C/EBPδ downstream target genes which have been shown to function in survival. To our knowledge, the function of C/EBPδ in survival has not been

investigated, although previous studies have documented an important role for

C/EBPβ in survival in a variety of cell types (131, 162, 270, 319, 338). This led us

to investigate the potential pro-survival function of C/EBPδ in prostate cancer

cells. Work in our lab has shown that IL-6 treatment induced STAT3 activation,

C/EBPδ expression and growth arrest in LNCaP cells (241). We hypothesized

that under these conditions, LNCaP cells would be resistant to doxorubicin

induced apoptosis since doxorubicin targets dividing cells. LNCaP cells

pretreated with IL-6 were resistant to doxorubicin induced apoptosis compared to

untreated control cells (Fig. 4.3). Interestingly, doxorubicin increased C/EBPδ

protein levels independent of STAT3 activation (Fig. 4.4). This finding was

unique, as previous work in our lab had shown that STAT3 activation was

required for the induction of C/EBPδ under G0 growth arrest conditions (122, 123,

241, 258, 259). Subsequent analysis demonstrated that doxorubicin did not affect

C/EBPδ mRNA levels, indicating that doxorubicin increased C/EBPδ levels in a

transcriptionally independent manner (Fig. 4.5 A). Thus, it is possible that

C/EBPδ protein levels increased following doxorubicin treatment as the result of

increased translation of the C/EBPδ mRNA and/or stabilization of the C/EBPδ

protein. We hypothesize that doxorubicin regulates C/EBPδ expression by

protein stabilization. Previous reports from our lab have demonstrated that the

113 C/EBPδ protein has a relatively short half-life (~120 min.) (63, 259). In addition, work from other labs has demonstrated that doxorubicin regulates p300 protein stability by regulating the activation of the p38 MAPK pathway (221). It is possible that doxorubicin functions in a similar manner to regulate C/EBPδ levels.

Current investigations in the lab are focused on the mechanism whereby doxorubicin regulates C/EBPδ expression.

The observation that C/EBPδ levels increase in response to doxorubicin is significant and suggests that C/EBPδ plays an important role in apoptosis.

Reduction of C/EBPδ protein levels increased doxorubicin induced apoptosis suggesting that C/EBPδ functioned as an anti-apoptotic gene (Fig. 4.6). Despite this observation, the mechanism whereby C/EBPδ protects LNCaP cells from doxorubicin induced apoptosis is not completely clear. C/EBPδ expression may induce growth arrest, rendering LNCaP cells resistant to doxorubicin induced apoptosis. Following this hypothesis, siRNA mediated C/EBPδ knockdown would result in the inability of LNCaP cells to growth arrest in response to doxorubicin addition, rendering them more sensitive to doxorubicin. While we cannot rule this possibility out at this time, our microarray and ChIP on Chip data suggest that

C/EBPδ can directly regulate the expression of a variety or pro-survival genes

(Table 4.1). The pro-survival function of C/EBPδ may be independent of its growth arrest function. Interestingly, siRNA mediated knockdown of C/EBPδ reduced expression of the anti-apoptotic protein bcl-xl, suggesting that C/EBPδ regulates bcl-xl gene expression (Fig. 4.6 B). Initial promoter analysis has

114 identified two potential C/EBP binding sites within 1 KB of the bcl-xl transcription start site. In addition, previous studies in LNCaP cells demonstrated that IL-6 induced STAT3 activation increases bcl-xl expression (154). Work from our lab demonstrated that IL-6 induced STAT3 activation induces C/EBPδ expression in

LNCaP cells (241). Thus, activated STAT3 may up regulate bcl-xl expression by inducing C/EBPδ. It is also possible that C/EBPδ regulates bcl-xl expression indirectly by inducing thioredoxin 2 (TRX2) expression. As previously mentioned,

TRX2 has been shown to inhibit apoptosis by inducing bcl-xl expression (312).

These results suggest that C/EBPδ may play an important role in protecting prostate cancer cells from apoptosis by inducing the expression of pro-survival genes.

In contrast to LNCaP cells, doxorubicin treatment of mammary epithelial cells reduced C/EBPδ levels in a dose dependent manner (Fig. 4.7 A). It is possible that the expression of C/EBPδ in MCF-12A cells may be regulated similar to the expression of survivin in LNCaP cells. In the presence of doxorubicin, pro-survival gene expression must be down regulated in order for

MCF-12A cells to undergo apoptosis. Consistent with this hypothesis, overexpression of C/EBPδ inhibited doxorubicin induced apoptosis (Fig. 4.7 B).

In conclusion, this study identified a number of previously uncharacterized

C/EBPδ downstream target genes involved in the regulation of growth arrest. In addition, we identified a novel mechanism of C/EBPδ regulation in response to doxorubicin treatment and demonstrated that C/EBPδ may play a significant role

115 in cell survival. Ongoing studies are investigating the mechanism of C/EBPδ regulation in response to doxorubicin and the further characterization of the role of C/EBPδ in survival.

116 CHAPTER 5

PERSPECTIVES AND FUTURE WORK

The IL-6/STAT3/C/EBPδ growth arrest pathway and prostate cancer

At the time of these studies, the ability of IL-6 to induce growth arrest and

STAT3 activation in LNCaP cells had been described, however, no growth arrest

specific downstream effector genes had been identified (Fig. 5.1). We identified

C/EBPδ as a STAT3 target gene induced in response to IL-6 mediated growth

arrest. C/EBPδ expression is rapidly and persistently induced in LNCaP cells in

response to IL-6 (Fig. 5.1). Characterization of the IL-6/STAT3/C/EBPδ growth

arrest pathway in various prostate cancer cell lines demonstrated that defects in

this pathway rendered cells resistant to IL-6 induced growth arrest. Subsequent

studies utilizing IL-6 overexpressing LNCaP cells demonstrated that cells

exposed to IL-6 for prolonged periods of time constitutively expressed activated

STAT3 and C/EBPδ but were no longer growth inhibited, further suggesting that

the loss of this growth arrest pathway may play an important role in prostate

cancer progression. It is unclear how prostate cancer cells lose the ability to growth arrest in response to IL-6. It is possible that prolonged exposure to IL-6 results in the non-specific activation of other cellular signaling pathways.

Previous studies have demonstrated that constitutive activation of the PI-3 117 kinase/Akt pathway mediates STAT3 transcriptional activity of growth promoting

rather than growth inhibitory genes (10). Future experiments will examine the

effects of prolonged IL-6 exposure on intracellular signaling pathways. In addition, the identification of the defects that have occurred in the IL-

6/STAT3/C/EBPδ pathway in the various prostate cancer cell lines studied in this dissertation may provide further insights into the role of this signaling pathway in prostate cancer progression.

Figure 5.1. Summary of dissertation studies. Pathways depicted in gray represent previously known pathways. Newly identified findings from this dissertation are depicted in black.

118 It is of interest that serum and growth factor withdrawal induced growth

arrest in a STAT3/C/EBPδ independent manner in prostate cancer cells.

Previous work from our lab identified C/EBPδ as a growth arrest specific gene induced in response to serum and growth factor withdrawal or contact inhibition in human and mouse mammary epithelial cells (204-206, 258). This suggests that activation of the STAT3/C/EBPδ growth arrest pathway may be specific to IL-

6 family cytokine treatment in prostate cancer cell lines. It is also possible that serum and growth factor withdrawal results in G1 rather than G0 growth arrest in

prostate cancer cells lines. Recently our lab has utilized a novel pyronin Y

staining technique which allows us to distinguish G0 from G1 arrest (257). This

technique may be employed in LNCaP cells to determine the phase in which

cells growth arrest in response to serum and growth factor withdrawal. In

addition, future studies may examine the ability of other IL-6 family cytokines to

induce growth arrest in prostate cancer cells.

Prostate cancer metastasis to bone

Having established a role for the IL-6/STAT3/C/EBPδ growth arrest

pathway in prostate cancer cells, we wanted to determine if this pathway played

a role in prostate cancer metastasis to bone. Previous studies have

demonstrated that prostate cancer frequently metastasizes to bone and the

proliferative growth fraction of prostate cancer bone metastases is significantly

reduced (Fig. 5.1)(12, 23, 48, 229). Our results demonstrated that the

STAT3/C/EBPδ growth arrest pathway is activated in response to osteoblast

derived IL-6, suggesting that this pathway may play an important role in the 119 reduced growth rates observed clinically in prostate cancer metastases (Fig. 5.1).

Interestingly, previous studies demonstrated that prostate cancer cell lines harboring defects in the STAT3/C/EBPδ signaling pathway (Du145, PC-3) develop bone metastases more effectively than LNCaP cells (76). In addition, work by Culig and coworkers has demonstrated that LNCaP cells generated after prolonged exposure to IL-6 have increased tumor volumes compared to control cells (268). These data suggest that the STAT3/C/EBPδ growth arrest pathway may play an important role in prostate cancer metastasis to bone. Future studies will utilize the Xenogen IVIS Light Imaging System to investigate the specific function of STAT3 and C/EBPδ in prostate cancer metastasis to bone. LNCaP cells stably expressing STAT3 and C/EBPδ siRNA constructs will be injected into nude mice and their ability to metastasize to bone compared to parental LNCaP cells using the IVIS system.

The function of C/EBPδ in prostate cancer growth arrest

Our initial studies demonstrated that C/EBPδ overexpression suppressed prostate cancer cell growth in three different prostate cancer cell lines. Further studies utilized microarray and ChIP on Chip analyses to identify C/EBPδ downstream target genes. To our knowledge, our analyses are the first to investigate the function of C/EBPδ on a genome wide scale. We identified several candidate C/EBPδ downstream target genes which have been reported to function in growth arrest. RT-PCR, northern blot and chromatin immunoprecipitations must be performed on an individual gene basis to further

120 verify the importance of these genes as C/EBPδ targets. In addition, further

studies are required to investigate the induction of these candidate genes in

other cell lines (prostate and mammary) in response to C/EBPδ overexpression.

Future experiments will also investigate the potential interactions between

C/EBPδ and key cell cycle regulatory proteins and the significance of these

interactions in prostate cancer cell growth inhibition.

Doxorubicin induction of C/EBPδ protein levels

Investigation into the survival function of C/EBPδ identified a novel

mechanism of C/EBPδ regulation by doxorubicin. Our studies demonstrated that doxorubicin increased C/EBPδ protein levels in a trancriptionally independent manner in prostate cancer cells. This observation provides insight into a potentially novel mechanism of C/EBPδ regulation in response to apoptotic stresses in prostate cancer cells. Future studies will investigate the mechanism of this regulation. In addition, the induction of C/EBPδ in response to other apoptotic stresses including hypoxia and UV irradiation will also be examined in prostate cancer cells. Interestingly, we demonstrated that doxorubicin downregulated C/EBPδ expression in mammary epithelial cells, suggesting that the mechanism of C/EBPδ regulation in response to doxorubicin may be cell type specific.

121 The novel function of C/EBPδ in survival

Previous studies have demonstrated that both C/EBPα and C/EBPβ play important pro-survival roles in various cell types (24, 25, 131, 270, 319, 324,

338). Our studies are the first to describe a pro-survival function for C/EBPδ. In our initial screen we identified bcl-xl as a potential downstream target of C/EBPδ, as knockdown of C/EBPδ levels reduced bcl-xl expression. Current studies in our lab have also demonstrated that C/EBPδ can bind to the bcl-xl promoter in human mammary epithelial cells and induce gene expression. Further analyses will examine the potential role of C/EBPδ in regulating bcl-xl expression. In addition, we will also investigate additional pro-survival downstream target genes upregulated in response to C/EBPδ overexpression. The ability of C/EBPδ to induce growth arrest as a potential mechanism to evade doxorubicin induced apoptosis will also be assessed.

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