Defining Anti-Tumorigenic and Anti-Inflammatory

DEFINING ANTI-TUMORIGENIC AND ANTI-INFLAMMATORY

EFFECTS MEDIATED BY ESTROGEN RECEPTOR BETA IN

COLON EPITHELIAL CELLS

------

A Dissertation Presented to

the Faculty of the Department of Biology and Biochemistry

University of Houston

------

In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy

------

Trang Vu

March 2015 DEFINING ANTI-TUMORIGENIC AND ANTI-INFLAMMATORY

EFFECTS MEDIATED BY ESTROGEN RECEPTOR BETA IN

COLON EPITHELIAL CELLS

Trang Vu

APPROVED:

Dr. Cecilia Williams, Chairman

Dr. Chin-Yo Lin

Dr. Preethi Gunaratne

Dr. Mong-Hong Lee, The University of Texas MD Anderson Cancer Center

Dean, College of Natural Sciences and Mathematics

DEFINING ANTI-TUMORIGENIC AND ANTI-INFLAMMATORY

EFFECTS MEDIATED BY ESTROGEN RECEPTOR BETA IN

COLON EPITHELIAL CELLS

------

A Dissertation Presented to

the Faculty of the Department of Biology and Biochemistry

University of Houston

------

In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy

------

Trang Vu

March 2015

iii

Abstract

Despite its slow development and our capacity for early detection using endoscopy, colorectal cancer remains the second leading cause of cancer death in the United

States. Epidemiological studies indicate a role for estrogen in protecting against colorectal cancer. Estrogen receptor β (ERβ) has been found to be the main ER in the colon. Cell line studies have implicated that ERβ has anti-tumorigenic and antiproliferative effects on colon cancer cells lines while human epidemiological data suggest that polymorphisms in the ERβ gene are associated with greater cancer risk and lower survival.

Overexpression of ERβ changes the miRnome. Here we show that ERβ can alleviate the progression of colon cancer by inhibiting proliferation through decreasing MYC transcription and therefore decreasing levels of miR-17-92 (OncomiR-1). This is reflected in a decrease in cell number, decrease in migration, and increase in apoptosis.

Furthermore, addition of miR-17 using miRNA mimics reverses the effects of ERβ. This demonstrates that ERβ influences not only the transcriptome, but also the miRnome and that the miRnome can be potential targets for novel treatment approaches.

ERβ upregulates miR-205 and downregulates PROX1. This study uncovers the pathway in which ERβ downregulates the transcription factor PROX1 by upregulating miR-205 directly. MiR-205 then targets PROX1 mRNA for degradation. This results in the cells

iv adopting a more adhesive phenotype and reduces the metastatic potential. This study uncovers the potential for ERβ to be anti-metastatic in addition to being anti-proliferative and anti-inflammatory.

ERβ modulates the NFkB inflammatory cascade. One of ERβ’s attributes is that it has anti-inflammatory capabilities. Using a whole genome approach, this study reveals that

ERβ can modulate the NFkB inflammatory network. In SW480 cells, ERβ downregulates

NFkB transcription factors and induces apoptosis. In HT29 cells, ERβ prevents p65 subunit of NFkB from translocating to the nucleus. Overall, ERβ attenuates the NFkB signaling cascade by attenuating genes related to inflammation while enhancing genes related to apoptosis and cellular defenses. This makes ERβ a useful target for anti- inflammatory drug treatments for patients suffering from chronic inflammatory diseases.

v Table of Contents 1. Introduction ...... 1 1.1 Colon cancer ...... 1 1.2 Development of colon cancer ...... 2 1.3 Chronic inflammation and colon cancer ...... 5 1.3.1 Tumor necrosis factor α ...... 6 1.3.2 NFkB pathway ...... 7 1.4 The role of estrogen receptor β in colon cancer ...... 9 1.5 The role of micro-RNAs in colon cancer ...... 11 2. Materials and methods ...... 13 2.1 Cell culture ...... 13 2.2 RNA extraction ...... 13 2.3 Complementary DNA synthesis of mRNA and miRNA populations and quantitative real-time-PCR ...... 14 2.4 Transfection with ERβ plasmids, luciferase plasmids, siRNAs, miRNA mimics and miRNA inhibitors ...... 15 2.5 Estradiol treatments ...... 16 2.6 Western blot analysis ...... 16 2.7 Luciferase assay ...... 16 2.8 Generation of tissue-specific knockout mice and collection of samples ...... 17 3. Chapter 1: ERβ expression induces changes in the microRNA pool in human colon cancer cell lines ...... 18 3.1 Introduction ...... 18 3.2 Supplemental materials and methods ...... 19 3.2.1 Receptor quantification ...... 19 3.2.2 Cell counting and viability assays ...... 20 3.2.3 qPCR primers ...... 20 3.2.4 Immunoblot ...... 21 3.2.5 Migration wound-healing assay ...... 21 3.2.6 Apoptosis assay ...... 22 3.3 Results and Discussion ...... 22 3.3.1 Exogenously expressed ERβ are within physiological ranges ...... 22 3.3.2 ERβ affects miRNAs through repression of MYC ...... 24 3.3.3 MiR-17 re-expression can override the ERβ-mediated reduction of proliferation ...... 26 3.3.4 ERβ-mediated repression of miR-17 can enhance apoptosis ...... 28 3.3.5 MiR-17 mediates the upregulation of NCOA3 and CLU after ERβ expression ...... 29 3.3.6 ERβ modulates miR-200a/b and epithelial-mesenchymal transition markers 32 3.3.7 Cell-specific effects of ERβ ...... 34 3.1.4 Conclusion ...... 35 4. Chapter 2: ERβ decreases metastatic potential of colon cancer cells through the upregulation of miR-205 and its direct targeting of PROX1 ...... 37 4.1 Introduction ...... 37 4.2 Supplemental materials and methods ...... 38

vi 4.2.1 The Cancer Genome Atlas clinical data ...... 38 4.2.2 Immunoblot ...... 39 4.2.3 qPCR primers ...... 39 4.2.4 Generation of tissue-specific knockout mice and collection of samples ...... 40 4.2.5 Correlation analyses ...... 41 4.2.6 Cell adhesion ...... 41 4.2.7 Zebra fish migration assay ...... 41 4.3 Results and Discussion ...... 42 4.3.1 Expression of ERβ and miR-205 are anti-correlated with expression of PROX1 in both primary colon and tumor cell lines ...... 42 4.3.2 Tissue-specific knockout of ERβ decreases miR-205 and increases PROX1 in the colon of mice ...... 44 4.3.3 ERβ upregulates miR-205 expression in different colon cancer cell lines ...... 46 4.3.4 MiR-205 represses PROX1 expression in colon cancer cells ...... 49 4.3.5 Downregulation of PROX1 by miR-205 regulates cell adhesion and metastasis in vitro and in vivo ...... 50 4.4 Conclusion ...... 52 5. Chapter 3: ERβ mediates anti-inflammatory effects via modulation of the NFκB networks ...... 53 5.1 Introduction ...... 53 5.2 Supplementary material and methods ...... 55 5.2.1 Microarray and data analysis and mining ...... 55 5.2.2 Immunoblotting ...... 55 5.2.3 qPCR primers ...... 56 5.3 Results and Discussion ...... 57 5.3.1 ERβ modulates NFkB RELA(p65) nuclear translocation in colon cancer cell lines ...... 57 5.3.2 ERβ inhibits TNFα activation of NFκB-response element in SW480 cells ...... 60 5.3.3 TNFα induces genome-wide transcription in colon cancer cell lines ...... 62 5.3.4 TNFα and ERβ induces apoptosis in SW480 cells ...... 65 5.3.5 ERβ modulates NFkB transcription factors in SW480 colon cancer cells ...... 67 5.3.6 ERβ modulates genome-wide transcription in colon cancer cell lines ...... 71 5.3.7 ERβ modulation on genome-wide transcription at 24 hours ...... 78 5.4 Conclusion ...... 80 6. Concluding remarks and future directions ...... 83 7. Bibliography ...... 88

vii List of manuscripts

Included in this dissertation

K. Edvardsson*, T. Nguyen-Vu*, Ström, A., Gustafsson J-Å, and Cecilia Williams. Estrogen receptor beta expression induces changes in the microRNA pool in human colon cancer cells. Carcinogenesis. 2013 Jul; 34(7): 1431-41. *Equal contributors

Nguyen-Vu, T., Wang, J., Mesmar, F., Mukhopadhyay, S., Saxena, A., Gustafsson, J-Å, Bondesson, M., and Cecilia Williams. ERβ decrease metastatic potential of colon cancer cells through the upregulation of miR-205 and its direct targeting of PROX1. Manuscript in preparation.

Nguyen-Vu, T., Saxena, A., and Cecilia Williams. ERβ mediates anti-inflammatory effects via modulation of the NFkB networks. Manuscript in preparation.

Other publications

T. Nguyen-Vu, L.-L. Vedin, Jonsson, P., Lin, J. Z., Candelaria, L. P., Addanki, S., Williams, C., Gustafsson, J.- Å., Steffensen, K. R., and Chin-Yo Lin. Liver X receptor ligands inhibit breast cancer cell proliferation through an E2F-mediated mechanism. Breast Cancer Res. 2013 Jun 28; 15(3): R51.

Katchy, A., Pinto, C., Jonsson, P., Nguyen-Vu, T., Pandelova, M., Schramm, K.-W., Gustafsson J-Å, Bondesson, M., and Cecilia Williams. Co-exposure to Phytoestrogens and Bisphenol A mimic estrogenic effects in a sub-additive manner. Toxicol Sci. 2014 Mar;138(1):21-35.

Emma H. Wall, Hewitt, S. C. Case, L. K., Nguyen-Vu, T., Korach, K. S., Teuscher, C., and Chin-Yo Lin. 2014. Genetic Control Of Ductal Morphology, Eastrogen-Induced Outgrowth, and Gene Expression In Mouse Mammary Gland. Endocrinology. 2014 Aug;155(8):3025-35.

Nicholes Candelaria, Sridevi Addanki, Nguyen-Vu, T., and Chin-Yo Lin. 2014. Anti- proliferative Effects and Mechanisms of Liver X Receptor Ligands in Pancreatic Ductal Adenocarcinoma Cells. PLoS One. 2014 Sep 3;9(9).

R. L. Smith, K. Liu, Candelaria, N. R., Nguyen-Vu, T., Rogerson, C. M., Johnson, W. E., Cheung, E., Bajic, V. B., and Chin-Yo Lin. 2014. RUNX1 is involved in estrogen receptor functions and breast cancer cell proliferation. (Submitted).

Frank Bearoff, Laure K. Case, Trang Nguyen-Vu, Emma H. Wall, Julie A. Dragon, Naresha Saligrama, Roxana del Rio, Chin-Yo Line, Jiri Forejt, Elizabeth P. Blankenhorn, and Cory Teuscher. QTL Analysis of Myelin Oligodendrocyte Glycoprotein-Induced Expreimental Allergic Encephalomyelitis Using B6/PWD Consomic Strains of Mus musculus. 2014. Manuscript in progress.

viii

1. Introduction

1.1 Colon cancer

Colon cancer is defined as cancer that originates in the colon. Colon cancer is often grouped together with colorectal cancer (CRC), a term that describes both colonic and rectal cancers depending on the origin of the location of the cancer (1). Despite a slow development and our capacity for early detection using endoscopy, colorectal cancer remains the second leading cause of cancer death in the United States. The American

Cancer Society estimates that in 2015, there will be 93,090 new cases of colon cancer and about 49,700 estimated deaths (1). Worldwide, in 2012, there were over 1.4 million cases of colorectal cancer of which 54% of these cancers occurred in developed countries. Colorectal cancer is also projected to affect 2.4 million people worldwide by

2035 (2).

Risk factors for developing colon cancer include, but are not limited to: diet, physical activity, alcohol, smoking, age, obesity, personal history of inflammatory bowel disease, and family history of colorectal cancer or adenomatous polyps (1). While current preventative approaches of removing polyps, and long-term aspirin treatment is used, the efficacy is low and a truly preventative or targeted therapy against early stages of colon cancer remains to be developed.

1.2 Development of colon cancer

The development of colon cancer is a slow process that involves accumulations of small genetic and epigenetic alterations which changes the normal colonic mucosal environment and eventually leads to carcinoma (3,4). Unlike other cell types in the body, the colonic mucosa is constantly being formed and broken down. The colonic epithelial cells originate from stem cells at the base of the crypt and as they migrate towards the villi, they differentiate into mature cells. As cells grow from the crypt out towards the villus in this maturation process, mutations can accumulate and instead of eventually undergoing apoptosis and sloughing off in the intestinal lumen, these cells further proliferate resulting in colon polyps or adenomas. Adenomas are not cancerous, but further accumulation of mutations and recruitment of inflammatory factors can encourage the microenvironment to become primed for tumorigenesis. Even though genetic mutations are the cause of colon cancer, non-genetic factors can also exacerbate the disease. Chronic inflammation can promote colorectal tumor growth by allowing these mutations to further accumulate and promote carcinogenesis.

2 For diagnostic purposes, colon cancer is separated into two categories, hereditary and non-hereditary or sporadic colon cancer. Hereditary colon cancer accounts for 5% of overall cancer cases (5) suggesting that lifestyle and environmental factors have a greater influence on colon cancer development. Hereditary colorectal cancers can include syndromes such as Hereditary Non-Polyposis Colon Cancer (HNPCC), Familial

Adenomatous Polyposis (FAP), and MYH-Associated Polyposis (MAP). These arise from mutations in the chromosome instability pathway (CIS), K-ras, BRAF/V600E, adenomatous polyposis coli (APC) mutation, and/or p53 mutations. Patients suffering from hereditary colon cancers are usually diagnosed earlier life compared to those that suffer from sporadic colon cancer which is usually found in patients older than 50 years of age (6). Even though hereditary colon cancer is more rare than sporadic colon cancer, there is no correlation between family history and survival of patients with hereditary colon cancers (7).

One of the most well-known and well-studied forms of hereditary colon cancer is Familial

Adenomatous Polyposis (FAP). FAP arises when there is a mutation in the

Adenomatous polyposis coli (APC) gene. APC is a tumor suppressor gene that is also known as the “gatekeeper gene” in colon carcinogenesis (8-11). There can be multiple mutations in the APC gene which leads to differences in severity of FAP in colon cancer patients (12). Adding to the complexity of APC is that certain mutations in the APC gene are inherited, while other mutations can be sporadic. It is the latter that is responsible for the non-inherited colon cancer cases. APC regulates the breakdown of β-catenin in the canonical Wnt/β-catenin signaling pathway. Canonical Wnt signaling pathway plays an important role in the regulation of cell proliferation, angiogenesis and apoptosis, but

3 more importantly, in the case of colon cancer, Wnt/β-catenin maintains the intestinal homeostasis (13). Wnt pathway is a signal transducer. In the absence of an activating ligand, β-catenin is phosphorylated and targeted for degradation. When an activating ligand is present, it can bind to the membrane receptor Frizzled. Frizzled in turn activates the cytosplasmic protein Disheveled. Disheveled then blocks the action of the

β-catenin degradation complex. APC is one of the components that can bind to β- catenin and target it for degradation. When the APC gene is mutated, APC can no longer bind to β-catenin and target the complex for degradation (Figure 1.1).

Accumulation of β-catenin leads to activation of genes in the Wnt signaling pathway.

Thus, loss of APC function can lead to initiation and progression of colon cancer via

Wnt/β-catenin signaling pathway.

Figure 1.1. Wnt/β-catenin signaling cascade. Mutated APC cannot bind β-catenin and target the complex for degradation resulting in β-catenin accumulation in the cytoplasm, which then translocates to the nucleus to form transcription factor complexes that turn on genes in the Wnt signaling pathway.

4 Unlike hereditary colon cancer development, for sporadic colon cancer, age is the most important risk factor for developing the disease (6,14). As mentioned, certain APC mutations are not inherited, thus APC also plays an important role in the development of sporadic colon cancer cases. Fearon and Vogelstein described a suppressor pathway in the development of colon cancer (3). In this canonical suppressor pathway, there is accumulation of mutations in the classical oncogenes and tumor suppressor genes such as K-ras, APC, or p53. In fact, in 70% of cases of sporadic colon cancers, APC gene is mutated (15,16) causing cells to avoid apoptosis therefore creating an opportunity for mutations to further accumulate.

1.3 Chronic inflammation and colon cancer

As mentioned, one of the risk factors for developing colon cancer is personal history of inflammatory bowel disease (IBD). Inflammatory bowel disease, which includes ulcerative colitis (UC) and Crohn’s disease (CD), describes the condition in which the colon area is in a constant state of inflammation. Thus, another major risk factor for development of colon cancer is inflammation. Inflammation itself is helpful, but chronic inflammation is one of the hallmarks of tumor growth and promotion, and correlates with poor prognosis in many different types of cancer including breast (17) and colon cancer

(18-20). Acute inflammation in response to infection or injury activates immune cells to recruit cytokines, chemokines, and additional immune cells to eradicate foreign pathogens while promoting healing and regrowth of the infected area. Thus acute inflammation involves apoptotic as well as proliferative processes. Prolonged inflammation can cause over-activation of immune cells and therefore opportunistic pathogens can invade and damage the gut environment (21). Prolonged inflammation

5 can cause cellular mutations to occur that interfere with the balance of apoptosis and proliferation that occurs in acute inflammation. Cells in these chronically inflamed sites have usually evaded apoptosis and are proliferative. Inflammation affects all stages of colon cancer development from initiation to progression and finally metastasis. Thus, controlling or minimizing chronic inflammation can be the key to reducing colon cancer development (22). In fact, long-term use of aspirin has been shown to reduce colon cancer risk (23) and mortality (24).

1.3.1 Tumor necrosis factor α

An important player in inflammation is the cytokine tumor-necrosis factor α (TNFα).

TNFα is a proinflammatory cytokine produced by immune as well as tumor cells. During chronic inflammation, TNFα was found to be dysregulated. TNFα thus can mediate tumor progression through proliferation, invasion, and metastasis of cells. In estrogen receptor positive breast cancer cells, TNFα was found to be regulated in a positive feedback loop with TNFα in which estradiol via estrogen receptor α (ERα), kept levels of

TNFα high in the tumor microenvironment (25). In colon cancer, TNFα is responsible for activation of the NOXO1 gene which adds to a more inflammatory environment causing many of the symptoms of inflammatory bowel and Crohn’s diseases (26). TNFα works through NFkB transcription family of transcription factors. TNFα can bind via two receptors, TNFR1 and TNFR2. Once the cytokine binds its receptor, a conformation change occurs in the receptor, releasing the inhibitory subunits and activating subunits there can bind and initiate activation of NFkB pathway, activation of MAPK pathway, or induction of apoptosis (27).

6 1.3.2 NFkB pathway

As mentioned, TNFα can activate NFkB pathway leading to cellular inflammatory responses. NFkB is a family of transcription factors that include two classes: I and II.

Class I NFkB transcription factors include NFkB1 and NFkB2, these are also known as p105 and p100 or the cleaved names p50 and p52, respectively. NFkB1 and NFkB2 subunits are synthesized as the precursor p105 and p100. Class I NFkB transcription factors are activated in the non-canonical NFkB pathway. These two subunits lack a transcription activator therefore they are part of an inhibitory NFkB transcription factor complex. The precursors can only be activated when they are bound to an NFkB subunit that has a transactivating domain and the subunits must be cleaved into their shorter, p50 and 52, forms. P50 and p52 can also bind to each other, forming a heterodimer, but because there is no transactivating domain, they must be bound to a third protein BCL3, which contains a transactivating domain, to be active (Figure 1.2, Pathway 3).

The second class of NFkB transcription factors consists of RELA (p65), RELB, and REL

(c-REL). These three subunits share a rel homology domain and contain a transactivating domain in their C-termini. Class II NFkB transcription factors are kept as dimers in the in the cytoplasm and are inhibited by inhibitor-kB subunits (IkB). Once

NFkB activation is initiated, the IkB subunits are phosphorylated, released and degraded by IkB kinases (IKKs) from the NFkB dimers and these translocate to the nucleus to activate transcription (28) (Figure 1.2).

7 Introduction to NF-jB TD Gilmore 6682 Canonical Pathway Non-canonical Pathway Pathway 3

NEM

NEM NEMO NIK IKK ? α β P P

P αα P p105 p50 Proteasomal l B degradation p105 RelA P processing p50 p100 P RelB p50 p100 p50 processing RelA p50 p52 RelB

Nucleus Nucleus Nucleus Bcl-3

B B B

Figure 2 NF-kB signal transduction pathways. In the canonical (or classical) NF-kB pathway, NF-kB dimers such as p50/RelA are maintained in the cytoplasm by interaction with an independent IkB molecule (often IkBa). In many cases, the binding of a ligand to a 1 cell surface receptor (e.g., tumor necrosis factor-receptor (TNF-R) or a Toll-like receptor)With recruits permission adaptors (e.g.,from TRAFs Nature and Publishing RIP) to Group the cytoplasmic domain of the receptor. In turn, these adaptors often recruit an IKK complex (containing the a and b catalytic subunits and two molecules of the regulatory scaffold NEMO) directly onto the cytoplasmic adaptors (e.g., by virtue of the K63-ubiquitin- binding activity of NEMO). This clustering of molecules at the receptor activates the IKK complex. IKK then phosphorylates IkB at two serine residues, which leads to its K48 ubiquitination and degradation by the proteasome. NF-kB then enters the nucleus to turn on target genes. The auto-regulatory aspect of the canonical pathway, wherein NF-kB activates expression of the IkBa gene that leads Figure 1.2to resequestration NFkB transcription of the complex in thefactors cytoplasm by theand newly pathways. synthesized IkB proteinCanonical is not shown. NFkB The non-canonical activation (or requires alternative) pathway is largely for activation of p100/RelB complexes during B- and T-cell organ development. This pathway differs from the canonical pathway in that only certain receptor signals (e.g., Lymphotoxin B (LTb), B-cell activating factor (BAFF), CD40) phosphorylationactivate thisof pathwayinhibiting and because-kB subunits, it proceeds through which an IKK releases complex that p50/RelA contains two transcription IKKa subunits (but factors not NEMO). that In the translocate non- the canonical pathway, receptor binding leads to activation of the NF-kB-inducing kinase NIK, which phosphorylates and activates an IKKa complex, which in turn phosphorylates two serine residues adjacent to the ankyrin repeat C-terminal IkB domain of p100, nucleus andleading activate to its partial transcription. proteolysis and liberation Non of-canonical the p52/RelB complex. pathway Other distinct requires NF-kB pathways cleavage no doubt and exist. maturation For example, of p100 in Pathway 3, p50 (or p52) homodimers enter the nucleus, where they become transcriptional activators by virtue of interaction with the IkB-like co-activator Bcl-3 (or IkBz). How Pathway 3 is regulated is not known. In all three pathways, various post-translational subunit. Pathwaymodiﬁcations 3 (e.g.,is cleavage phosphorylation, of p105 acetylation subunit and prolyl into isomerization) the shorter of the NF- isoform;kB subunits activation can modulate theirrequires transcriptional a third subunit activity (see Perkins, 2006, this issue). that has a transactivating subunit to turn on gene transcription. mechanisms of action and how some may prove signaling pathway, physiology and disease, therapy and important for the treatment of human diseases. simple model systems. Basic biochemistry Although we have learned a great deal about Future perspectives the intracellular NF-kB signaling pathway, we still have very rudimentary knowledge about the dynamics Even with the many advances since 1999, there continue of the NF-kB pathway in cells in tissue culture and know essentially nothing about these dynamics in whole to be several unsolved mysteries in the saga of NF-kB. If I use my crystal ball to predict what the next 7 years organisms. In most cell types and signaling conditions, have in store, I suspect that progress will be made in the it is still not known what is the contribution of following four general areas: basic biochemistry of the speciﬁc NF-kB complexes (e.g., p50-RelA vs p52-c-Rel vs c-Rel-c-Rel) to given physiological responses or how

Oncogene

1 Gilmore TD. Introduction to NF-κB: players, pathways, perspectives. Oncogene. 2006;25(51):6680–6684. doi:10.1038/sj.onc.1209954.

1.4 The role of estrogen receptor β in colon cancer

Epidemiological studies indicate a role for estrogen in protecting against colorectal cancer. Men are at a higher risk for colon cancer, exhibiting an earlier on-set than pre- menopausal women (29), and among patients with irritable bowel syndrome men are

60% more likely to develop colorectal disease than women (30). After menopause, cancer incidences for women are the same as for men (31), however, when placed on hormone replacement therapy, women’s colon cancer incidence decreases (32). Meta- analysis of 20 case-control cohort studies show that use of oral contraceptives is associated with a lower colon cancer incidence (33). In one case, administration of oral contraceptives to a patient with familial adenomatous polyposis (FAP) caused polyps to regress completely (34). Experimentally, 17β-estradiol (E2) has been demonstrated to reduce the formation of preneoplastic lesions in mice (35). Thus it appears that estrogen levels can play a critical role in the development and progression of colon cancer.

The effect of estrogen is mediated by the estrogen receptors (ERs). ERs are type I nuclear receptors that, like NFkB transcription factors, reside in the cytoplasm. Once activated by ER-binding ligands, the nuclear receptors dimerize and translocate into the nucleus to activate transcription. ERs bind to DNA at an estrogen-response-element

(ERE) upstream of genes and control that gene’s transcription (36,37). Depending on cofactors that bind on the ER-DNA complex, the binding can be activating or repressing in nature. ERs do not necessarily have to bind directly to their EREs on DNA. Another

9 mechanism of regulation of ERs is thru tethering to other transcription factors present on

DNA such as AP-1 (38,39) or RUNX1 (40) (Figure 1.3).

Figure 1.3. Mechanism of ER-DNA binding. ERs are activated by ER-binding ligands and dimerize. Once dimerization occurs, they translocate to the nucleus and can either activate or inhibit transcription depending on recruitment of coactivators or corepressors. ERs can bind directly to their estrogen response element

(ERE) or tether to another transcription factor to regulate gene transcription.

There are two subtypes, ERα and ERβ, that are encoded by two distinct genes. ERβ has been reported to have ligand-dependent and ligand-independent activities (41). That is,

ERβ can be activated, dimerized, and translocated to the nucleus all in the absence of ligand. While ERα mediates many of the negative effects of estrogen, e.g., proliferation of breast cancer cells, ERβ has been associated with more favorable effects in both men and women (36). Due to the differences in their ligand binding domains, ERα and ERβ can be differentially activated by selective estrogen receptor modulators (SERMs). ERβ is thus a potential target for compounds that are structurally similar to estrogen, but that do not activate ERα.

The transcriptional effect of ERα is well studied in breast cancer cells (42,43). In colon cancer, however, ERα is not widely expressed. The predominant ER in normal colonic epithelium is ERβ, as determined both at the protein (44) and messenger RNA (mRNA) levels (45). Immunohistochemistry has further shown decreased expression of ERβ during colon cancer progression (46). Polymorphism in the ERβ gene affects colon cancer risk and survival (47-49). In vivo animal studies have demonstrated that ERβ has been shown to be antiproliferative in both colon cancer cells (50-53) and in xenografts

(51). Genome-wide analysis of protein-coding genes allowed us to identify multiple ERβ- mediated anti-tumorigenic and anti-inflammatory effects of ERβ through regulations of microRNAs (miRNAs) and modulations of the NFκB inflammatory cascade. Thus ERβ plays an important role in colon carcinogenesis.

1.5 The role of micro-RNAs in colon cancer

MiRNAs are short (~22 nt), single-stranded non-coding RNA regulators. They regulate gene expression through complementary binding between the miRNA 5’-sequence and the 3’-untranslated region (3’-UTR) of the target mRNA. Such binding mediates degradation of the mRNA transcript and/or suppression of its translation. MiRNA genes are transcribed by RNA polymerase II into primary miRNAs. The nuclear protein DGCR8 and the enzyme Drosha introduce a cleavage to liberate a hairpin loop from the primary miRNAs. The resulting hairpin, precursor miRNA (pre-miRNA), is exported from the nucleus to the cytoplasm. The protein Dicer then cleaves the loop, leaving a short, double-stranded duplex. One strand of the duplex is released and often degraded, whereas the other strand, the mature miRNA, is incorporated into the Argonaute

11 containing miRNA-induced silencing complex, which facilitates the interaction between the mature miRNA and its mRNA target. A miRNA can target several different genes and many of these targets are often in the same pathway (54). As one mRNA can also be targeted by several miRNAs, the total impact of miRNAs on the transcriptome and proteome can be significant. Currently, there are almost 2,500 miRNA genes in the human genome (55) and together they may target and affect the expression of up to

60% of all protein-coding genes. MiRNAs have been linked to tumorigenesis due to their frequent localization at fragile sites and common breakpoint regions (56) and both oncomiRs and tumor suppressor miRNAs have been identified (57). Altered levels of miRNAs are common in tumors, including those of the colon (58-61). MiRNAs are also currently being used as a diagnostic marker in human cancers (62,63) as well as therapeutic targets in clinical trials.

In our current study, we propose ERβ as a crucial component in regulating the proliferative and inflammatory processes that occur during colon carcinogenesis. We will show three mechanisms of ERβ that include: 1) antiproliferation and pro-apoptotic mechanism through down regulation of MYC and thus downregulation of miR-17-92 miRNA cluster; 2) increased cellular adhesion and decreased metastasis through downregulation of PROX1 via upregulation of miR-205; and 3) attenuation of inflammatory response and increased apoptosis through modulation of NFkB transcription factor subunits and genome-wide transcriptomic changes.

2. Materials and methods

2.1 Cell culture

Cell lines were obtained from American Type Culture Collection (Manassas, VA). Full- length ERβ was stably expressed via lentivirus transduction in three mixed cell populations of SW480, HT29, and HCT116, with empty vectors as control, as described in (64). Stably transfected cells (SW480, HT29, and HCT116) were cultured in RPMI-

1640 (Invitrogen, Carlsbad, CA) with 5% fetal bovine serum (FBS) and 1% penicillin- streptomycin (PEST) (Invitrogen). SW403 cells were cultured in RMPI-1640 (Invitrogen) with 10% FBS and 1% PEST. SW620 cells were cultured in DMEM (Invitrogen) with

o 10% FBS and 1% PEST. All cells were kept at 37 C with 5% CO2.

2.2 RNA extraction

Total RNA was extracted with TRIzol (Invitrogen) and purified with miRNeasy spin columns (Qiagen, Chatsworth, CA), according to standard protocol. On-column DNA I

13 digestion was used to remove remaining genomic DNA. Quantitative and qualitative

RNA analyses were performed using NanoDrop 1000 spectrophotometer and the Agilent

2100 BioAnalyzer (Agilent techanologies, Palo Alto, CA), respectively. Samples with

RNA integrity > 9.5 were used.

2.3 Complementary DNA synthesis of mRNA and miRNA populations and quantitative real-time-PCR

For mRNA analysis, complementary DNA was synthesized from total RNA from either 1

µg (cell lines) or 500 ng (mouse tissue) using either Superscript III (Invitrogen) and random hexamer primers, as described in (41) or using IScript cDNA Synthesis Kit (Bio-

Rad) according to manufacturer’s protocol.

For miRNA analysis, poly(A) tails were added to 1 µg (cell lines) or 500 ng (mouse tissue) of total RNA with NCode miRNA First-Strand cDNA Synthesis Kit (Invitrogen) followed by complementary DNA synthesis using corresponding universal primer, according to manufacturer’s protocol.

For the real-time PCR reaction of miRNA population, a corresponding universal real-time reverse PCR primer from the kit and a forward primer based on the mature miRNA sequence were used. U6 was used a reference gene. For mRNA analysis, 18S or 36B4 were used as reference genes. Real-time-PCR reaction were done using either Fast

SYBR-Green Master mix (Applied Biosystems) or iTaq Universal SYBR Green Supermix

(Bio-Rad) according to conditions specified by the manufacturer. Real-time-PCR

14 reactions were performed on 7900HT Fast Real-Time PCR System (Applied

Biosystems). All prier pairs were checked with melting curve analysis. Relative expression was calculated as fold-change in relation to control using the ΔΔCt-method.

Unpaired two-tailed t-test was used to test significance between two parallel treatment groups of identical origin and considered significant if p < 0.05.

2.4 Transfection with ERβ plasmids, luciferase plasmids, siRNAs, miRNA mimics and miRNA inhibitors

FugeneHD (Promega, San Obispo, CA) was used for transient transfection of full-length

ERβ, ERβ-DNA-binding mutant (ERβ-DBD-mutant), or luciferase plasmids. Cells were cultured for 24 hours then plasmids were mixed with Opti-MEM (Life Technologies,

Grand Island, NY) and FugeneHD according to manufacturer’s protocol. All siRNAs, miRNAs mimics, and miRNA inhibitors, were purchased through Dharmacon (GE

Healthcare, Lafayette, CO). Transfections were performed using DharmaFECT reagents according to manufacturer’s instructions. In brief, cells were seeded in six-well or 12-well plates for 24 hours then transfected in antibiotic-free medium with DharmaFECT I (0.1% of total volume) and siRNAs, miRNA mimics, miRNA inhibitors, and appropriate controls.

Meidum was changed to complete medium 24 hours after transfection, and the cells were cultured for additional times before harvest depending on individual experiments.

All experiments were performed in triplicates.

15 2.5 Estradiol treatments

After cells were cultured for 24 hours, medium was changed to phenol-free medium with

5% dextran-coated-charcoal-treated (DCC) FBS and 1% PEST 24 hours before 10 nM estradiol (E2) treatments. For E2 treatments involving transfections, transfections were done as described above. After 24 hours transfections, cells were washed 3 times with phosphate buffered saline (PBS) and E2 were done as described.

2.6 Western blot analysis

Cells were lysed with RIPA lysis buffer. Protein concentrations were determined using

Qubit Protein Assay Kit (Invitrogen) along with the Qubit 2.0 Fluorometer (Invitrogen).

Approximately 50 or 100 µg of total protein were loaded onto a 10% polyacrylamide gel.

Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were transferred to either polyvinylidene difluoride (Millipore, Billerican, MA) or nitrocellulose membrane (Bio-Rad). The membrane was blocked with either 5 or 10% non-fat milk in Tris-buffered saline Tween-20 and then incubated with appropriate primary antibodies in Tris-buffered saline Tween-20 overnight. The membrane was then reprobed with appropriate secondary antibodies conjugated with horseradish peroxidase for 2 hours. Blots were processed using ECL kit (Thermo Fisher Scientific, Rockford, IL) and exposed to film.

2.7 Luciferase assay

Luciferase plasmids and appropriate plasmid controls were transfected as previously described. 24 hours after transfection, cells were washed with PBS and lysed using

16 Passive Lysis Buffer (Promega). If ligand was used as part of treatment, 24 hours after transfection of plasmid, cells were washed 3 times with PBS, changed to appropriate

10% DCC-FBS medium, treated with appropriate ligand for an additional 4 hours then lysed. Lysates were then mixed with reagents in Dual-Luciferase Reporter Assay

System (Promega) and read on [plate reader]. All experiments were done in triplicates and significance determined using unpaired two-tailed t-test. Significance determined by p < 0.05.

2.8 Generation of tissue-specific knockout mice and collection of samples

B6.129X1-Esr2tm1Gust mice were crossed over multiple generations with B6.SJL-

Tg(Vil-cre)997Gum/J mice (Jackson Laboratory, Bar Harbor, Maine) to generate intestinal-specific ERβ knockout mice (ERβiKO). Mice were genotyped using PCR with

DNA extracted from either an ear punch or tail clipping at 6 weeks of age. Animals were housed in controlled environment at 20oC with illumination schedule of 12 hours light, 12 hours dark. Standard pellet food and water were provided ad libitum. Animals and procedures performed on animals in this study were approved by Institutional Animal

Care and Use Committee at University of Houston, Houston, Texas. At 12-20 weeks of age, mice were sacrificed and samples were collected by scraping the epithelial cells of the colon and stored in Trizol reagent and RNA isolation, cDNA synthesis, and qPCR analyses were done as previously described.

3. Chapter 1: ERβ expression induces changes in the

2 microRNA pool in human colon cancer cell lines

3.1 Introduction

Previously, studies have described the effects of ERβ on the transcriptome in colon cancer cell lines (41), and that ERβ’s effect on the transcriptome leads to antitumorigenic and anti-proliferative effects in colon cancer cell lines. Here, we examine the genome-wide effects of ERβ on the miRnome of colon cancer cell lines. We hypothesize that ERβ in part mediates its anti-tumorigenic and anti-proliferative effect through miRNAs.

2 Work in this chapter has been published in: K. Edvardsson*, T. Nguyen-Vu*, Ström, A., Gustafsson J-Å, and Cecilia Williams. Estrogen receptor beta expression induces changes in the microRNA pool in human colon cancer cells. Carcinogenesis. 2013 Jul; 34(7): 1431-41. *Equal contributors.

18 A key component of cellular control and processes is the interplay between miRNAs and nuclear receptors (65-72). Estrogen via the nuclear receptor ERα, has been implicated to have an effect on miRNAs in breast cancer cells (73-76). More recently, ERβ was also indicated to affect miRNAs in breast cancer (77). Since then, there have been few studies examining the effects of ERβ via miRNAs in the colon. In this study, we examined changes in the miRNA pool and subsequent downstream effects of these changes in cells where ERβ has been overexpressed (41,64). In order to estimate the impact of ERβ on the miRnome, we have performed focused functional studies of selected miRNAs repressed by ERβ.

3.2 Supplemental materials and methods

3.2.1 Receptor quantification

Receptor expression was quantified using radiolabeled ligand-binding assay. Cells were plated at 500,000 cells per well in six-well plates, incubated at 37oC for 24 hours and washed with PBS three times. Cells were then incubated with 0.5 nM [3H]-estradiol in the absence or presence of 1 µM unlabeled estradiol (to measure total and unspecific binding, respectively) in 10% DCC-fetal bovine serum for 3 hours at 37oC. Cells were then washed with PBS three times, lysed with lysis buffer and lysates were mixed with

LSC-cocktail (Emulsifier-Safe; Packard BioScience, Waltham, MA). Radioacivity was measured using liquid scintillation counter (LS-6000-SC; Beckman-Coulter, Brea, CA).

Experiments were performed in six replicates and measurements of total protein and total cell number were performed in parallel.

3.2.2 Cell counting and viability assays

Cells were plated in 6-well plates 24 hours prior to any transfections and/or treatments.

Following trypan blue staining, the cells were counted with the Countess automated cell counter (Invitrogen). All experiments were performed in triplicates.

3.2.3 qPCR primers

For detection of miRNA, a universal reverse primer provided in the NCode miRNA First-

Strand cDNA Synthesis Kit (Invitrogen) was used. The following primers were used for qPCR analysis if miRNA expression: miR-19a: F: 5’-TCCTGTGCAAATCTATGCAAAAC-3’ miR-19b: F: 5’-TCCTGTGCAAATCTATGCAAAAC-3’ miR-17: F: 5’-CATCAAAGTGCTTACAGTGCAGG-3’ miR-18a: F: 5’-CGTTAAGGTGCATCTAGTGCAGA-3’ miR-92a: F: 5’-TATTGCACTTGTCCCGGCCTG-3’ miR-20a: F: 5’-TAAAGTGCTTATAGTGCAGGTAG-3’ miR-200a: F: 5’-ACGCCACAATTAACAAGCCACATCGTTAC-3’

For detection of mRNA, the following primer pair were used:

MYC: F: 5’-CCACGTCTCCACACATCAGC-3’

R: 5’-CTTGGCAGCAGCAGGATAGTCCTT-3’

NCOA3: F: 5’-GCCTGGCTTTGAAGACATAATCCG-3’

R: 5’-TCTTGATAGTGACGCTTCTGGGAC-3’

20 CLU: F: 5’-TTCCAGGACAGGTTCTTCAC-3’

R: 5’-GGGACTTGGGAAAGAAGAAG-3’

ZEB1: F: 5’-CGCTTTACCTCTCTGAAAGAAC-3’

R: 5’-TCATGTGACGTTCAAGTTGG-3’

E-cadherin: F: 5’-AAAGGGCCTCTACGGTTTCAT-3’

R: 5’-CGTGACTTTGGTGGAAAACT-3’

3.2.4 Immunoblot

Primary antibody against NCOA3 (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:500 dilution; E-cadherin (Santa Cruz Biotechnology) used at 1:1,000 dilution. Both antibodies were incubated overnight.

3.2.5 Migration wound-healing assay

Cell migration was measured using in vitro scratch assays. Cells were seeded in 12-well plates and were allowed to attach for 24 hours after which an artificial gap was created in the confluent cell monolayer with a pipette tip. Cells treated with miRNA mimics or controls were transfected 24 hours before the gap introduction. The medium was replaced with fresh medium after the cells were washed twice with pre-warmed PBS to remove the debris and to smooth the edges of the scratch. Initial images of the scratch

(0 hours) were taken with Olympus 1X51 inverted microscope, after which the cells were

o incubated in a 5% CO2 cell incubator at 37 C. Plates were taken out of the incubator intermittently to measure cell migration; images of the scratch were taken at 4 and 8 hours following the initial scratch and image acquisition. Distance of migration and area

21 covered by migrating cells were analyzed quantitatively from the acquired images by using Olympus cellSens digital imaging software and ImageJ software, respectively.

3.2.6 Apoptosis assay

SW480 control cells were transfected with miR-17 inhibitor and control for 48 hours, then treated with vehicle (dimethyl sulfoxide, DMSO) or the DNA-damaging agent cisplatin

(cis-diammineplatinum(II) dicholoride) (Sigma-Aldrich, St. Louis, MO) with a final concentration of 10 µg/mL for 24 hours. Cells were stained with trypan blue and counted as described previously. Statistical analysis was determined with Chi-square test between amount of live and dead control cells with endogenous miR-17 expression, and cells were transfected with miR-17 inhibitor, treated with either DMSO or cisplatin.

3.3 Results and Discussion

3.3.1 Exogenously expressed ERβ are within physiological ranges

In colon cancer cell lines with overexpressed ERβ, using radiolabeled ligand-binding assays we determined that the levels of expression in these cell lines are within physiological ranges. Cell lines with or without ERβ were incubated with 1 nM [3H]- estradiol in the absence or presence of 1000 nM unlabeled estradiol and total and unspecific binding was recorded, as shown in Figure 2.1. The resulting expression levels were calculated and correspond to 3,770 ± 940 receptors/cell (6.3 fmol/106 cells) for stably transfected SW480-ERβ cells and 2,470 ± 597 receptors/cell (4.1 fmol/106 cells)

22 A) Intestinal tract expresses nuclear ER

Duodenum Small intestine Small intestine Large intestine Rectum (normal) (normal) (normal) (normal) (normal) Intestinal tract does not for stably transfected HT29-ERβ cells and 3,325 ± 307 receptors/cell (5.5 fmol/106 cells) Colorectal carcinoma express any ER for transiently transfect SW620, which falls within physiological ranges when compared with expression of ERα in breast cancer cell lines (78,79). Parental or control cell lines Large intestine did not show significant levels of binding, supportingSmall intestine the lack of endogenous ERβ (normal) (normal) expression. Thus, the exogenously introduced ERβ is expressed at physiologically relevant levels and possess ligand-binding abilities. ER ER ER Colorectal Colorectal cancer, female cancer, male

B) SW480 HT29 3,769 receptors/cell 2,468 receptors/cell * * 1000 1000

800 800 6 6 600 600

400 400 DPM/10 cells 200 DPM/10 cells 200

Control Control ER ER Control Control ER ER [E2*] [E2*]+[E2] [E2*] [E2*]+[E2] [E2*] [E2*]+[E2] [E2*] [E2*]+[E2]

SW620 3,325 receptors/cell 800 *

600 6

400

DPM/10 cells 200

Control Control ER ER [E2*] [E2*]+[E2] [E2*] [E2*]+[E2]

Figure 2.1 Exogenously expressed ERβ are within physiological ranges. Stably expressed ERβ in

SW480 and HT29 colon cancer cell lines show ERβ receptor at physiological levels. Transiently expressed

ERβ in SW620 colon cancer cell line also show ERβ receptor at physiological levels.

23 3.3.2 ERβ affects miRNAs through repression of MYC

Results from TaqMan Low-Density Arrays show that upon ERβ overexpression in

SW480 cell line, the majority of miRNAs are downregulated. We could confirm that 25 miRNAs were upregulated and 3 miRNAs were downregulated. Of the 25 miRNAs upregulated, 6 miRNAs were from the miR-17-92 polycistronic miRNA cluster (Figure

2.2).

Figure 2.2 miRNA expression of miR-17-92 cluster. miR-17-92 cluster is downregulated in SW480 cell line when ERβ is overexpressed. Estrogen treatment had no effects on the levels of miRNAs.

ERβ regulation of miRNAs can be through indirect mechanisms. Although the ERs can regulate genes distant from their chromatin-binding site (80), we can assume that miRNAs are also regulated indirectly. Previously, it was observed that the proto- oncogene MYC is strongly downregulated following ERβ expression in these colon cancer cell lines as well as other cell lines (41,42,64). Since ERβ binds the MYC promoter within 70 kb, this indicates that it is possible that MYC is directly regulated by

ERβ (81,82). MYC is a key regulator of miRNAs (83-85), which contributes to its

24 oncogenic properties (86). We observed that when ERβ is, overexpressed, MYC levels are decreased in SW480 cells (Figure 2.3).

Figure 2.3 MYC expression levels following ERβ overexpression in SW480 colon cancer cell line.

Overexpression of ERβ leads to downregulation of MYC on the mRNA level.

It has been noted that several miRNAs are regulated by MYC including the miR-17-92 cluster (83,84). The overexpression of miR-17-92 cluster has, in colon cancer, been associated with MYC gene amplification and overexpression (87) and associated with poor survival (88). Therefore, it is plausible that in our system, the downregulation of miR-17-92 cluster is an indirect a consequence of ERβ downregulation of MYC. To confirm this pathway, we used small interfering RNA to silence MYC. The silencing of

MYC was efficient and reduced its mRNA by 50% within 48 hours of small interfering

RNA transfection (Figure 2.4). This resulted in a downregulation of miR-17 and miR-19a at 72 hours after transfection. Other miRNAs in the miR-17-92 cluster were not affect perhaps due to different processing of individual miRNAs. MiR-200a/b, on the other hand, was upregulated upon MYC silencing, whereas miR-221 was unaffected (data not

25 shown). We conclude that MYC does not contribute to the ERβ-mediated repression of miR-200a/b and miR-221, but that the miR-17-92 cluster and miR-106 are transcriptionally downregulated as a consequence of the ERβ-mediated downregulation of MYC.

Figure 2.4 Knockdown of MYC via siRNA results in downregulation of miRNAs in the miR-17-92 cluster. Knockdown of MYC in SW480 cells using siRNA was successful in reducing MYC mRNA levels at

48 hours. In turn, at 72 hours of MYC siRNA treatment, miR-17 and miR-19a is subsequently downregulated as well.

3.3.3 MiR-17 re-expression can override the ERβ-mediated reduction of proliferation

Expression of ERβ resulted in an antiproliferative effect in the colon cancer cells.

TaqMan Low-Density Array analysis and qPCR confirmation showed that the entire miR-

17-92 cluster was downregulated following ERβ expression (Figure 2.2). The members of the miR-17-92 cluster (also called Oncomir-1) are implicated in colon cancer (88) and have been related to enhanced cell proliferation (89,90). Since miR-17 was

26 downregulated when SW480 cells were treated with siRNA against MYC, we selected it to test whether the downregulation of this cluster had a significant role in the ERβ- mediated effect on proliferation. We reintroduced miR-17, using a miR-17 miRNA mimic, into ERβ-expressing SW480 cells and investigated whether this could oppose the ERβ- mediated repression of growth. Figure 2.5 shows that transfection with miR-17 mimic, reverses the effects of MYC and ERβ resulting in an increase cell proliferation. Thus, part of the antiproliferative effect attributed to ERβ in colon cancer cell line SW480 is through downregulation of MYC and subsequent downregulation of miR-17.

Figure 2.5 Transfection of miR-17 mimic increases cell proliferation in SW480 cells. Overexpression of miR-17 using miRNA mimics opposes antiproliferative effects of ERβ in SW480 colon cancer cell line.

3.3.4 ERβ-mediated repression of miR-17 can enhance apoptosis

Another aspect of the miR-17-92 cluster besides being proliferative is that this

ploycistron miRNA cluster can also suppress apoptosis (89). Since ERβ increases

apoptosis in normal colonocytes (91) and intestines (92), we hypothesize that ERβ may

mediate this apoptotic effect in SW480 cells through suppression of miR-17-92 cluster.

SW480 Control Non-targeting siRNA A) SW480 Control Although we did not observe a significantSW480 effect ER on apoptosisB) 1.25 by inhibiting miR-17 in 1.4 SW480 ER 1.2 MYC siRNA

1.2 SW480 cells grown under regular conditions, we noted a clear effect1 on cell death when 1

* 1 0.8 the cells were challenged with DNA-damaging cisplatin treatment,0.75 56% of the miR-17- ** 0.8 *** 0.6 ** silenced cells died (P-value < 0.001) (Figure 2.6). Our results demonstrate0.5 that inhibition 0.6 *** 0.4 Fold-change 0.4 of miR-17 sensititized the cells to** cisplatin-induced apoptosis. This leads us to conclude Fold-change 0.25 0.2 *** *** 0.2 that ERβ, through decreased miR-17 expression can*** enhance apoptosis after DNA 0 0 0 damage in colon cells. MYC MYC miR-17 miR-19a miR-19a miR-19b miR-17 miR-18a miR-92a miR-20a 48h 72h C) SW480 ER D) E) NCOA3 900,000 * 70 SW480 Control * 2 60 *** 700,000 control ER 50 1.5 500,000 40 NCOA3 30 1 300,000 20 0.5 B-actin Live cell counts Dead cells (%) Fold-change 100,000 10 0 Mimic miR-17 Inhibitor miR-17 SW480 SW480

control mimic control inhibitor control ER F) Figure 2.6 Inhibition of miR-17 leads toG) more cellular apoptosis in SW480 cells. DownregulationH) of SW480 Control 2.5 miR-17 using miRNA inhibitor along with cisplatin treatments leads to higher cell death in SW480 cells. 2.5 Inhibitor control Mimic control 2 ** miR-17 inhibitor miR-17 mimic 2 1.5 *** 1.5 28 1

1 Fold-change 0.5 *** *** Fold-change 0.5 0 CLU NCOA3 0 CLU NCOA3 SW480 ER

inhibitor miR-17 control inhibitor NCOA3

B-actin 3.3.5 MiR-17 mediates the upregulation of NCOA3 and CLU after ERβ expression

Besides proliferative and antiapoptotic effects of miR-17, we wanted to further dissect potential downstream targets of this miRNA to infer other potential functional effects.

Two predicted targets were implicated in colorectal cancer, the nuclear receptor coactivator NCOA3 (SRC-3) and the clusterin protein, CLU. NCOA3 may be a direct target of miR-17, based on their complementing nucleotide sequences with one conserved 8-mer site at position 714-720 of NCOA3 3’-UTR and once conserved 7-mer- m8 site at position 1296-1302 (Figure 2.7).

Figure 2.7. miRNA and mRNA sequence alignment of miR-17 and NCOA3. miR-17 may directly target

NCOA3 based on their complementing sequences.

In human colon cancer HCT116 cells, miR-17 has been reported to repress CLU levels, which contributed to stimulated angiogenesis and cell growth (93). Both NCOA3 and

CLU were upregulated in ERβ-expressing cells compared with controls (41). We confirmed that NCOA3 was upregulated both at the mRNA and protein level (Figure 2.8).

This led to the hypothesis that ERβ, through MYC and miR-17, regulate a cascade of

CLU and NCOA3 activity. As NCOA3 regulates the transcriptional activity of ERβ

(94,95), this may also constitute a feed-forward loop.

29 SW480 Control Non-targeting siRNA A) SW480 Control SW480 ER B) 1.25 1.4 SW480 ER 1.2 MYC siRNA

1.2 1 1

* 1 0.8 0.75 ** 0.8 *** 0.6 ** 0.5 0.6 *** 0.4 Fold-change 0.4 ** Fold-change 0.25 *** 0.2 0.2 *** *** 0 0 0 MYC MYC miR-17 miR-19a miR-19a miR-19b miR-17 miR-18a miR-92a miR-20a 48h 72h C) SW480 ER D) E) NCOA3 900,000 * 70 SW480 Control * 2 60 *** 700,000 control ER 50 1.5 500,000 40 NCOA3 30 1 300,000 20 0.5 B-actin Live cell counts Dead cells (%) Fold-change 100,000 10 0 Mimic miR-17 Inhibitor miR-17 SW480 SW480 control mimic control inhibitor control ER F) G) Figure 2.8 NCOA3 is upregulated withH) overexpressed ERβ in SW480 cell lines. Overexpression of ERβ SW480 Control 2.5 in SW480 leads to upregulation of NCOA3 at the transcriptional (mRNA) as well as translational (protein) 2.5 Inhibitor control Mimic control levels. 2 ** miR-17 inhibitor miR-17 mimic 2 1.5 *** 1.5 To further confirm that the regulation 1 of NCOA3 and CLU are directly due to ERβ-

1 Fold-change mediated downregulation of the miR-0.517- 92 cluster,*** SW480 cells*** (absent of ERβ) were Fold-change 0.5 transfected with a miR-17 miRNA inhibitor.0 Inhibition of miR-17 in SW480 cells using CLU NCOA3 0 miRNA inhibitor led to the upregulation of CLU and NCOA3 (Figure 2.9A) and CLU NCOA3 SW480 ER conversely, when miR-17 was overexpressed in SW480-ERβ cells, using miR-17 mimic, inhibitor miR-17 CLUcontrol and NCOA3inhibitor levels were correspondingly decreased (Figure 2.9B). This confirms

NCOA3that the upregulation of CLU and NCOA3 in ERβ-expressing SW480 cells is a direct

consequence of the repressed miR-17 levels. B-actin

30 SW480 Control Non-targeting siRNA A) SW480 Control SW480 ER B) 1.25 1.4 SW480 ER 1.2 MYC siRNA

1.2 1 1

control mimic control inhibitor control ER F) G) H) A SW480 Control B 2.5 2.5 Inhibitor control 2 Mimic control ** miR-17 inhibitor miR-17 mimic 2 1.5 *** 1.5 1

1 Fold-change 0.5 *** *** Fold-change 0.5 0 CLU NCOA3 0 CLU NCOA3 SW480 ER

inhibitor miR-17 control inhibitor NCOA3

B-actin

Figure 2.9 MiR-17 directly regulates levels of CLU and NCOA3. SW480 cells transfected with miR-17

inhibitor and miR-17 mimic. A) Overexpression of miR-17 inhibitor increases mRNA levels of CLU and

mRNA as well as protein levels of NCOA3. B) Overexpression of miR-17 mimic decreases mRNA levels of

CLU and NCOA3.

As NCOA3 also activates ERβ and functions as a histone acetyltransferase, which acts

on the chromatin level, making target genes more accessible for the transcriptional

machinery. The upregulation of NCOA3 is likely to be beneficial in a clinical perspective

because overexpression of NCOA3 is associated with prolonged overall survival and

decreased probability of death in colon cancer (96). Although theses results suggest that

ERβ increases DNA repair in colon cells, it is the axis miR-17/CLU that is responsible for

increased apoptotic capacity and that other ERβ-mediated mechanisms are responsible

for increased DNA repair. Thus, we have dissected a pathway where ERβ regulates its

own coactivator through MYC and miR-17.

31 3.3.6 ERβ modulates miR-200a/b and epithelial-mesenchymal transition markers

Besides changes on the transcriptional level by ERβ, we also noted morphological changes to the cell shape of ERβ-expressing cells as well (Figure 2.10A).

Cells that overexpress ERβ adopt a more stretched out and adhesive phenotype. To test whether this morphological change has any functional effects, we used a migration wound-healing assay to measure cell migration. Indeed, ERβ-expressing cells exhibit slower migration compared to controls (Figure 2.10B).

AA) B)B 12 10 8 6 ** 4 2 Gap decrease (%) 0 SW480 control SW480 ER SW480 Control SW480 ER C) 3.5 D) E) 1.4 Figure 2.10 Effects SW480 of ERβ Control on morphology (E2) and migrationControl in SW480** cels. A) Overexpression of ERβ 1.2 3 *** SW480 ER (E2) 8 miR-200a mimic

1 changes2.5 the morphology of SW480 cells to adopt a moremiR-200b stretched mimic out, adhesive phenotype. B) Adhesive 6 ** 0.8 * phenotype2 is reflected by reduced migration in ERβ expressing cells as measured with scratch wound 4 0.6 assay.1.5 0.4

Fold-change 2 Relative miR-200a fold-change

Fold-change 1 0.2 *** *** 0 0.5 ** 0 ZEB1 E-cadherin control ER 0 MiRNAs are also known to contribute to cellularControl changesmiR-200a miR-200b in morphology and migration.HT-29 ZEB1 E-cadherin mimic mimic Two miRNAsControl thatER were changed by ERE-cadherinβ in our cells are: miR-200a and miR-200b. The

E-cadherin miR-200-family of miRNAs has been known to repress epithelial-mesenchymal transition B-actin B-actin (EMT) through the downregulation of repressors of E-cadherin expression, ZEB1 and

ZEB2 in breast cancer cells (97). In our colon cancer cell lines, we observe that the

32 downregulation of miR-200a/b in SW480 cells coincided with an upregulation of ZEB1 and a downregulation of E-cadherin (Figure 2.11A). To confirm that the changed levels of ZEB1 and E-cadherin were a result of the ERβ-mediated repression of miR-200a/b, A) B) we re-introduced miR-200a/b into SW480-ERβ cells individually, using miRNA12 mimics. 10 We show that both repressed ZEB1 levels and high E-cadherin levels in ERβ8- expressing 6 ** colon cancer cells is a consequence of miR-200a/b levels (Figure 2.11B). 4 2 Gap decrease (%) 0 SW480 control SW480 ER SW480 Control SW480 ER C)A 3.5 D)B E) 1.4 SW480 Control (E2) Control ** 1.2 3 *** SW480 ER (E2) 8 miR-200a mimic

1 2.5 miR-200b mimic 6 ** 0.8 * 2 4 0.6 1.5 0.4

Fold-change 2 Relative miR-200a fold-change

Fold-change 1 0.2 *** *** 0 0.5 ** 0 ZEB1 E-cadherin control ER 0 Control miR-200a miR-200b HT-29 ZEB1 E-cadherin mimic mimic

Control ER E-cadherin

E-cadherin

B-actin B-actin

Figure 2.11 ERβ expression affects epithelial-mesenchymal transition markers ZEB1 and E-cadherin.

A) ERβ overexpression leads to increased ZEB1 mRNA levels and decreased E-cadherin mRNA and protein levels. B) Overexpression of miR-200a and miR-200b individually is responsible for decreased ZEB1 mRNA levels and increased E-cadherin levels both on mRNA and protein levels.

Because the miR-200 cluster has de facto ERβ-binding sites 78 kb upstream and 70 kb

A) downstream of theB) primary miRNA chromosomal region (81), it is possible that ERβ 12 directly represses miR10-200a/b which are responsible these morphological changes. To 8 further support our data,6 in another colon cancer** cell line, HT29, stably expression of 4 ERβ leads to downregulation2 of miR-200a (Figure 2.12). Gap decrease (%) 0 SW480 control SW480 ER SW480 Control SW480 ER C) D) E) 3.5 1.4 SW480 Control (E2) Control ** 1.2 3 *** SW480 ER (E2) 8 miR-200a mimic

1 2.5 miR-200b mimic 6 ** 0.8 * 2 4 0.6 1.5 0.4

Fold-change 2 Relative miR-200a fold-change

Fold-change 1 0.2 *** *** 0 0.5 ** 0 ZEB1 E-cadherin control ER 0 Control miR-200a miR-200b HT-29 ZEB1 E-cadherin mimic mimic Control ER E-cadherinFigure 2.12 MiR-200a levels in HT-29 colon cancer cell line. HT-29 colon cancer cell line stably

E-cadherin expressing ERβ downregulates miR-200a. B-actin B-actin

3.3.7 Cell-specific effects of ERβ

Thus far, we have explored effects of ERβ mainly in the colon cancer cell line SW480.

SW480 colon cancer cell line is a non-metastatic cell line. To examine whether ERβ

might have the same described effect in advanced colon cancer, we selected the

metastatic colon cancer cell line SW620 for subsequent experiments. The SW620 colon

cancer cell line originates from the same patient as SW480, but the main difference is

that SW620 was isolated from a metastatic lymph node one year later. Thus the major

34 difference between SW480 and SW620 is that the former is non-metastatic and the latter is metastatic. Although transient transfection of ERβ resulted roughly the same number of ERβ receptors/cell (SW480: 3,769 receptors/cell; SW620: 3,325 receptors/cell)

(Figure 2.1) we did not observe any regulation on MYC or miRNAs that were seen as changed in SW480 cells. It is possible that ERβ does not mediate the same protective mechanism in more advanced and invasive stages, or that stable expression of ERβ is required to observe the same changes as seen in SW480.

3.1.4 Conclusion

The aim of this study was to investigate whether ERβ expression impacts miRNA regulation in colon cancer cells and to begin dissecting its potential influence through functional studies. Here, we present evidence that introduction of ERβ in SW480 colon cancer cells induces significant changes in the miRNA pool and that these changes contribute in part to the antiproliferative and antitumorigenic properties of ERβ. Thus, miRNAs themselves play an important role in colon carcinogenesis and ERβ also plays an important role in regulating these miRNAs. Re-introduction of ERβ resulted in subsequent mRNA regulations and functional effects affecting proliferation, apoptosis, and migration mainly through downregulation of MYC, which in turns controls the miR-

17-92 miRNA cluster. Morphological changes in cell shape due to ERβ were also through miRNAs: 200a/b, which controls E-cadherin. We propose that the capacity of miRNAs to target multiple pathways help elucidate the genome-wide anti-tumorigenic regulations mediated by ERβ in colon cancer cells. As ERβ appears to have a significant potential in preventing or attenuating colon cancer development, our results provide

35 insights into the mechanism whereby ERβ acts in the colon and can aid in the development of better diagnostic tools, prevention and treatment of colon cancer.

4. Chapter 2: ERβ decreases metastatic potential of colon cancer cells through the upregulation of miR-205 and its direct targeting of PROX1

4.1 Introduction

Previous studies have found that ERβ could regulate inflammation, apoptosis, and cell adhesion pathways along with microRNAs (miRNAs) in colon cancer cells (41,98).

Particularly, we noted that expression of ERβ in the colorectal cancer cell line SW480 downregulated the prospero homeobox 1 (PROX1) (41). PROX1 is critical in organ development during embryogenesis, and its dysregulation have been correlated with several different cancers (99). It is upregulated in colon adenomas and associated both with a poor grade of tumor differentiation and with worse outcome, especially in women

(100). PROX1 is associated with the transition from benign adenoma to carcinoma in

37 vivo, and its silencing reduces size and incidence of human colorectal tumor xenografts

(101). PROX1 has also been shown to promote EMT in colon cancer cells by inhibiting

E-cadherin (102). Thus, the regulation of PROX1 by ERβ may be a key mechanism whereby ERβ protects against development of colorectal carcinoma.

The mechanism of how ERβ regulates PROX1 in colon cancer cells has not been well- studied. This regulation could be at the transcript or post-transcriptional level. We have previously found that the microRNA (miRNA) miR-205 is significantly upregulated upon expression of ERβ in SW480 cells (98). The function of miR-205 is relatively unknown in colon cancer, but low miR-205 in colorectal cancer has been correlated with invasion into lymphatic vessels (103). MiRNAs can control gene expression through complementary binding between the miRNA 5’-sequence and the 3’-untranslated region

(3’-UTR) of the target mRNA which mediates degradation of the mRNA transcripts and/or suppression of its translation. In this study, we hypothesize that part of ERβ’s anti-tumorigenic properties is through silencing of PROX1 via upregulation of miR-205.

4.2 Supplemental materials and methods

4.2.1 The Cancer Genome Atlas clinical data

RNA-sequencing and microarray data from patient colon tumors and adjacent normal tissues were downloaded from TCGA data portal. 233 samples were from colon adenocarcinoma samples and 21 were from normal control colon samples. Significance was determined by two-tailed t-test.

4.2.2 Immunoblot

Primary antibody against PROX1 (Upstate Biotechnology, Lake Placid, NY) was used at

1:1,000 dilution for incubation overnight.

4.2.3 qPCR primers

For detection of miRNA primers, a universal reverse primer provided in the NCode miRNA First-Strand cDNA Synthesis Kit (Invitrogen) was used. For detection of miR-

205: F: 5’-TGCGGCCCAGTGTTTAGACTATC-3’ and pre-miR-205: F: 5’-

CCTCAGACAATCCATGTGCT-3’.

Primers used for mRNA detection were as follows:

Human PROX1: F: 5’-TCATAAAGTCCGAGTGCGGC-3’

R: 5’-TGGGTGACAATCCTTCCTGC-3’

Human ERβ exon 1: F: 5’-AGGAATAAGATGGGCGATTG-3’

R: 5’-AGCAGAGCAAGCACATTCAT-3’

Mouse PROX1: F: 5’-AACGAAAGAGAGATGGCCCC-3’

R: 5’-TGGAAACTCTGTTGCTGCTG-3’

Mouse ERβ exon 1: F: 5’-TCACGAATACTCAGCCATGA-3’

R: 5’-GAAGTTGGCCATAGCACATT-3’

Mouse ERβ exon 3: F: 5’-GCCCTGTTACTACTAGTCCAAGC-3’

R: 5’-CAGGACCAGACACCGTAATG-3’

39 Primers used for genotyping of mice were as follows:

B6.129X1-Esr2tm1Gust F: 5’-ATCAGCCCATGGGCAGAGTGT-3’

R: 5’- CCAGATGCATAATCACTGCAGACG-3’

B6.SJL-Tg(Vil-cre)997Gum/J F: 5’-GTGTGGGACAGAGAACAAACC-3’

R: 5’-ACATCTTCAGGTTCTGCGGG-3’

4.2.4 Generation of tissue-specific knockout mice and collection of samples

B6.129X1-Esr2tm1Gust mice were crossed over multiple generations with B6.SJL-

Tg(Vil-cre)997Gum/J mice (Jackson Laboratory, Bar Harbor, Maine) to generate intestinal-specific ERβ knockout mice (ERβiKO). Mice were genotyped using PCR with

40 4.2.5 Correlation analyses

Correlation for ERβ and PROX1, and ERβ and miR-205 were done using Pearson correlation, significance was determined if p < 0.05. Correlation for PROX1 and miR-205 were done using Spearman correlation, significance was determined if p < 0.05.

4.2.6 Cell adhesion

Cells were treated as described above. 96-well plates were coated with collagen and treated cells were trypsinized, counted, and plated on collagen-coated plates. After 1 hour, plates were washed and standard MTS assay (Promega, San Luis Obispo,

California) was used to determine amount of cells left on plate.

4.2.7 Zebra-fish migration assay

We used green-fluorescent-protein-tagged (GFP-tagged) zebra fish embryos to study tumor metastasis. Different cell lines have been labeled in vitro with 2 µM of lipophilic dye CM-Dil (Invitrogen, Carlsbad, CA, USA). Approximately 5 nL of 5x106 of labeled cells have been injected in the perivitelline cavity of embryos 48 hours post-fertilization.

The embryos are checked 3 hours post-injection; poorly injected embryos (i.e. direct blood stream injection) have been removed from the study. Fish embryos were incubated in 32oC incubator. Tumor dissemination in fish body (mainly in tail) was monitored using the fluorescent microscope 48 hours after initial injection. Fisher’s exact test was used to calculate p-value.

41 4.3 Results and Discussion

4.3.1 Expression of ERβ and miR-205 are anti-correlated with expression of PROX1 in both primary colon and tumor cell lines

Our previous studies in SW480 cells suggested that overexpression of ERβ could increase miR-205 levels (98) and decrease PROX1 levels (41) in colon cancer cells. To determine whether this relationship is relfective in clinical samples, we searched RNA- sequencing and microarray data from The Cancer Genome Atlas (TCGA) dataset and compared the expression of ERβ, miR-205 and PROX1 in patient samples of colorectal tumors with adjacent normal tissues (Figure 3.1). This is consistent with earlier findings that in the colon, ERβ expression is decreased in the cancerous state compared to non- cancerous state. In the same data set, miR-205 shows the same trend as ERβ while

PROX1 levels show the opposite trend.

Figure 3.1. Expression of ERβ, miR-205, and PROX1 in clinical samples. RNA expression of ERβ, miR-

205, and PROX1 in normal and cancerous tissues taken from patients. Dataset downloaded from TCGA. P- values for ERβ, p < 0.001; hsa-miR-205, p = 0.005; PROX1, p < 0.001.

We further investigated whether this correlation holds true for different colon cancer cell lines. As shown in Figure 3.2, miR-205 levels are in general anti-correlated with PROX1 in 5 different colon cancer cell lines, SW480, HT29, HCT116, SW403, and SW620, respectively (Figure 3.2, left and center panels). SW403 and SW620 show the lowest miR-205 levels and the highest PROX1 protein levels (Figure 3.2, left and right panels), consistent with our hypothesis that miR-205 targets PROX1. Even through SW403

PROX1 mRNA levels show less PROX1 than in SW480, while PROX1 is higher, this may be due to stability of PROX1 protein in different colon cancer cell lines (Figure 3.2 center and right panels). Collectively, these data support the overall regulation that ERβ is correlated with miR-205 expression, and that both ERβ and miR-205 is anti-correlated with PROX1 in colon cancer both in cell lines and in clinical samples.

Figure 3.2 miR-205 and PROX1 levels are anti-correlated. miR-205 and PROX1 levels are anti-correlated on the RNA and protein levels in 5 different colon cancer cell lines: SW480, HT29, HCT116, SW403, and

SW620.

43 Our results from the clinical samples along with other studies (44,46,104) clearly indicate that ERβ possesses anti-proliferative effects and that as cancer progresses, ERβ expression is reduced (Figure 3.1, left panel, p < 0.001). Separate studies have shown that high levels of PROX1 promotes the progression from benign to malignant colorectal tumors (101), and indeed in a study based on 643 European colorectal cancer patients,

PROX1 was an important prognostic indicator for colorectal cancer-specific survival

(100). We also observe this phenomenon in which PROX1 levels are higher in cancerous tissues as compared with controls (p < 0.001) (Figure 3.1, right panel). MiR-

205 downregulation has been implicated in EMT in cell line studies (97,98,105), but has not been clearly described as a prognostic marker in colon cancer. From our analysis, miR-205 levels are indeed low in cancer tissues (p = 0.005) as compared with controls.

Overall, we observe both in clinical samples and in colon cancer cell lines that ERβ and miR-205 are loss in the cancerous state while PROX1 levels are increased. Thus, we propose that there exist an ERβ-miR-205-PROX1 pathway and it plays an important role in colon cancer development.

4.3.2 Tissue-specific knockout of ERβ decreases miR-205 and increases PROX1 in the colon of mice

To explore whether this ERβ-miR-205-PROX1 pathway holds true in vivo and whether this pathway is dependent on ERβ, we measured the levels of ERβ, miR-205, and

PROX1 in intestine-specific ERβ knockout mice (ERβiKO) (Figure 3.3). The Villin/Cre- driven knockout of ERβ exon 3 (Esr2fl/fl, Vil-Cre), which disrupts the coding frame and generates multiple stop codons in the ERβ mRNA in colonic epithelial cells, was

44 confirmed by expression analysis (qPCR) (Figure 3.3A). Because knockout of ERβ exon

3 causes non-sense-mediated decay of the ERβ transcript, we also measured levels of

ERβ exon 1 (Figure 3.3B) to ensure that ERβ is indeed not present in the epithelial cells of these knockout mice. As expected, the expression levels of ERβ exon 3 are dramatically decreased in ERβiKO mice confirming the knockout. MiR-205 levels are significantly downregulated (Figure 3.3C) and PROX1 levels are increased (Figure

3.3D).

A B

C D

Figure 3.3 Expression of ERβ exon 3, ERβ exon 1, miR-205 and PROX1 levels in intestinal-specific

iKO ERβ knock-out mice (ERβ ). A) Levels of ERβ exon 3 are significantly lower in ERβiKO mice as compared to controls (p = 0.04). B) Levels of ERβ exon 1 are significantly lower in ERβiKO as compared to controls (p = 0.04). C) Levels of miR-205 in ERβiKO are lower than in control mice (p = 0.05). D) PROX1 mRNA levels show an increasing trend in ERβiKO than in controls.

45 Table 3.1 below shows the correlation between ERβ, PROX1, and miR-205 RNA in intestinal epithelial ERβ knockout mice. ERβ and PROX1 are significantly anti-correlated

(R2=-0.38) while ERβ and miR-205 are significantly positively correlated (R2=0.51). Most notably, miR-205 and PROX1 levels are significantly anti-correlated (R2=-0.045). These findings are consistent with the data from human clinical samples and colon cancer cell lines, and strongly support that ERβ regulates the expression of miR-205 and PROX1 in colonic epithelial cells.

Table 3.1. Correlation between ERβ, PROX1, and miR-205 in ERβiKO colon tissue. Expression of ERβ and PROX1 are anti-correlated; Expression of ERβ and miR-205 are correlated; Expression of PROX1 and miR-205 are anti-correlated. Comparisons between ERβ and PROX1, ERβ and miR-205 were analyzed using Pearson correlation. Comparison between PROX1 and miR-205 were analyzed using Spearman correlation. Significance was determined as p < 0.05.

PROX1 miR-205 -0.38 0.51 Correlation coefficient ERβ 0.038 0.021 p-value -0.046 Correlation coefficient PROX1 0.048 p-value

4.3.3 ERβ upregulates miR-205 expression in different colon cancer cell lines

We had previously noted that expression of ERβ resulted in upregulation of miR-205 in

SW480 cell line and that a putative ERβ-binding site is located 104 kb upstream of the miR-205 based on genome-wide chip sequencing studies performed in human breast

46 cancer cells (81). To further explore the generality of this mechanism, we tested three more colon cell lines with and without expression of ERβ (HT29, SW403, and SW620, respectively). None of these three cell lines expresses detectable amounts of ERβ. HT29 was stably transduced with ERβ (as previously characterized) (41,98) while SW403 and

SW620 were transiently transfected with ERβ. In cells with overexpression of ERβ, miR-

205 was found to be upregulated in all cell lines (Figure 3.5), consistent with our previous observation in SW480 cells. As estrogen receptors can bind to cis-regulatory

DNA either directly through its DNA-binding domain (DBD) or via a tethering mechanism, we tested the effect of an ERβ mutated in the DBD region. In both SW403 and SW620 cells, this mutation inhibited the increase of miR-205 levels (Figure 3.5), suggesting the upregulation of miR-205 is dependent on direct DNA binding of ERβ. Notably, in SW403 cells, even with ERβ-DBD-mutant, miR-205 is still upregulated. This may be due to an

ERβ-tethering mechanism much like ERα tethering to other transcription factors such as

FOXA1, Ap2g, GATA3 and RUNX1 to regulate transcription of genes (reviewed in

(106)). MiR-205 upregulation is almost completely abolished when transfected with ERβ-

DBD-mutant in SW620 cells. This shows that there are other miR-205 regulatory pathways that are specific to each colon cancer cell line.

Figure 3.5 ERβ upregulates miR-205 in colon cancer cell lines HT29, SW403 and SW620. HT29 cells with stable ERβ expression show increased miR-205 levels. SW403 and SW620 cell lines transiently transfected with ERβ also show increases of miR-205 miRNA. Transfection with ERβ-DBD mutant decreases miR-205 levels in SW403 and SW620 cell lines.

To further examine the mechanism of ERβ upregulation of miR-205, we also measured levels of primary- (pri-miR-205) and precursor-miR-205 (pre-miR-205) and whether their expression was also influenced by estradiol (E2). In stably transfected SW480 cells, ERβ upregulates both the pri-miR-205 and pre-miR-205 transcripts (Figure 3.6). We did not see any effects of E2 treatment on the transcript levels. In the transiently transfected cell line SW620, we observed upregulation of pri-miR-205 transcript by ERβ, but not in the precursor-miR-205 levels (Figure 3.6). This could be due to the processing of the miRNA transcript in the cell where precursor-miR-205 has a shorter half-life. Estradiol also did not affect pri- or pre-miR-205 transcripts.

Figure 3.6 Primary- and precursor-miR-205 levels in SW480 and SW620 colon cancer cells. Primary and precursor miR-205 levels are upregulated in the presence of ERβ in SW480 cells. Only primary-miR-

205 levels are upregulated in SW620 cells transiently transfected with ERβ.

4.3.4 MiR-205 represses PROX1 expression in colon cancer cells

The anti-correlative relationship between miR-205 and PROX1 mRNA levels imply that miR-205 may affect PROX1 expression directly since miRNAs are known to regulate many different targets. To determine whether increased levels of miR-205 can repress

PROX1, SW480 and HT29 cells were transfected with miR-205 miRNA mimic. Protein levels 48 hours after miR-205 mimic transfections showed that PROX1 levels had decreased significantly in both cell lines (Figure 3.7). These data show that miR-205 regulates PROX1 expression in colon cancer cells.

Figure 3.7 PROX1 protein levels in SW480 and HT29 cells. SW480 and HT29 cells were transfected with miR-205 and levels of PROX1 are thus downregulated.

4.3.5 Downregulation of PROX1 by miR-205 regulates cell adhesion and metastasis in vitro and in vivo

PROX1 is upregulated in colon adenomas and associated both with a poor grade of tumor differentiation and with worse clinical outcome (100). Previous studies have shown that knockdown of PROX1 in SW480 colon cancer cells using siRNA resulted in increased expression of genes related to cell adhesion, without affecting proliferation

(101). Thus, the adverse role of PROX1 in colon cancer includes its anti-adhesion effects. To characterize the functional effects of ERβ and miR-205, we measured cell adhesion ability using collagen assays in metastatic SW620 cells transfected with miR-

205 mimic. We observe that transfection with miR-205 mimic into metastastic SW620 cell line leads to decreased levels of PROX1 mRNA (Figure 3.8, left panel) and that this in turns makes the cells more adhesive to the collagen medium (Figure 3.8, right panel), suggesting that in SW620 cells with low PROX1 levels, cells have become more adhesive, and less metastatic.

Figure 3.8 miR-205 transcriptional and functional effects in SW620 cells. Left panel, miR-205 mimic downregulates PROX1 mRNA levels. Right panel, miR-205 mimic causes SW620 cells to become more adhesive.

ERβ overexpression in SW480 has been reported to reduce migration as measured by wound-healing assay (98) and here we demonstrated that downregulation of PROX1 via miR-205 mimic increases cell adhesion in vitro. To demonstrate the effect of the ERβ- miR-205-PROX1 pathway on metastasis in vivo, we used the zebrafish model for cell migration (in collaboration with Fahmi Mesmar). Colon cancer cell lines (SW480, HT29,

+/- ERβ, +/- miR-205 mimic) were injected into zebrafish embryos. The migration into the circulatory system was observed after 48 hours. Table 3.2 shows that both ERβ and miR-205 exhibited a significant anti-metastatic potential in both cells lines SW480 and

HT29. SW480 cells with overexpressed ERβ had 20.8% metastatic cells as compared to

36.2% (p-value = 0.020). Similarly, HT29 cells that contained ERβ also had roughly half the percentage of metastatic cells (11.0% and 25.9%, respectively, p-value = 0.008).

When both cell lines were transfected with miR-205 mimic, the percentage of metastatic

51 cells drops to below 10% (9.2% or SW480 and 5.8% for HT29, p-value 0.006 and 0.001, respectively). Using zebrafish as an in vivo assay for metastasis is advantageous in that we can treat a high number of embryos in a relative short amount of time (107,108).

Thus, we have demonstrated that the ERβ-miR-205-PROX1 pathway is responsible for both cell adhesion and metastasis in vitro and in vivo.

Table 3.2 ERβ and miR-205 reduces metastasis in vivo. SW480 and HT29 cells with either ERβ and ERβ in combination with miR-205 exhibit less metastasis in vivo. Significance determined as p < 0.05.

4.4 Conclusion

ERβ is widely known to have anti-tumorigenic effects in various different cancers. In this current study, we have reported anti-metastatic effects of ERβ in colon cancer through this ERβ-miR-205-PROX1 pathway both in vitro and in vivo. Of course more studies need to be done to investigate the detailed mechanisms of ERβ regulation on processing of miR-205 transcript in different colon cancer cell lines. Since miRNAs are known to target multiple mRNAs, it would also be noteworthy to look at other targets of miR-205 and whether they contribute to anti-proliferative and anti-metastasis effects of ERβ-miR-

205. Lastly, in addition to miR-205, other miRNAs can also silence PROX1. It could be interesting to look at PROX1 regulation by miRNAs that are not influenced by ERβ.

5. Chapter 3: ERβ mediates anti-inflammatory effects via modulation of the NFκB networks

5.1 Introduction

One of the major risk factors for development of colon cancer is inflammation.

Inflammation itself is crucial, but chronic inflammation is one of the hallmarks of tumor growth and promotion and correlates with poor prognosis in many different types of cancer including breast (17) and colon cancer (18-20). Thus, controlling or minimizing chronic inflammation can be the key to reducing colon cancer development. In fact, long- term use of aspirin has been shown to reduce colon cancer risk (23) and mortality (24).

It has been previously reported that NFkB-mediated inflammatory response is involved in cancer development in colon (109,110) and breast (111-113). ERα and NFkB have been shown to interact both in a repressive and synergistic manner in a number of

53 cancers. In the case of breast cancer, there is extensive cross-talk between NFkB and

ERα where ERα and NFkB can act synergistically to enhance transcription of genes such as BIRC3 to protect breast cancer cells against apoptosis (114). Cooperative binding of ERα and NFkB to adjacent response elements leads to major increase of the multidrug transporter gene ABCG2, whose expression is associated with the development of drug-resistance (115). Also, 17β-estradiol (E2) can increased TNFα mRNA and protein levels in ERα-positive breast cancer cell lines through ERα (25).

Like ERα, ERβ has also been shown to modulate NFkB signaling. In rat aortic smooth muscle cells (RASMCs), treatment with an ERβ agonist was able to attenuate TNFα- induced inflammatory response by downregulation of mRNA expression of inflammatory mediators such as P-selectin, ICAM-1, VCAM-1, MCP-1, and CINC-2β resulting in a less inflamed state (116). This was achieved via recruitment of RELA(p65) unit to the promoter region of IkBα, thus upregulating the levels of the inhibitor of NFkB activation

(117).

Our preliminary findings (41) demonstrated that NFkB-regulated IL-6 is strongly downregulated by overexpression of ERβ in colon cancer cell line SW480. Although overall, ERβ attenuates IL-6 and in turn affects NFkB signaling, the mechanism of regulation on the gene level is not known. Here, we further investigate and describe the modulation of NFkB activity by ERβ in colon cancer cell lines SW480 and HT29. We hypothesize that ERβ may attenuate inflammation by modulating NFκB signaling.

54 5.2 Supplementary material and methods

5.2.1 Microarray and data analysis and mining

RNA (250 ng) was converted to cRNA using the Illumina TotalPRep-96 RNA

Amplification kit (Ambion, Carlsbad, CA). Complimentary-RNA was used for hybridization onto the Illumina Whole-Genome Gene Expression Direct Hybridization microarray (Illumina, San Diego, CA), and 25,559 probes from the microarray were included for analysis. Probes for multiple genes were eliminated. The R software running lumi and limma packages were used to determine differentially expressed genes.

Normalized intensity values were log-2 transformed. To correct for false discovery, we implemented the Benjamini-Hochberg correction (118). An additional filter for differentially expressed genes was set at 1.3-fold change in expression in either direction. Array data have been deposited in the NCBI Gene Expression Omnibus database [GSE65979]. Bioinformatic analyses of enriched gene sets were made in

Pathway Studio (Ariadne Genomics, Rockville, MD). Fisher’s exact test was applied to determine significantly enriched pathways. Gene Ontology (GO) categories used were provided within the software. Heat maps were generated using the Eisen Cluster and

Treeview software.

5.2.2 Immunoblotting

Primary antibody against p65, lamin B1 and PARP (Cell Signaling) were used at 1:1,000 dilution for incubation overnight. Quantification of protein levels was analyzed using the

55 densitometric analysis package in ImageJ software (version 10.2) on triplicate samples of western blots.

5.2.3 qPCR primers

Primers used for mRNA detection were as follows:

Human RELA: F: 5’-GGAGCACAGATACCACCAAG-3’

R: 5’-GAGCTCAGCCTCATAGAAGC-3’

Human RELB: F: 5’-ACATATCCGTGGTGTTCAGC-3’

R: 5’-ATTGACAGTCACGGGCTCT-3’

Human NFKB1: F: 5’-CCGCTTGGGTAACTCTGTTT-3’

R: 5’-TGGGGTGGTCAAGAAGTAGT-3’

Human NFKB2: F: 5’-GATCTGCGCCGTTTCTGT-3’

R: 5’-CCGCTGCCTCTGAAGTTTT-3’

Human BCL3: F: 5’-CATTTACTCTACCCCGACGA-3’

R: 5’-GTGTCTCCATCCTCATCCAC-3’

Human ABTB2: F: 5’-GGGTGTGGAGGAAAGTGATG-3’

R: 5’-CTCAGCGCTGTAGTACATGG-3’

Human FOXO1: F: 5’-CAAGAGGGTCCTGGAGACA-3’

R: 5’-CTAGCATTTGAGCTGGTTCG-3’

Human CDKN1A: F: 5’-AGGTGGACCTGGAGACTCTCAG-3’

R: 5’-TCCTCTTGGAGAAGATCAGCCG-3’

Human PMAIP1: F: 5’-CTGGAAGTCGAGTGTGCTAC-3’

R: 5’-AGGTTCCTGAGCAGAAGAGT-3’

Human SGPP2: F: 5’-CTCCTCCCCTCCAGTTGTAA-3’

56 R: 5’-AGATAAGGAGGGTGAAGGCA-3’

Human EFNA1: F: 5’-CTGGTGGAGCATGAGGAGTA-3’

R: 5’-CGCTGGAACTTCTCAGACAG-3’

Human JUNB: F: 5’-GGCAGCTACTTTTCTGGTCA-3’

R: 5’-GTAAAAGTACTGTCCCGGGG-3’

Human GCH1: F: 5’-AGCTGAACCTCCCTAACCTG-3’

R: 5’-TAGCCCTTGGTGAAGAACTG-3’

Human ICAM1: F: 5’-TGACGAAGCCAGAGGTCTCAG-3’

R: 5’-AGCGTCACCTTGGCTCTAGG-3’

5.3 Results and Discussion

5.3.1 ERβ modulates NFkB RELA(p65) nuclear translocation in colon cancer cell lines

From preliminary data, ERβ was reported to be responsible for downregulation of inflammatory networks through upregulation of CAV1 and downregulation of IL-6 (41).

NFkB is a family of transcription factors that are translocated to the nucleus upon stimulation. Therefore, it is possible that one of the ways through which ERβ affects the inflammatory cascade is via interference of the nuclear translocation of the NFkB transcription factor. In the absence of inflammatory signaling molecules, RELA(p65) subunits are kept inactive in the cytoplasm by IK-complexes. When there is stimulation by cytokines or chemokines, RELA(p65) subunits are released from the inhibitory complexes through phosphorylation and then translocated to the nucleus where it can

57 act as a transcription factor to regulate gene transcription. In SW480 and HT29 cells, in the absence of TNFα, most of the RELA(p65) subunits are in the cytoplasmic portion of the cell lysate (Figure 4.1, lanes 1-4, 9-12). Upon treatment with TNFα, we can see that the RELA subunit is now translocated to the nuclear portion of the cell lysate in both cell lines (Figure 4.1, lanes 4 and 12). This confirms that in these colon cancer cell lines,

TNFα does indeed stimulate RELA(p65) translocation to the nucleus. We then wanted to know whether ERβ affects RELA(p65) nuclear translocation. Interestingly, in SW480 cells, without any TNFα treatment, there is more RELA(p65) in the nucleus of ERβ- expressing SW480 cell line than in control SW480 cells without ERβ expression (Figure

4.1, lanes 2 and 6).

In HT29 cells, the presence of ERβ itself does not enhance nuclear translocation of

RELA subunit like in SW480 cells (Figure 4.1, lanes 10 and 14) rather ERβ inhibits the nuclear translocation by TNFα treatment. ERβ-mediated effect on RELA(p65) translocation is unique to each cell line. When treated with TNFα, we do observe nuclear translocation of RELA(p65) subunit in SW480 cells, but it is independent of ERβ (Figure

5.1, lanes 6 and 8). In contrast however, in HT29 cells, ERβ attenuates the nuclear translocation of the RELA(p65) subunit when cells are treated with TNFα (Figure 4.1, lanes 12 and 16).

Clearly in SW480 and HT29, ERβ itself and ERβ in conjunction with TNFα, play different roles in RELA(p65) nuclear translocation. In a study on RASMCs, ERβ attenuated the

NFkB response by recruitment of RELA(p65) unit to the promoter region of IkBα, thus upregulating the levels of the inhibitor of NFkB activation (117). Therefore, it is plausible

58 that nuclear RELA(p65) is recruited to the promoter region of the inhibitor of NFkB activation and not the inflammatory response genes in SW480 ERβ expressing cells in the absence of TNFα stimulation. This would result in more nuclear RELA(p65), but an attenuated inflammatory cellular response. In HT29 cells, ERβ inhibited the translocation of the RELA(p65) subunit. What is unknown is whether this inhibition is dependent on the phosphorylation of IK-complexes. ERβ may inhibit phosphorylation and degradation of IkBα, the subunit that is responsible for holding RELA(p65) inactive in the cytoplasm or ERβ may inhibit the kinase activity of IKK-NEMO complex that is responsible for phosphorylation of IkBα.

Figure 4.1 RELA(p65) nuclear translocation in SW480 and HT29 cell lines. TNFα and ERβ effects on

RELA nuclear translocation in SW480 and HT29 colon cancer cell lines.

59 5.3.2 ERβ inhibits TNFα activation of NFκB-response element in

SW480 cells

Previous published studies have described the estrogenic effects of ERα on the NFkB inflammatory response in breast cancer cell lines (114,119). We also observed that in

SW480 cells with ERβ overexpressed, in the absence of TNFα, there was more nuclear

RELA(p65) subunit in the nucleus (Figure 4.1, lane 6). We wanted to examine whether the presence of ERβ in SW480 would interfere with the NFkB response. When SW480 cells were transfected with an NFkB-RE luciferase reporter construct, we see that TNFα, in the absence of ERβ, can elicit a luciferase response, but when ERβ is present, the response is no longer present (Figure 4.2). This suggests that in SW480 cells, the presence of ERβ can attenuate the NFkB response. Therefore, ERβ, through direct or indirect mechanisms, can block NFkB transcription factors from binding to their response elements. From our nuclear translocation results, SW480 cells with stably expressing

ERβ, had more RELA(p65) subunit in the nuclear fraction even in the absence of TNFα

(Figure 4.1, lanes 2 and 6). Along with these luciferase assays, it is possible that even with RELA(p65) subunits in the nucleus, ERβ can interfere with RELA(p65) transcription factor binding. Studies in human endothelial cells have actually shown that ERβ can recruit RELA(p65) to the promoter region of the IkBα gene while inhibiting binding of the

NFkB transcription factor to MCP-1 and CINC-2β genes (117), resulting in attenuation of

TNFα-induced inflammation.

What is also surprising is that the combination of E2 and TNFα in absence of ERβ can diminish the TNFα response in SW480 cells. It is possible that in our control cells lacking

ERβ, there are other estrogenic responses like the membrane bound G protein-coupled

60 estrogen receptor (GPER). GPER has functions independent from ERα and ERβ. In patients suffering from irritable bowel syndrome (IBS), colonic biopsies revealed high expression of GPER in mast cells and no expression of either ERα or ERβ (120). It has been shown that GPER can bind E2 and mediate non-genomic signaling. In ERα- positive MCF-7 cells, GPER antagonizes ERα resulting in cell growth inhibition (121). In the breast cell line MCF10A, activation of GPER by estrogenic compounds induced growth in these non-tumorigenic cells (122). Even though GPER is membrane bound protein and our NFkB-response element luciferase is in the nucleus, through indirect mechanisms, GPER can still interfere with the NFkB response. Clearly, in our colon cancer cell lines, we cannot exclude the possibility of GPER playing a role in inflammatory processes involving estrogen signaling.

Figure 4.2 NFkB luciferase response. In SW480 cells, the ERβ reduces the luciferase activation in control cells treated with TNFα. The combination of E2 and TNFα also reduces the luciferase signal.

61 5.3.3 TNFα induces genome-wide transcription in colon cancer cell lines

Studies in breast cancer cell lines have dissected the transcriptomic changes of ERα and NFkB in breast cancer cell lines (114). It is known that for certain groups of genes,

ERα can inhibit NFkB from binding to DNA, but for other groups of genes, binding of

ERα and NFkB can be synergistic (114,123). In fact, in the more aggressive luminal B type breast cancer, ERα and NFkB act in concert to upregulate the anti-apoptotic gene

BIRC3 (124). Another study used TNFα stimulation on human microvascular endothelial cells (HMECs) and human umbilical vein endothelial cells (HUVECs) reported that after

4 hours of TNFα treatment, up-regulated genes included those controlling cytokines, chemokines, and adhesion molecules. These were all involved in inflammation, apoptosis and immune systems (125). This study on HMEC and HUVEC was a quantitative-PCR-based study examining expression of 55 selected genes, but the TNFα response in these cells is independent of ERs. To our knowledge, there have been no genome-wide studies looking at the transcriptomic changes in colon cancer cell lines

SW480 and HT29 and the effects of ERβ in combination with TNFα. To better understand the genome-wide regulation of ERβ on the inflammatory processes, we utilized bead microarray to better assess changes in the transcriptome of colon cancer cell lines SW480 and HT29 with and without ERβ overexpression in combination with

TNFα to examine the influence of the combinatory effect of ERβ and TNFα has on the transcriptome.

When SW480 and HT29 (control cells lacking ERβ) were treated with TNFα for 2 hours, a total of 206 and 774 genes were significantly (Figure 4.3). Of these TNFα responsive

62 genes, 108 genes were changed in both cell lines. These 108 genes here referred to as core TNFα genes. Of these 108, all but 7 genes were upregulated, suggesting that the addition of TNFα to the cells has stimulatory effect. We note that in addition, 98 genes changed by TNFα were unique to SW480 and 666 genes changed were unique to HT29, suggesting that in addition to common TNFα responsive genes, there exist TNFα- responsive genes that are cell line specific. Also, there are more uniquely changed genes in HT29 cell line compared to SW480. Table 4.1 lists the top most differentially expressed genes in the core TNFα responsiveVenn Diagram group.

SW480 HT29

98 108 666

Figure 4.3 TNFα responsive genes in SW480 and HT29 cell lines. A total of 872 genes were differentially expressed following 2 hours of TNFα treatment in SW480 and HT29 cell lines. 108 genes were commonly differentially expressed, while 98 were uniquely changed in SW480 and 666 genes were uniquely changed in HT29.

Unique objects: All = 872; S1 = 206; S2 = 774

63 Table 4.1 Top 20 core TNFα responsive genes. List of genes that are most differentially upregulated in both cell lines SW480 and HT29 following 2 hours of TNFα treatment.

Gene SW480,*fold%change p,value HT29,*fold%change p,value CCL20 29.12726699 0.023 57.32352662 1.36E+04 BIRC3 17.325711 0.013 32.95757308 1.05E+03 IL8 14.21802386 0.022 56.67956017 2.42E+05 CD83 13.23600289 0.006 9.856950423 1.26E+04 CXCL1 12.81989263 0.012 25.5078676 6.73E+06 BCL3 12.356765 0.017 9.850537642 2.56E+05 TNFAIP3 12.11116604 0.007 14.56277754 6.60E+05 CSF2 10.84231895 0.041 7.473323731 3.05E+04 SDC4 7.371006997 0.037 9.296629269 2.34E+05 TNFAIP2 6.864500659 0.016 12.30169257 2.37E+05 NFKBIA 6.462717293 0.007 12.92003479 6.93E+05 NFKBIZ 6.072459547 0.022 1.502024946 5.59E+03 CX3CL1 5.721017416 0.021 11.90498617 1.73E+03 SLC25A24 5.612434527 0.020 14.62114138 2.42E+05 RELB 5.05774507 0.007 6.939140534 2.65E+04 NUAK2 4.696245742 0.037 5.729643159 9.48E+04 PLAU 4.689748056 0.006 4.002086435 1.05E+02 IL32 4.083372165 0.018 3.891112033 3.81E+04 CXCL6 4.021849774 0.044 2.097241547 1.94E+03 IRF1 3.620823629 0.018 6.897811992 1.40E+03

To better understand genes that are regulated by TNFα, we compared our 108 core

TNFα responsive genes in SW480 and HT29 cells with TNFα-treated MCF-7 cells from a previous study in breast cancer cell line (114). Results show that 18 genes are in common between the difference cancer cell lines (Figure 4.4). Of these 18 genes, 15 have a RELA(p65) binding site in the promoter region, suggesting that these genes are direct targets of NFkB.

Figure 4.4 TNFα-responsive genes in colon and breast. TNFα-responsive genes in both SW480 and

HT29 colon cancer cell lines compared with TNFα-responsive genes in breast cancer cell line MCF7.

5.3.4 TNFα and ERβ induces apoptosis in SW480 cells

To understand the overall functions of TNFα-responsive genes that are common in both colon cancer cell lines, we looked at the enriched gene ontologies of each group using

Pathway Studio software. Table 4.2 lists the overrepresented themes in the core TNFα responsive genes. The ontologies that are enriched are known inflammatory, immune, and apoptotic responses attributed to TNFα activation of the NFkB pathway.

Table 4.2 Gene Ontology for core TNFα-responsive genes. Overrepresented themes for genes that are differentially expressed and commonly regulated in SW480 and HT29 cells.

Gene$Ontology$category p/value inflammatory+response 5.08E410 apoptotic+process 8.05E409 immune+response 8.69E409 positive+regulation+of+reactive+oxygen+ species+metabolic+process 9.95E409

One of the main characteristics of colon cancer is the abundant of inflammation found in the area of tumor growth. Acute inflammation is helpful as it can induce apoptosis in the inflamed cells, but chronic inflammation can stimulate proliferation, cause cellular mutations to accumulate, which can drive tumorigenesis. Chronic inflammation is a result of increased proinflammatory factors (i.e., cytokines and interleukins) that promote proliferation of intestinal epithelial cells. It has been reported that TNFα can induced a wide range of cellular effects that leads to a more inflammatory and tumorigenic state, but TNFα can also trigger apoptosis through TNFR1 (126) and TRAILs (127). Thus,

TNFα can have dual roles in exerting proapoptotic and antiapoptotic functions. ERβ is known to be antitumorigenic by modulating the transcriptome to become more apoptotic.

In colon cancer cell lines studies, when ERβ-expressing SW480 were challenged with cisplatin, cells were more apoptotic compared to control cells (98). Gene ontologies reveal that apoptosis is enriched. We wanted to further examine whether treatment with

TNFα induces apoptosis and whether ERβ has any influence on TNFα-driven apoptosis.

In SW480 cell line, treatment with TNFα in the presence of ERβ induces apoptosis as measured with PARP cleavage after 72 hours. Figure 4.5 shows that ERβ in combination with TNFα treatment significantly enhances PARP cleavage, and therefore, triggers the cell the undergo apoptosis. TNFα or ERβ alone was not enough to significantly upregulate the PARP cleavage. This is in line with previous studies where the presence of ERβ along with a DNA-damaging agent cisplatin made cells more susceptible to apoptosis (98).

66 A

Figure 4.5 TNFα and ERβ induces apoptosis. A) ERβ and 72 hours TNFα treatment separately induces apoptosis through PARP cleavage in SW480 cell line. B) Densitometry data for cleaved PARP western blot band, p = 0.004.

5.3.5 ERβ modulates NFkB transcription factors in SW480 colon cancer cells

Indeed the NFkB cascade is an intricate balance of heterodimers of the different NFkB subunits (128). In the canonical NFkB activation pathway, the heterodimer RELA/p50 subunits are held inactive in the cytoplasm. Upon TNFα signaling and phosphorylation, it translocates to the nucleus to activate transcription (Figure 4.1). In SW480 cells, we observe that NFkB subunits RELA and RELB are transcriptionally upregulated after 2 hours of TNFα treatment, but with when ERβ is present, this upregulation is no longer

67 present (Figure 4.5). This suggests that ERβ can attenuate the NFkB response be downregulating mRNA levels of the NFkB transcription factors. A search in the promoter regions of RELA and RELB genes reveal that 9 kB upstream of the transcription start- site of RELB lies a potential ERβ binding site (Chr19: 45478166-45478295) (81).

Therefore, it is plausible that attenuation of RELB by ERβ may be through direct DNA- binding. ChIP assays would be able to determine ERβ binding to the promoter region of

RELB in the presence and absence of TNFα. We could not find potential ERβ binding sites in the regulatory region of RELA. However, ERs have been shown to regulation transcription through tethering mechanisms (40) or through a third mechanism where it regulates another transcription factor like MYC (98).

Figure 4.5 mRNA expression of NFkB subunits following TNFα treatment. RELA and RELB mRNA expression in control and ERβ SW480 cells after TNF treatment.

To add to the complexity of NFkB signaling, NFkB1 (p50) and NFkB2 (p52) subunits can heterodimerize to form another NFkB transcription factor complex. Since NFkB1 and

NFkB2 subunits lack a transactivating domain, this heterodimer serves as a transcriptional repressor. However, NFkB1/NFkB2 can bind to a third transcription factor

B-cell lymphoma 3 protein, BCL3, and activate transcription. In SW480 cells,

68 quantitative-PCR confirmation shows that when TNFα is present, NFkB1 and NFkB2 mRNA levels are unchanged. However, when TNFα and ERβ are both present, the mRNA levels are upregulated (Figure 4.6, left and middle panel), suggesting that the heterodimer of NFkB1/NFkB2 is serving as a transcriptional repressor. NFkB2 subunit is highly upregulated in the presence of both TNFα and ERβ. Upstream of NFkB2, is a potential ERβ binding site (Chr10: 104153638-104153847) (81) along with multiple

RELA(p6) binding sites. It is therefore plausible that ERβ and RELA(p65) can bind synergistically to upregulate NFkB2. A luciferase assay to measure potential activation by the two transcription factors would confirm out hypothesis.

The NFkB1/NFkB2 can become activated when bound to BCL3. In the same cells, BCL3 mRNA levels are increased with TNFα treatment, but when both TNFα and ERβ are present, this leads to the downregulation of BCL3 when compared to treatment with

TNFα alone, however, levels are still high when compared to untreated SW480 cells that do not express ERβ (Figure 4.6, right panel). Therefore, in SW480 cells, the presence of

ERβ downregulates TNFα response by downregulating the NFkB activating subunits:

RELA, RELB, and BCL3; while upregulating the heterodimer transcriptional repressor

NFkB1 and NFkB2.

Figure 4.6 mRNA levels of non-canonical NFkB subunits. mRNA levels of NFkB1, NFkB2 and BCL3 in

SW480 cell line.

Since we have observed the effect of ERβ on NFkB transcription factors, we wanted to know whether NFkB could affect an ER-response. When SW480 cells were transfected with a TATA-ERE luciferase reporter construct, we see that E2 elicits a luciferase response. The combination of E2 and TNFα has no additive effect (Figure 4.7).

Figure 4.7 TATA-ERE luciferase response. E2 activates ERE transactivation luciferase response in

SW480 cells.

70 5.3.6 ERβ modulates genome-wide transcription in colon cancer cell lines

To better appreciate the effect of ERβ on the NFkB cascade, we examined how TNFα responsive genes were affected by the presence of ERβ in SW480 and HT29 cells. In

SW480 cell line, 208 genes were significantly changed by TNFα. These genes, when

ERβ is present along with TNFα, can be clustered into three categories: enhanced, attenuated, or unchanged. Figure 4.8 shows a heatmap representative along with qPCR confirmation of some of these groups of genes changed by TNFα and/or ERβ in SW480 cells. In addition, we have measured the NFkB transcription factors that were changed by ERβ (Figure 4.5 and 4.6).

A B

Figure 4.8 Clustered heatmap and qPCR confirmation of genes changed by TNFα and/or ERβ in

SW480 cells. A) Heatmap representative of genes significantly changed by TNFα, with and without ERβ, red represents upregulation, green represents downregulation; B) qPCR confirmation of genes represented in each group: attenuated or enhanced by ERβ in the presence of TNFα.

To better understand the transcriptomic changes, we searched for common ontologies of the overall TNFα regulated genes in SW480 (208 genes total), then separately, we examined the group of genes attenuated and enhance by ERβ. For the 208 genes that were TNFα responsive in SW480 after 2 hours treatment, these fall within normal inflammatory responses in the cell (Table 4.3A). For the group of genes that were attenuated in the presence of TNFα and ERβ combined, responses to chemical and organic substances ontologies are overrepresented, but these do not show a clear network that ERβ could be influencing (Table 4.3B). When we look at the ontologies for the enhanced genes, we see that the category immune response is overrepresented

(Table 4.3C). This suggests that ERβ further turns on the immune response in the cell.

72 Table 4.3 Gene Ontology categories for TNFα-regulated genes in SW480 cells after 2 hours TNFα treatment. A) Gene ontologies for TNFα-regulated genes fall within known inflammatory responses. B)

Gene ontologies for genes that were TNFα-responsive, but where attenuated in the presence of ERβ. C)

Gene ontologies for genes that were TNFα-responsive, but where enhanced in the presence of ERβ.

A TNFα%regulated Gene$Ontology$category p/value inflammatory%response 5.08E;10 apoptotic%process 8.05E;09 immune%response 8.69E;09 positive%regulation%of%reactive%oxygen%species%metabolic% process 9.95E;09

B ERβ%attenuated Gene$Ontology$category p/value response%to%organic%substance 4.96E;03 response%to%oxygen;containing%compound 5.09E;03 cellular%response%to%chemical%stimulus 7.93E;03 cellular%response%to%organic%substance 1.12E;02 response%to%chemical 6.98E;02

C ERβ%enhanced Gene$Ontology$category p/value regulation%of%response%to%stimulus 7.74E;04 positive%regulation%of%macromolecule%metabolic%process 2.14E;03 positive%regulation%of%cellular%metabolic%process 3.69E;03 negative%regulation%of%cellular%process 4.76E;03 immune%response 6.56E;03

Similarly, we looked at the same type of regulatory effect of TNFα with and without ERβ in HT29 cells. HT29 cells had a stronger response to TNFα than SW480 cells with 774 genes changed by TNFα. With the addition of ERβ, we see that similar to SW480, there are groups of genes that are attenuated and enhanced by the combination of TNFα and

73 ERβ. Figure 4.9 shows a heatmap representative of the regulation of TNFα and TNFα in combination with ERβ (Figure 4.9A) and qPCR confirmation of some of the attenuated and enhanced genes (Figure 4.9B).

A B

Figure 4.9 Clustered heatmap and qPCR confirmation of genes changed by TNFα at 2 hours, with and without ERβ in HT29 cells. A) Heatmap representative of genes significantly changed by TNFα with and without ERβ, red represents upregulation, green represents downregulation; B) qPCR confirmation of genes represented in each group: attenuated or enhanced by ERβ in the presence of TNFα.

74 GO analysis for HT29 cells show categories that are different from the known inflammatory responses as in SW480 cells. Overrepresented genes in HT29 are mainly involved with cellular proliferation and apoptosis (Table 4.4).

75 Table 4.4 Gene Ontology categories for TNFα responsive genes in HT29 cells after 2 hours TNFα treatment. A) Gene ontologies for TNFα responsive genes fall within categories pertaining to growth and proliferation of cells. B) Gene ontologies for genes that were TNFα responsive, but where attenuated in the presence of ERβ. C) Gene ontologies for genes that were TNFα responsive, but where enhanced in the presence of ERβ.

A TNFα%regulated Gene$Ontology$category p/value regulation%of%transcription,%DNA8dependent 4.28E823 negative%regulation%of%apoptotic%process 5.12E814 negative%regulation%of%cell%proliferation 1.51E811 transforming%growth%factor%beta%receptor%signaling% pathway 1.64E810 angiogenesis 1.65E810

B ERβ%attenuated Gene$Ontology$category p/value cellular%macromolecule%metabolic%process 1.96E-05 cellular%metabolic%process 2.53E-05 macromolecule%metabolic%process 1.01E-04 primary%metabolic%process 1.75E-04 regulation%of%cellular%macromolecule%biosynthetic% process 2.51E-04

C ERβ%enhanced Gene$Ontology$category p/value organ%development 1.64E-04 system%development 1.20E-03 negative%regulation%of%transcription%from%RNA% polymerase%II%promoter 1.57E-03 biological%regulation 2.43E-03 regulation%of%cell%proliferation 3.74E-03

We have examined TNFα and TNFα in combination with ERβ responses in SW480 and

HT29 cells at 2 hours after treatment separately. To focus in on core TNFα genes and how they may be affected in both cell lines, we extracted the 108 core TNFα genes and clustered these genes accordingly. Figure 4.10 is a clustered heatmap of these 108 genes in both cell lines in response to TNFα and TNFα in combination with ERβ in both cell lines. Most of the core TNFα are upregulated and remain upregulated even when

ERβ is present. The few genes that were downregulated by TNFα remain downregulated.

Figure 4.10 Clustered heatmap of core TNFα genes. Heatmap representative of genes significantly changed by TNFα with and without ERβ, in SW480 and HT29 cell lines, red represents upregulation, green represents downregulation.

5.3.7 ERβ modulation on genome-wide transcription at 24 hours

We have seen and describe the short-term (2 hours) TNFα effects on transcription in

SW480 and HT29 cells. We wanted to know whether these effects by TNFα and TNFα in combination with ERβ were regulated at a longer time-point, 24 hours.

In SW480 cells, after 24 hours of TNFα treatment, we see that the number of genes that are TNFα responsive increased from 206 to 313, 55 genes that were TNFα responsive at 2 hours remained responsive at 24 hours (Figure 4.11A). This suggests that there are secondary and even tertiary effects of TNFα in the cell and these could translate to cellular or biological changes. We searched the gene list for common ontologies and at

24 hours, immune response is still an overrepresented group (Figure 4.11B). At 2 hours after TNFα treatment, inflammatory response is overrepresented in the gene ontologies

(Table 4.3A), but at 24 hours, we see that inflammatory response is no longer an overrepresented category, but present are immune and defense responses (Figure

4.11B). Figure 4.11C show a heatmap of the 55 common genes between 2 and 24 hours time-point. From the heatmap, we can see that most of the genes that were upregulated strongly by TNFα at 2 hours are not as strongly responsive after 24 hours. However, when ERβ is present at 24 hours with TNFα, there is a small subset of genes that are enhanced. These genes are upregulated similar to that at 2 hours.

78 A

B GO term P-value Response to stress 1.25E-14 Defense response 1.26E-13 Response to stimulus 3.82E-13 Resposne to biotic stimulus 7.65E-13 Immune response 1.40E-12

Figure 4.11 SW480 TNFα-responsive genes after 24 hours TNFα treatment. A) Venn diagram of TNFα- responsive genes at 2 and 24 hours in SW480 cells. B) Gene ontology categories of overrepresented genes that are responsive at 24 hours. C) Heatmap of 55 common genes between 2 and 24 hours time-point.

In HT29 cells, after 24 hours, we see that the number of genes that are TNFα- responsive decreased from 774 to 373 (Figure 4.12A). At 24 hours, 105 genes that were

TNFα responsive at 2 hours remained responsive. This suggests that the primary effects of TNFα on the transcriptome at 2 hours are still present at 24 hours. Gene ontology showed that processes related to cellular defense, immunity and responses to cytokines are still overrepresented (Figure 4.12B). Figure 4.12C show a heatmap of the 105 common genes between 2 and 24 hours time-point. The attenuating and enhancing effects of ERβ are not very clear at 24 hours due to the small number of genes.

79 A

B GO#term Gene$Ontology$category p/value response#to#cytokine 1.16E-31 defense#response 8.28E-28 immune#response 2.49E-26 immune#system#process 1.49E-25 cytokine3mediated#signaling#pathway 5.31E-23

Figure 4.12 HT29 TNFα-responsive genes after 24 hours TNFα treatment. A) Venn diagram of TNFα- responsive genes at 2 and 24 hours in HT29 cells. B) Gene ontology categories of overrepresented genes that are responsive at 24 hours. C) Heatmap of 105 common genes between 2 and 24 hours time-point.

5.4 Conclusion

One of the major risk factors for colon cancer is inflammation. Inflammation itself is helpful, but chronic inflammation is one of the hallmarks of tumor growth especially in the case for colon cancer. Inflammation affects all stages of colorectal cancer development including initiation, promotion, progression, and metastasis. Thus, controlling or minimizing inflammation can be the key to reducing colon cancer development. Using genome-wide techniques to study transcriptional changes in colon cancer cell lines

SW480 and HT29, we demonstrate that ERβ plays a role in modulating the inflammatory response brought by TNFα treatment of these cells.

Our results show that ERβ attenuates the inflammatory response of NFkB in a cell- specific manner. In SW480 cells, there is actually more RELA(p65) subunit of NFkB in the nucleus of ERβ expressing cells (Figure 4.1). However in these same cells, attenuation of inflammatory responses was through upregulation NFkB inhibitory subunits: NFKB1 and NFKB2, and downregulation of the activating subunit BCL3 (Figure

4.6). Also, in SW480 cells, ERβ was able to interfere with NFkB response element

(Figure 4.2) suggesting that through direct or indirect mechanism, ERβ can inhibit NFkB transcription factor binding on the DNA. The presence of ERβ along with TNFα treatment also induces apoptosis through PARP cleavage in SW480 cells (Figure 4.5).

In addition, ERβ overall attenuates genes that are TNFα responsive at 2 hours while it enhanced genes that were related to cellular inflammatory responses. At 24 hours, more genes were differentially regulated when compared with 2 hours. These genes reflected cellular responses that were related to cellular defense and immune responses.

In HT29 cells, we also saw that ERβ attenuates the overall inflammatory responses.

What is different is that in the presence of ERβ, there was less nuclear p65 (Figure 4.1, lanes 10 and 14). This suggests that ERβ attenuates the NFkB response by interfering with localization of NFkB transcription factor subunits. What is surprising is that despite

ERβ affecting nuclear localization of the p65 subunit, there were more genes responsive to TNFα in HT29 than in SW480 cells at 2 hours. These genes were mainly involved with processes such as proliferation and apoptosis. At 24 hours, there were fewer genes that were TNFα responsive when compared with 2 hours. Gene ontology revealed that most of the genes responsive at 24 hours were cytokine and cellular defense responses.

Overall, our data show that ERβ has potential regulatory effects on NFkB signaling by attenuation of TNFα responses. This may present a novel preventative or therapeutic approach for the treatment of inflammatory-associated colorectal cancers.

6. Concluding remarks and future directions

Colorectal cancer remains the second leading cause of cancer death in the United

States amongst men and women. Preventative and targeted therapies against early stages of colon cancer remains to be developed. Epidemiological studies have implicated the antiproliferative and anti-inflammatory potential of using ERβ as a target for preventative and targeted therapy in the early stages of cancer. Here we have demonstrated an important role of ERβ in colon cancer development. Colon cancer has two facets: proliferation and inflammation. We have shown that as cancer develops, ERβ is lost while inflammation is increased. In order to dissect the molecular mechanisms of

ERβ, we used colon cancer cell lines to examine changes in the miRnome as well as transcriptome.

83 In chapter one, using colon cancer cell lines, we examined the antiproliferative effect of

ERβ through changes in the miRnome. ERβ was responsible for downregulation of miR-

17-92 cluster. MiR-17-92 cluster, also known as oncomiR-1, is responsible for promotion of cancers including breast, leukemia, and brain. In SW480 colon cancer cell line with

ERβ overexpression, miR-17-92 was significantly downregulated. Upstream of the miRNA cluster is a MYC binding site. We noted that the regulation of ERβ on miR-17-92 was through MYC downregulation. Thus, we have uncovered a regulation pathway where antiproliferative effects of ERβ occur through downregulation of MYC and subsequently, downregulation of miR-17-92 cluster. This suggests that ERβ has protective effects against colon cancer by inhibiting proliferation. What is difficult is that as colon cancer progresses, expression of ERβ is lost. Since ERβ is a nuclear receptor, it is possible to treat patients with ERβ-specific ligands as a preventative measure or in very early stages of colon cancer to maintain the antiproliferative effects of ERβ. During latter stages of cancer where expression of ERβ is lost, it is possible to target miR-17-92 cluster with miRNA inhibitors to decrease proliferation of transformed colonocytes. In liver cancer, systemic administration of miR-34a resulted in decreased tumor growth and regression (129). It is possible then to treat colon cancer patients with miR-17-92 inhibitors. Since miR-17-92 is a poly-cistronic miRNA, one could use a single miRNA or multiple miRNAs in this pathway in treatments. Because the nature of miRNAs is that they can target multiple mRNAs, further studies would have to be done looking at the specific miRNA down mRNA targets as well as potential off target effects.

In chapter two, again using colon cancer cell lines, we examined another branch of ERβ regulation of miRNAs, this time through miR-205. In chapter one, we saw that ERβ was

84 responsible for down-regulation of miR-17-92 cluster through downregulation of MYC.

Thus, ERβ controls an mRNA that in turns controls miRNAs. In this chapter, ERβ upregulates miR-205, therefore, it is ERβ that controls a miRNA that in turns controls an mRNA. In a previous study, overexpression of ERβ downregulated the transcription factor PROX1. MiR-205 can actually bind complementary to the 3’-UTR of PROX1 and target the mRNA for degradation. In our study, we show that upregulation of miR-205 by

ERβ is via direct DNA binding and that high levels of miR-205 is responsible for downregulation of PROX1 and thus makes cancerous cells more antiprolierative and more adhesive, less metastatic. This suggests that not only is ERβ responsible for decrease in proliferation, in cells that are becoming metastatic, ERβ can slow or inhibit these cells. Most of the time, when colon cancer reaches the metastatic stage, expression of ERβ is ablated. Therefore, treating patients with ERβ ligands would have very little efficacy since there is no receptor to target. However, one could use miRNA mimics to upregulate levels of miR-205 or siRNA technologies to downregulate transcription factor PROX1. Similarly to miRNA treatments described for the first chapter, one could treat patients with miR-205 mimic. What is of concern is that high levels of miR-205 in colon cancer is beneficial, but in the case of breast cancer, miR-205 was found at high levels in patient serum (130) and in human ovarian cancer cell lines, miR-205 was responsible for promoting invasion through VEGF (131). Clearly for miR-

205 and miRNAs that serve dual functions in different cancers, possible treatments would have to be targeted to tissue-specific sites where off-target effects are minimal.

Studies using colon cancer animal models with miR-205 mimic as treatment would be beneficial to observe the potential off-target effects in other tissues like breast or ovaries.

85 Lastly, in chapter three, using a whole-genome approach, we observe that ERβ is responsible for attenuating the inflammatory response. While our first two chapters focused on specific pathways, this chapter looks at the overall transcriptomic changes that are attributed to ERβ in an inflammatory state. We found that ERβ has cell line specific mechanisms to attenuate the NFkB cascade. In SW480 cells, ERβ downregulated NFkB transcription factors subunits and enhanced apoptosis. In HT29 cells, ERβ inhibited nuclear translocation of NFkB p65 subunit. This suggests that ERβ does possess anti-inflammatory capabilities, but it is cell line specific. Inflammation is one of the hallmarks of cancer and many patients suffer from inflammatory bowel diseases years before the onset of cancer. In this instance, using ERβ-specific ligands in conjunction with NSAIDs to reduce inflammation would be effective in treating these ailments. Reducing inflammation helps to prevent the further transformation of colonocytes, but in cases where colon cancer is in the latter stages and ERβ is no longer present, one could target the downstream effectors of the NFkB cascade. ERβ has been shown to attenuate inflammation by increasing levels of the IkBα, the inhibitory subunit, or by inhibiting the nuclear translocation of RELA(p65). Further studies into the mechanism of how ERβ recruits RELA(p65) to the IkBα promoter or how ERβ prevents nuclear translocation would have to be done.

Although cell-line studies like ours is an crucial basic step to understanding the genetic changes that occur during colon cancer development, higher-order studies like xenografts, knock-out mouse technology, drug treatment trials, etc., are still needed. We did observe some of the same transcriptomic changes in our ERβiKO mice that we saw in our cell lines suggesting that transcriptomic changes reported here are indeed a result of

86 MiR-205 Mediates Downregulation of Transcription Factor PROX1 in Colon Cancer Trang Nguyen-Vu, Jun Wang, Weimin Xiao, Cecilia Williams

INTRODUCTION. Transcription factor prospero homebox 1 In ERb expressing SW480 cells, PROX1 levels are lower than in Expression levels of PROX1 and miR-205 levels in human colon (PROX1) is critical in organ development during controls. When miR-205 mimic miRNAs are transfected into adenoma samples show inverse relationship between PROX1 embryogenesis. Dysregulation of PROX1 has been correlated these cells, levels of PROX1 are further decreased. PROX1 mRNA and miR-205 (Figure 5). with diferent cancers including brain, breast, liver, pancreas, levels are directly regulated by miR-205 levels (Figure 2). colon, and esophagus. Previous studies have reported that in colon cancer cell lines, ERb exerts antitumorigenic efects PROX1 in SW480 Control ERb

1.2 80 through downregulation of PROX1 (1). In a followup study, we * 4 1 * 60 have found ERb can upregulate miR-205 (2). Interestingly, 3 PROX1 0.8 2 40 miR-205 can target PROX1 mRNA through base-pair binding in 0.6 1 20 the 3’-UTR region of PROX1 mRNA (3). In this current study, we 0.4 0.2 0 0

further explore miR-205 regulation on PROX1 and it’s 0 implications in colon cancer development. Figure 2. Expression of PROX1 mRNA is directly MiR-205 Mediates Downregulationinfuenced by levels of miR-205 in SW480 cells. of Transcription Factor Conserved miRNA hsa-miR-205/PROX1 alignment In a third colon cancer cell line HT-29, addition of miR-205 3’ guCUGAGGCCA--CCUUACUUCCu 5’ hsa-miR-205 Trang Nguyen-Vu, Jun Wang, WeiminFigure 5. Inverse relationship Xiao, of PROX1 Cecilia and miR-205 in colonWilliams cancer clinical samples. PROX1 in Colondecreased Cancer levels of PROX1 on the mRNA (Figure 3a) and the INTRODUCTION. Transcription factor prospero homeboxprotein 1 In levels ERb expressing (Figure3b). SW480 cells, PROX1 levels are lower than in 5’ aaGUCUCUAUUAGCAAUGAAGGg 3’ PROX1 Expression levels of PROX1 and miR-205 levels in human colon (PROX1) is critical in organ development during controls. When miR-205 mimic miRNAs are transfected into adenoma samples show inverse relationship between PROX1 embryogenesis. Dysregulation of PROX1 has been correlated these cells, levels of PROX1 are further decreased. PROX1 mRNA CONCLUSIONS. As colon cancer progresses, ERb expression is a) and miR-205 (Figure 5). MATERIALS & METHODS.with diferent Using cancers colon including cancer brain, cell breast, lines SW480liver, pancreas, levels are directly regulated byPROX1 miR-205 levelsControl (Figure 2). lost. Currently, no colon cancer cell lines express measurable colon, and esophagus. Previous studies have reported that in 1.2 ERb and HT-29 with stable ERb expression and addition colon cell 1 levels of ERb. When ERb is reexpressed in these colon cancer cell colon cancer cell lines, ERb exerts antitumorigenic efects 0.8 PROX1 in SW480 Control lines, PROX1 mRNA levels are decreased while miR-205 levels are 0.6 ERb

line SW403 with no ERb expression, we measured levels of 1.2 80 through downregulation of PROX1 (1). In a followup study, we 0.4 * 4 1 * increased. Previous studies have60 shown ERb directly ERβ. A big hurdle inmiR-205 studying and colon PROX1. havecancer found To and further ERb ER canβ is examine upregulate that as thecancer miR-205 regulation progresses, (2). Interestingly, of ER β 0.2 3 PROX1 0 0.8 downregulation2 of PROX1 and this downregulation40 is associated miR-205 on PROX1,miR-205 we transfected can target PROX1 SW480 mRNA and through HT-29 base-pair cells with binding in 0.6 1 20 is lost. Thus it is important to look at thetwo 3’-UTR different region aspects: of PROX1 ERmRNAβ signal (3). In this before current ER study,β is welost 0.4 with decreased proliferation. ERb also upregulates miR-205. In miR-205 mimics and measured PROX1 levels on the mRNA and 0.2 0 0

further explore miR-205 regulation on PROX1 and it’s 0 this study, we have shown that miR-205 can directly infuence protein levels. In SW403 cell lines, we transiently transfected b) and downstream ERβ signaling cascade.implications Since in colon nuclear cancer development. receptors are transcription + + ERbFigure 2. Expression of PROX1 mRNA is directly expression of PROX1. Transfection experiments using miR-205 with ERb plasmid vector and measured changes in miR-205. miR-205infuenced by levels of+ miR-205 in SW480 +cells. Conserved miRNA hsa-miR-205/PROX1 alignment mimics were successful in downregulating PROX1 levels in factors that can bind ligand, it is possible to administer ERβ-specific ligands as a PROX1 In a third colon cancer cell line HT-29, addition of miR-205 diferent colon cancer cell lines. Clinical data also show that ERb RESULTS. Regulation3’ guCUGAGGCCA--CCUUACUUCCu of 5’ hsa-miR-205 Figure 5. Inverse relationship of PROX1 and miR-205 in colon cancer clinical samples. b) miR-205 in SW403 decreased levels of PROX1 on the mRNA (Figure 3a) and the expression is lost as cancer progresses while PROX1 levels are preventative measurePROX1 for those is through individuals ERb (ESR2) at risk for colon3.5 cancer. Once*** colon cancer protein levels (Figure3b). 5’ aaGUCUCUAUUAGCAAUGAAGGg3 3’ PROX1 increased. Clinical data also refects the inverse relationship and miR-205 (Fig. 1a). 2.5 Figure 3. Expression of PROX1 mRNA is directly infuenced by miR-205 in HT-29 cells both on 2 the mRNA (a) and protein (b) levels. CONCLUSIONS.between As PROX1 colon cancerand miR-205. progresses, ERb expression is develops and ERβ isTransient no longer ERb detected, transfection it is possiblein to1.5 activate downstream signaling a) 1 PROX1 Control lost. Currently, no colon cancer cell lines express measurable MATERIALS & METHODS. Using colon cancer cell lines SW480 1.2 ERb SW403 cells increased 0.5 1 levels ofFUTURE ERb. When DIRECTIONS. ERb is reexpressed To in furtherthese colon investigate cancer cell transcriptional and HT-29 with stable ERb0 expression and addition colon cell cascade of ERβ withmiR-205 miRNA/siRNA levels (Fig. 1b). therapeutics Four and/or Control suppressingERb inflammatoryIn clinical samples, 0.8expression of ERb is downregulated as colonlines, PROX1 mRNA levels are decreased while miR-205 levels are line SW403 with no ERb expression, we measured levels of 0.6 regulation of PROX1 by miR-205, we will use luciferase assay c) cancer progresses 0.4whereas PROX1 levels are increased (Figureincreased. Previous studies have shown ERb directly diferent colon miR-205 cancer and cell PROX1. To further examine the regulation of 0.2 measuring miR-205 binding to 3’UTR region of PROX1 mRNA. 0

processes using NSAIDs and other drugs. Figure 5.1Correlation shows of PROX1 the and overall miR-205 levels regulation in of miR-205 on PROX1, we transfected SW480 and HT-29 cells with4). downregulation of PROX1 and this downregulation is associated lines show inverse colon cancer cell lines 4.5 with decreased proliferation. ERb also upregulates miR-205. In relationship betweenmiR-205 PROX1 mimics and4 measured PROX1 levels on the mRNA and ERb and PROX1 levels in clinical samples PROX1 300 REFERENCES. ERβ in colon epithelial cells as well asprotein the impact levels. In on SW4033.5 cellular cell effects.lines, we transiently transfected this study, we have shown that miR-205 can directly infuence 3 b) ERb and miR-205 levels (Fig. 1c). 250 + + 2.5 ERb expression(1) K. of PROX1. Edvardsson, Transfection et al. experiments (2011) Estrogen using miR-205 receptor b induces with ERb plasmid vector and measured changes in miR-205. + + 2 miR-205PROX1 200 mimicsantiin were f successfulammatory in and downregulating antitumorigenic PROX1 networks levels in in colon cancer a) 1.5 PROX1 1 diferent colon cancer cell lines. Clinical data also show that ERb miR-205 150 RESULTS. Regulation0.5 of cells. Mol. Endocrinol., 25, 969-979. b) miR-205 in SW403 expression is lost as cancer progresses while PROX1 levels are 0 PROX1 is through ERb (ESR2) 3.5 *** 100 A6enua.on(of(the( Decreased(cell(prolifera.on( SW403 SW480 (2) K. Edvardsson, T. Nguyen-Vu, et. al. (2013) Estrogen receptor 3 increased. Clinical data also refects the inverse relationship and miR-205 (Fig. 1a). 2.5 inﬂammatory(cascade( and(migra.on( Figure50 3. Expression of PROX1 mRNA is directly infuenced by miR-205 in HT-29 cells both on beta expression induces changes in the microRNA pool in Figure 1. Regulation of ESR2 (ERb), PROX1,2 and miR-205. a) Inhibition the mRNA (a) and protein (b) levels. between PROX1 and miR-205. Transient ERb transfectionof PROX1 by ESR2 in and miR-205. b) miR-2051.5 levels in transiently ERb transfection in SW403 cells. c) Correlation1 of PROX1 and miR-205 0 human colon cancer cells. Carcinogenesis. Jul, 34(7): 1431-41. SW403 cells increased 0.5

levels in 4 diferent colon cancer cell lines. Control FUTURE DIRECTIONS. To further investigate transcriptional 0 Control miR(17$ERb Figure 4. Expression of ERb and PROX1 in normal and colon cancer clinical samples (3) microRNA.org miR-205 levels (Fig. 1b). FourMYC$ In clinical samples, expression of ERb is downregulated as colon regulation of PROX1 by miR-205, we will use luciferase assay diferent colon cancer cell c) cancer progresses whereas PROX1 levels are increased (Figure measuring miR-205 binding to 3’UTR region of PROX1 mRNA. NFkB$signaling$ Correlation of PROX1 and miR-205 levels in lines show inverse colon cancer cell lines 4). 4.5 relationship between PROX1 4 ERb and PROX1 levels in clinical samples PROX1 300 REFERENCES. CombinaAon$of$ERβ$agonist$ miRNAs3.5 $are$also$used$as$ and miR-205 levels (Fig. 1c). 3 ERb 250 (1) K. Edvardsson, et al. (2011) Estrogen receptor b induces along$with$NSAIDs$to$ 2.5 biomarkers$ 2 PROX1 a) 200 antiinfammatory and antitumorigenic networks in colon cancer control$inﬂammaAon$ 1.5 1 miR-205 150 0.5 cells. Mol. Endocrinol., 25, 969-979. 0 100 SW403 SW480 (2) K. Edvardsson, T. Nguyen-Vu, et. al. (2013) Estrogen receptor 50 beta expression induces changes in the microRNA pool in Figure 1. Regulation of ESR2 (ERb), PROX1, and miR-205. a) Inhibition of PROX1 by ESR2 and miR-205. b) miR-205 levels in transiently ERb transfection in SW403 cells. c) Correlation of PROX1 and miR-205 0 human colon cancer cells. Carcinogenesis. Jul, 34(7): 1431-41. levels in 4 diferent colon cancer cell lines. Control Figure 4. Expression of ERb and PROX1 in normal and colon cancer clinical samples (3) microRNA.org

Increased(cell(adhesion( and(decreased(metastasis(

PROX1$currently$being$used$ as$a$prognosAc$marker$

Figure 5.1 Overall schematic of cellular effects of ERβ. ERβ has anti-tumorigenic effects through regulation of miRNAs and PROX1 as well as anti-inflammatory effects through attenuation of the inflammatory cascade.

7. Bibliography

1. Cancer Facts & Figures 2015. 2014:1–56.

2. International WCRF. Colorectal cancer statistics. 2015.

3. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61(5):759–767.

4. Hisamuddin IM, Yang VW. Molecular Genetics of Colorectal Cancer: An Overview. Curr Colorectal Cancer Rep. 2006;2(2):53–59.

5. Penegar S, Wood W, Lubbe S, Chandler I, Broderick P, Papaemmanuil E, Sellick G, Gray R, Peto J, Houlston R. National study of colorectal cancer genetics. British Journal of Cancer. 2007;97(9):1305–1309. doi:10.1038/sj.bjc.6603997.

6. Cappell MS. Pathophysiology, clinical presentation, and management of colon cancer. Gastroenterol Clin North Am. 2008;37(1):1–24– v. doi:10.1016/j.gtc.2007.12.002.

7. Morris EJA, Penegar S, Whitehouse LE, QUIRKE P, Finan P, Bishop DT, Wilkinson J, Houlston RS. A retrospective observational study of

88 therelationship between family history andsurvival from colorectal cancer. British Journal of Cancer. 2013;108(7):1502–1507. doi:10.1038/bjc.2013.91.

8. Yang VW. APC as a checkpoint gene: The beginning or the end? Gastroenterology. 2002;123(3):935–939. doi:10.1053/gast.2002.35773.

9. Schlussel AT, Gagliano RA, Seto-Donlon S, Eggerding F, Donlon T, Berenberg J, Lynch HT. The evolution of colorectal cancer genetics-Part 1: from discovery to practice. J Gastrointest Oncol. 2014;5(5):326–335. doi:10.3978/j.issn.2078-6891.2014.069.

10. Bodmer WF, Bailey CJ, Bodmer J, Bussey HJ, Ellis A, Gorman P, Lucibello FC, Murday VA, Rider SH, Scambler P. Localization of the gene for familial adenomatous polyposis on chromosome 5. Nature. 1987;328(6131):614–616. doi:10.1038/328614a0.

11. Kwong LN, Dove WF. APC and Its Modifiers in Colon Cancer. Adv Exp Med Biol. 2009;656:85–106.

12. Nieuwenhuis MH, Vasen HFA. Correlations between mutation site in APC and phenotype of familial adenomatous polyposis (FAP): A review of the literature. Critical Reviews in Oncology/Hematology. 2007;61(2):153–161. doi:10.1016/j.critrevonc.2006.07.004.

13. Fevr T, Robine S, Louvard D, Huelsken J. Wnt/ -Catenin Is Essential for Intestinal Homeostasis and Maintenance of Intestinal Stem Cells. Molecular and Cellular Biology. 2007;27(21):7551–7559. doi:10.1128/MCB.01034-07.

14. Jo J, Nam CM, Sull JW, Yun JE, Kim SY, Lee SJ, Kim YN, Park EJ, Kimm H, Jee SH. Prediction of Colorectal Cancer Risk Using a Genetic Risk Score: The Korean Cancer Prevention Study-II (KCPS-II). Genomics Inform. 2012;10(3):175. doi:10.5808/GI.2012.10.3.175.

15. Schneikert J, Behrens J. The canonical Wnt signalling pathway and its APC partner in colon cancer development. Gut. 2007;56(3):417–425. doi:10.1136/gut.2006.093310.

16. Aoki K, Taketo MM. Adenomatous polyposis coli (APC): a multi-functional tumor suppressor gene. Journal of Cell Science. 2007;120(19):3327–3335. doi:10.1242/jcs.03485.

17. Baumgarten SC, Frasor J. Minireview: Inflammation: An Instigator of More Aggressive Estrogen Receptor (ER) Positive Breast Cancers. Molecular Endocrinology. 2012;26(3):360–371. doi:10.1210/me.2011-1302.

18. Terzić J, Grivennikov S, Karin E, Karin M. Inflammation and Colon Cancer. YGAST. 2010;138(6):2101–2114.e5. doi:10.1053/j.gastro.2010.01.058.

89 19. Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420(6917):860– 867. doi:10.1038/nature01322.

20. Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet. 2001;357(9255):539–545. doi:10.1016/S0140-6736(00)04046-0.

21. Guigoz Y, Dore J, Schiffrin EJ. The inflammatory status of old age can be nurtured from the intestinal environment. Curr Opin Clin Nutr Metab Care. 2008;11(1):13–20.

22. Grivennikov S, Karin E, Terzic J, Mucida D, Yu G-Y, Vallabhapurapu S, Scheller J, Rose-John S, Cheroutre H, Eckmann L, Karin M. IL-6 and Stat3 Are Requiredfor Survival of Intestinal Epithelial Cellsand Development of Colitis- Associated Cancer. Cancer Cell. 2009;15(2):103–113. doi:10.1016/j.ccr.2009.01.001.

23. Kim SE. The benefit-risk consideration in long-term use of alternate-day, low dose aspirin: focus on colorectal cancer prevention. Ann Gastroenterol. 2014;27(1):87–88.

24. Walker AJ, Grainge MJ, Card TR. Aspirin and other non-steroidal anti- inflammatory drug use and colorectal cancer survival: a cohort study. 2012;107(9):1602–1607. doi:10.1038/bjc.2012.427.

25. To SQ, Cheung V, Lazarus KA, Knower KC, Clyne CD. Molecular and Cellular Endocrinology. MOLECULAR AND CELLULAR ENDOCRINOLOGY. 2014;394(1-2):21–28. doi:10.1016/j.mce.2014.06.020.

26. Kuwano Y, Tominaga K, Kawahara T, Sasaki H, Takeo K, Nishida K, Masuda K, Kawai T, Teshima-Kondo S, Rokutan K. Free Radical Biology & Medicine. Free Radical Biology and Medicine. 2008;45(12):1642–1652. doi:10.1016/j.freeradbiomed.2008.08.033.

27. Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death and Differentiation. 2003;10(1):45–65. doi:10.1038/sj.cdd.4401189.

28. Gilmore TD. Introduction to NF-κB: players, pathways, perspectives. Oncogene. 2006;25(51):6680–6684. doi:10.1038/sj.onc.1209954.

29. Hendifar A, Yang D, Lenz F, Lurje G, Pohl A, Lenz C, Ning Y, Zhang W, Lenz HJ. Gender Disparities in Metastatic Colorectal Cancer Survival. Clinical Cancer Research. 2009;15(20):6391–6397. doi:10.1158/1078-0432.CCR-09- 0877.

30. Söderlund S, Granath F, Broström O, Karlén P, Löfberg R, Ekbom A, Askling J. CLINICAL—ALIMENTARY TRACT. YGAST. 2010;138(5):1697– 1703.e2. doi:10.1053/j.gastro.2010.02.007.

90 31. Grodstein F, Newcomb PA, Stampfer MJ. Postmenopausal hormone therapy and the risk of colorectal cancer: a review and meta-analysis. Am J Med. 1999;106(5):574–582.

32. Newcomb PA, Zheng Y, Chia VM, Morimoto LM, Doria-Rose VP, Templeton A, Thibodeau SN, Potter JD. Estrogen Plus Progestin Use, Microsatellite Instability, and the Risk of Colorectal Cancer in Women. Cancer Research. 2007;67(15):7534–7539. doi:10.1158/0008-5472.CAN-06-4275.

33. Fernandez E, La Vecchia C, Balducci A, Chatenoud L, Franceschi S, Negri E. Oral contraceptives and colorectal cancer risk: a meta-analysis. British Journal of Cancer. 2001;84(5):722–727. doi:10.1054/bjoc.2000.1622.

34. Giardiello FM, Hylind LM, Trimbath JD, Hamilton SR, Romans KE, Cruz- Correa M, Corretti MC, Offerhaus GJA, Yang VW. Oral contraceptives and polyp regression in familial adenomatous polyposis. Gastroenterology. 2005;128(4):1077–1080. doi:10.1053/j.gastro.2004.10.010.

35. Weige CC, Allred KF, Allred CD. Estradiol Alters Cell Growth in Nonmalignant Colonocytes and Reduces the Formation of Preneoplastic Lesions in the Colon. Cancer Research. 2009;69(23):9118–9124. doi:10.1158/0008-5472.CAN-09- 2348.

36. Thomas C, Gustafsson J-Å. The different roles of ER subtypes in cancer biology and therapy. Nature Publishing Group. 2011;11(8):597–608. doi:10.1038/nrc3093.

37. Heger Z, Zitka O, Krizkova S, Beklova M, Kizek R, Adam V. Molecular biology of beta-estradiol-estrogen receptor complex binding to estrogen response element and the effect on cell proliferation. Neuro Endocrinol Lett. 2013;34 Suppl 2:123–129.

38. Fogarty EA, Matulis CK, Kraus WL. Activation of estrogen receptor alpha by raloxifene through an activating protein-1-dependent tethering mechanism in human cervical epithelial cancer cells: a role for c-Jun N-terminal kinase. MOLECULAR AND CELLULAR ENDOCRINOLOGY. 2012;348(1):331–338. doi:10.1016/j.mce.2011.09.032.

39. Dahlman-Wright K, Qiao Y, Jonsson P, Gustafsson JA, Williams C, Zhao C. Interplay between AP-1 and estrogen receptor in regulating gene expression and proliferation networks in breast cancer cells. Carcinogenesis. 2012;33(9):1684–1691. doi:10.1093/carcin/bgs223.

40. Stender JD, Kim K, Charn TH, Komm B, Chang KCN, Kraus WL, Benner C, Glass CK, Katzenellenbogen BS. Genome-Wide Analysis of Estrogen Receptor DNA Binding and Tethering Mechanisms Identifies Runx1 as a Novel Tethering Factor in Receptor-Mediated Transcriptional Activation. Molecular and Cellular Biology. 2010;30(16):3943–3955. doi:10.1128/MCB.00118-10.

91 41. Edvardsson K, Ström A, Jonsson P, Gustafsson J-Å, Williams C. Estrogen Receptor β Induces Antiinflammatory and Antitumorigenic Networks in Colon Cancer Cells. Molecular Endocrinology. 2011;25(6):969–979. doi:10.1210/me.2010-0452.

42. Williams C, Edvardsson K, Lewandowski SA, Strom A, Gustafsson JA. A genome-wide study of the repressive effects of estrogen receptor beta on estrogen receptor alpha signaling in breast cancer cells. Oncogene. 2007;27(7):1019–1032. doi:10.1038/sj.onc.1210712.

43. Chang EC, Frasor J, Komm B, Katzenellenbogen BS. Impact of Estrogen Receptor β on Gene Networks Regulated by Estrogen Receptor α in Breast Cancer Cells. Endocrinology. 2006;147(10):4831–4842. doi:10.1210/en.2006- 0563.

44. Foley EF, Jazaeri AA, Shupnik MA, Jazaeri O, Rice LW. Selective loss of estrogen receptor beta in malignant human colon. Cancer Research. 2000;60(2):245–248.

45. Campbell-Thompson M, Lynch IJ, Bhardwaj B. Expression of estrogen receptor (ER) subtypes and ERbeta isoforms in colon cancer. Cancer Research. 2001;61(2):632–640.

46. Konstantinopoulos PA, Kominea A, Vandoros G, Sykiotis GP, Andricopoulos P, Varakis I, Sotiropoulou-Bonikou G, Papavassiliou AG. Oestrogen receptor beta (ERβ) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour's dedifferentiation. European Journal of Cancer. 2003;39(9):1251–1258. doi:10.1016/S0959-8049(03)00239-9.

47. Honma N, Yamamoto K, Ohnaka K, Morita M, Toyomura K, Kono S, Muramatsu M, Arai T, Ueki T, Tanaka M, Kakeji Y, Maehara Y, Okamura T, Ikejiri K, Futami K, Maekawa T, Yasunami Y, Takenaka K, Ichimiya H, Terasaka R. Estrogen receptor-β gene polymorphism and colorectal cancer risk: Effect modified by body mass index and isoflavone intake. Int J Cancer. 2012;132(4):951–958. doi:10.1002/ijc.27688.

48. Wu H, Xu L, Chen J, Hu J, Yu S, Hu G, Huang L, Chen X, Yuan X, Li G. Association of estrogen receptor beta variants andserum levels of estradiol with risk of colorectalcancer: a case control study. ??? 2012;12(1):1–1. doi:10.1186/1471-2407-12-276.

49. Passarelli MN, Phipps AI, Potter JD, Makar KW, Coghill AE, Wernli KJ, White E, Chan AT, Hutter CM, Peters U, Newcomb PA. Common Single- Nucleotide Polymorphisms in the Estrogen Receptor Promoter Are Associated with Colorectal Cancer Survival in Postmenopausal Women. Cancer Research. 2013;73(2):767–775. doi:10.1158/0008-5472.CAN-12-2484.

92 50. Martineti V, Picariello L, Tognarini I, Carbonell Sala S, Gozzini A, Azzari C, Mavilia C, Tanini A, Falchetti A, Fiorelli G, Tonelli F, Brandi ML. ERbeta is a potent inhibitor of cell proliferation in the HCT8 human colon cancer cell line through regulation of cell cycle components. Endocrine Related Cancer. 2005;12(2):455–469. doi:10.1677/erc.1.00861.

51. Hartman J, Lindberg K, Morani A, Inzunza J, Strom A, Gustafsson JA. Estrogen Receptor Inhibits Angiogenesis and Growth of T47D Breast Cancer Xenografts. Cancer Research. 2006;66(23):11207–11213. doi:10.1158/0008- 5472.CAN-06-0017.

52. Bielecki A, Roberts J, Mehta R, Raju J. Estrogen receptor-beta mediates the inhibition of DLD-1 human colon adenocarcinoma cells by soy isoflavones. Nutr Cancer. 2011;63(1):139–150. doi:10.1080/01635581.2010.516867.

53. Tu Z, Ma Y, Tian J, Li H, Akers W, Achilefu S, Gu Y. Estrogen receptor beta potentiates the antiproliferative effect of raloxifene and affects the cell migration and invasion in HCT-116 colon cancer cells. J Cancer Res Clin Oncol. 2012;138(7):1091–1103. doi:10.1007/s00432-011-1145-3.

54. Johnson CD, Esquela-Kerscher A, Stefani G, Byrom M, Kelnar K, Ovcharenko D, Wilson M, Wang X, Shelton J, Shingara J, Chin L, Brown D, Slack FJ. The let-7 MicroRNA Represses Cell Proliferation Pathways in Human Cells. Cancer Research. 2007;67(16):7713–7722. doi:10.1158/0008-5472.CAN- 07-1083.

55. Friedländer MR, Lizano E, Houben AJ, Bezdan D, ez-Coronel MNB, Kudla G, Mateu-Huertas E, Kagerbauer B, lez JG, Chen KC, LeProust EM, Mart EL, Estivill X. Evidence for the biogenesis of more than 1,000 novel human microRNAs. 2014;15(4):1–17. doi:10.1186/gb-2014-15-4-r57.

56. Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S, Shimizu M, Rattan S, Bullrich F, Negrini M, Croce CM. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA. 2004;101(9):2999–3004. doi:10.1073/pnas.0307323101.

57. Farazi TA, Spitzer JI, Morozov P, Tuschl T. miRNAs in human cancer. Ladanyi M, Hogendoorn PC, eds. J Pathol. 2010;223(2):102–115. doi:10.1002/path.2806.

58. Michael MZ, O' Connor SM, van Holst Pellekaan NG, Young GP, James RJ. Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol Cancer Res. 2003;1(12):882–891.

59. Nugent M, Miller N, Kerin MJ. MicroRNAs in colorectal cancer: Function, dysregulation and potential as novel biomarkers. European Journal of Surgical Oncology. 2020;37(8):649–654. doi:10.1016/j.ejso.2011.05.005.

93 60. Wu WKK, Law PTY, Lee CW, Cho CH, Fan D, Wu K, Yu J, Sung JJY. MicroRNA in colorectal cancer: from benchtop to bedside. Carcinogenesis. 2011;32(3):247–253. doi:10.1093/carcin/bgq243.

61. Hutchison J, Cohen Z, Onyeagucha BC, Funk J, Nelson MA. How microRNAs influence both hereditary and inflammatory-mediated colon cancers. Cancer Genetics. 2014;206(9-10):309–316. doi:10.1016/j.cancergen.2013.06.005.

62. Ahmed FE. miRNA as markers for the diagnostic screening of colon cancer. Expert Review of Anticancer Therapy. 2014;14(4):463–485. doi:10.1586/14737140.2014.869479.

63. Li T. MicroRNA-21 as a potential colon and rectal cancer biomarker. WJG. 2013;19(34):5615. doi:10.3748/wjg.v19.i34.5615.

64. Hartman J, Edvardsson K, Lindberg K, Zhao C, Williams C, Strom A, Gustafsson JA. Tumor Repressive Functions of Estrogen Receptor in SW480 Colon Cancer Cells. Cancer Research. 2009;69(15):6100–6106. doi:10.1158/0008-5472.CAN-09-0506.

65. Zhao C, Sun G, Li S, Shi Y. A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination. Nat Struct Mol Biol. 2009;16(4):365–371. doi:10.1038/nsmb.1576.

66. Bethke A, Fielenbach N, Wang Z, Mangelsdorf DJ, Antebi A. Nuclear Hormone Receptor Regulation of MicroRNAs Controls Developmental Progression. Science. 2009;324(5923):95–98. doi:10.1126/science.1164899.

67. Shah MS, Schwartz SL, Zhao C, Davidson LA, Zhou B, Lupton JR, Ivanov I, Chapkin RS. Integrated microRNA and mRNA expression profiling in a rat colon carcinogenesis model: effect of a chemo-protective diet. Physiol Genomics. 2011;43(10):640–654. doi:10.1152/physiolgenomics.00213.2010.

68. Smith LK, Shah RR, Cidlowski JA. Glucocorticoids Modulate MicroRNA Expression and Processing during Lymphocyte Apoptosis. Journal of Biological Chemistry. 2010;285(47):36698–36708. doi:10.1074/jbc.M110.162123.

69. Cochrane DR, Spoelstra NS, Howe EN, Nordeen SK, Richer JK. MicroRNA- 200c mitigates invasiveness and restores sensitivity to microtubule-targeting chemotherapeutic agents. Mol Cancer Ther. 2009;8(5):1055–1066. doi:10.1158/1535-7163.MCT-08-1046.

70. Eendebak RJAH, Lucassen PJ, Fitzsimons CP. Nuclear receptors and microRNAs: Who regulates the regulators in neural stem cells? FEBS Letters. 2011;585(5):717–722. doi:10.1016/j.febslet.2011.01.039.

71. Vrtačnik P, Ostanek B, Mencej-Bedrač S, Marc J. The many faces of

94 estrogen signaling. Biochem Med. 2014;24(3):329–342. doi:10.11613/BM.2014.035.

72. Gambari R. From microRNA functions to microRNA therapeutics: Novel targets and novel drugs in breast cancer research and treatment (Review). Int J Oncol. 2013. doi:10.3892/ijo.2013.2059.

73. Cochrane DR, Cittelly DM, Howe EN, Spoelstra NS, McKinsey EL, LaPara K, Elias A, Yee D, Richer JK. MicroRNAs link estrogen receptor alpha status and Dicer levels in breast cancer. HORM CANC. 2010;1(6):306–319. doi:10.1007/s12672-010-0043-5.

74. Bhat-Nakshatri P, Wang G, Collins NR, Thomson MJ, Geistlinger TR, Carroll JS, Brown M, Hammond S, Srour EF, Liu Y, Nakshatri H. Estradiol- regulated microRNAs control estradiol response in breast cancer cells. Nucleic Acids Research. 2009;37(14):4850–4861. doi:10.1093/nar/gkp500.

75. Maillot G, Lacroix-Triki M, Pierredon S, Gratadou L, Schmidt S, Benes V, Roche H, Dalenc F, Auboeuf D, Millevoi S, Vagner S. Widespread Estrogen- Dependent Repression of microRNAs Involved in Breast Tumor Cell Growth. Cancer Research. 2009;69(21):8332–8340. doi:10.1158/0008-5472.CAN-09- 2206.

76. Goh JN, Loo SY, Datta A, Siveen KS, Yap WN, Cai W, Shin EM, Wang C, Kim JE, Chan M, Dharmarajan AM, Lee AS-G, Lobie PE, Yap CT, Kumar AP. microRNAs in breast cancer: regulatory roles governing the hallmarks of cancer. Biol Rev Camb Philos Soc. 2015. doi:10.1111/brv.12176.

77. Paris O, Ferraro L, Grober OMV, Ravo M, De Filippo MR, Giurato G, Nassa G, Tarallo R, Cantarella C, Rizzo F, Di Benedetto A, Mottolese M, Benes V, Ambrosino C, Nola E, Weisz A. Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer. Oncogene. 2012;31(38):4196–4206. doi:10.1038/onc.2011.583.

78. Katzenellenbogen BS, Kendra KL, Norman MJ, Berthois Y. Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breast cancer cells grown in the short-term and long-term absence of estrogens. Cancer Research. 1987;47(16):4355–4360.

79. Webb P, Lopez GN, Greene GL, Baxter JD, Kushner PJ. The limits of the cellular capacity to mediate an estrogen response. Molecular Endocrinology. 1992;6(2):157–167. doi:10.1210/mend.6.2.1569962.

80. Carroll JS, Liu XS, Brodsky AS, Li W, Meyer CA, Szary AJ, Eeckhoute J, Shao W, Hestermann EV, Geistlinger TR, Fox EA, Silver PA, Brown M. Chromosome-Wide Mapping of Estrogen Receptor Binding Reveals Long- Range Regulation Requiring the Forkhead Protein FoxA1. Cell. 2005;122(1):33– 43. doi:10.1016/j.cell.2005.05.008.

95 81. Grober OM, Mutarelli M, Giurato G, Ravo M, Cicatiello L, De Filippo MR, Ferraro L, Nassa G, Papa MF, Paris O, Tarallo R, Luo S, Schroth GP, Benes V, Weisz A. Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation. BMC Genomics. 2011;12(1):36. doi:10.1186/1471-2164- 12-36.

82. Zhao C, Gao H, Liu Y, Papoutsi Z, Jaffrey S, Gustafsson JA, Dahlman- Wright K. Genome-Wide Mapping of Estrogen Receptor- -Binding Regions Reveals Extensive Cross-Talk with Transcription Factor Activator Protein-1. Cancer Research. 2010;70(12):5174–5183. doi:10.1158/0008-5472.CAN-09- 4407.

83. O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature. 2005;435(7043):839–843. doi:10.1038/nature03677.

84. Bui TV, Mendell JT. Myc: Maestro of MicroRNAs. Genes & Cancer. 2010;1(6):568–575. doi:10.1177/1947601910377491.

85. Kim JW, Mori S, Nevins JR. Myc-Induced MicroRNAs Integrate Myc-Mediated Cell Proliferation and Cell Fate. Cancer Research. 2010;70(12):4820–4828. doi:10.1158/0008-5472.CAN-10-0659.

86. Chang T-C, Yu D, Lee Y-S, Wentzel EA, Arking DE, West KM, Dang CV, Thomas-Tikhonenko A, Mendell JT. Widespread microRNA repression by Myc contributes to tumorigenesis. Nat Genet. 2007;40(1):43–50. doi:10.1038/ng.2007.30.

87. Diosdado B, van de Wiel MA, Droste JSTS, Mongera S, Postma C, Meijerink WJHJ, Carvalho B, Meijer GA. MiR-17-92 cluster is associated with 13q gain and c-myc expression during colorectal adenoma to adenocarcinoma progression. British Journal of Cancer. 2014;101(4):707–714. doi:10.1038/sj.bjc.6605037.

88. Yu G, Tang J-Q, Tian M-L, Li H, Wang X, Wu T, Zhu J, Huang S-J, Wan Y-L. Prognostic values of the miR-17-92 cluster and its paralogs in colon cancer. J Surg Oncol. 2011;106(3):232–237. doi:10.1002/jso.22138.

89. He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S, Powers S, Cordon-Cardo C, Lowe SW, Hannon GJ, Hammond SM. A microRNA polycistron as a potential human oncogene. Nature Publishing Group. 2005;435(7043):828–833. doi:10.1038/nature03552.

90. Zhang J, Xiao Z, Lai D, Sun J, He C, Chu Z, Ye H, Chen S, Wang J. miR-21, miR-17 and miR-19a induced by phosphatase of regenerating liver-3 promote the proliferation and metastasis of colon cancer. 2012;107(2):352–359. doi:10.1038/bjc.2012.251.

96 91. Yang L, Allred KF, Geera B, Allred CD, Awika JM. Sorghum Phenolics Demonstrate Estrogenic Action and Induce Apoptosis in Nonmalignant Colonocytes. Nutr Cancer. 2012;64(3):419–427. doi:10.1080/01635581.2012.657333.

92. Schleipen B, Hertrampf T, Fritzemeier KH, Kluxen FM, Lorenz A, Molzberger A, Velders M, Diel P. ER -specific agonists and genistein inhibit proliferation and induce apoptosis in the large and small intestine. Carcinogenesis. 2011;32(11):1675–1683. doi:10.1093/carcin/bgr188.

93. Dews M, Fox JL, Hultine S, Sundaram P, Wang W, Liu YY, Furth E, Enders GH, El-Deiry W, Schelter JM, Cleary MA, Thomas-Tikhonenko A. The Myc- miR-17 92 Axis Blunts TGF Signaling and Production of Multiple TGF - Dependent Antiangiogenic Factors. Cancer Research. 2010;70(20):8233–8246. doi:10.1158/0008-5472.CAN-10-2412.

94. Wong C-W, Komm B, Cheskis BJ. Structure−Function Evaluation of ER α and β Interplay with SRC Family Coactivators. ER Selective Ligands. Biochemistry. 2001;40(23):6756–6765. doi:10.1021/bi010379h.

95. Leo C, Li H, Chen JD. Differential mechanisms of nuclear receptor regulation by receptor-associated coactivator 3. J Biol Chem. 2000;275(8):5976–5982.

96. Grivas PD, Tzelepi V, Sotiropoulou-Bonikou G, Kefalopoulou Z, Papavassiliou AG, Kalofonos H. Estrogen receptor α/β, AIB1, and TIF2 in colorectal carcinogenesis: do coregulators have prognostic significance? Int J Colorectal Dis. 2009;24(6):613–622. doi:10.1007/s00384-009-0647-9.

97. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, Vadas MA, Khew-Goodall Y, Goodall GJ. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol. 2008;10(5):593–601. doi:10.1038/ncb1722.

98. Edvardsson K, Nguyen-Vu T, Kalasekar SM, Pontén F, Gustafsson JA, Williams C. Estrogen receptor expression induces changes in the microRNA pool in human colon cancer cells. Carcinogenesis. 2013;34(7):1431–1441. doi:10.1093/carcin/bgt067.

99. Elsir T, Smits A, Lindström MS, Nistér M. Transcription factor PROX1: its role in development and cancer. Cancer Metastasis Rev. 2012;31(3-4):793–805. doi:10.1007/s10555-012-9390-8.

100. Skog M, Bono P, Lundin M, Lundin J, Louhimo J, Linder N, Petrova TV, Andersson LC, Joensuu H, Alitalo K, Haglund CH. Expression and prognostic value of transcription factor PROX1 in colorectal cancer. British Journal of Cancer. 2011;105(9):1346–1351. doi:10.1038/bjc.2011.297.

101. Petrova TV, Nykänen A, Norrmén C, Ivanov KI, Andersson LC, Haglund C,

97 Puolakkainen P, Wempe F, Melchner von H, Gradwohl G, Vanharanta S, Aaltonen LA, Saharinen J, Gentile M, Clarke A, Taipale J, Oliver G, Alitalo K. Transcription Factor PROX1 Induces Colon Cancer Progression by Promoting the Transition from Benign to Highly Dysplastic Phenotype. Cancer Cell. 2008;13(5):407–419. doi:10.1016/j.ccr.2008.02.020.

102. Lu MH, Huang CC, Pan MR, Chen HH, Hung WC. Prospero Homeobox 1 Promotes Epithelial-Mesenchymal Transition in Colon Cancer Cells by Inhibiting E-cadherin via miR-9. Clinical Cancer Research. 2012;18(23):6416–6425. doi:10.1158/1078-0432.CCR-12-0832.

103. Orang AV, Safaralizadeh R, Feizi MAH, Somi MH. Diagnostic and Prognostic Value of miR-205 in Colorectal Cancer. Asian Pacific Journal of Cancer Prevention. 2014;15(9):4033–4037. doi:10.7314/APJCP.2014.15.9.4033.

104. JASSAM N, BELL SM, Speirs V, QUIRKE P. Loss of expression of oestrogen receptor beta in colon cancer and its association with Dukes' staging. Oncol Rep. 2005;14(1):17–21.

105. Wang X-Y, Wu M-H, Liu F, Li Y, Li N, Li G-Y, Shen S-R. Differential miRNA expression and their target genes between NGX6-positive and negative colon cancer cells. Mol Cell Biochem. 2010;345(1-2):283–290. doi:10.1007/s11010- 010-0582-7.

106. Candelaria NR, Liu K, Lin C-Y. Estrogen receptor alpha: Molecular mechanisms and emerging insights. J Cell Biochem. 2013;114(10):2203–2208. doi:10.1002/jcb.24584.

107. Teng Y, Xie X, Walker S, White DT, Mumm JS, Cowell JK. Evaluating human cancer cell metastasis in zebrafish. BMC Cancer. 2013;13(1):1–1. doi:10.1186/1471-2407-13-453.

108. Marques IJ, Weiss F, Vlecken DH, Nitsche C, Bakkers J, Lagendijk AK, Partecke L, Heidecke C-D, Lerch MM, Bagowski CP. Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model. BMC Cancer. 2009;9(1):128. doi:10.1186/1471-2407-9-128.

109. Janakiram NB, Rao CV. The role of inflammation in colon cancer. Adv Exp Med Biol. 2014;816:25–52. doi:10.1007/978-3-0348-0837-8_2.

110. Hofmanov xe1 JXI, Strakov xe1 N, Vaculov xe1 AHXLX, Tylichov xe1 Z, x160 afa x159 xed kov xe1 B, Skender B, Kozub xed k A. Review Article. Mediators of Inflammation. 2014:1–17. doi:10.1155/2014/848632.

111. Fouad TM, Kogawa T, Reuben JM, Ueno NT. The role of inflammation in inflammatory breast cancer. Adv Exp Med Biol. 2014;816:53–73. doi:10.1007/978-3-0348-0837-8_3.

98 112. Sims-Mourtada, Arnold K, Opdenaker L, Flynn D. Wound Healing and Cancer Stem Cells: Inflammation as a Driver of Treatment Resistance in Breast Cancer. CGM. 2015:1. doi:10.4137/CGM.S11286.

113. Harris RE. Cyclooxygenase-2 and the inflammogenesis of breast cancer. WJCO. 2014;5(4):677. doi:10.5306/wjco.v5.i4.677.

114. Frasor J, Weaver A, Pradhan M, Dai Y, Miller LD, Lin CY, Stanculescu A. Positive Cross-Talk between Estrogen Receptor and NF- B in Breast Cancer. Cancer Research. 2009;69(23):8918–8925. doi:10.1158/0008-5472.CAN-09- 2608.

115. Pradhan M, Bembinster LA, Baumgarten SC, Frasor J. Proinflammatory Cytokines Enhance Estrogen-dependent Expression of the Multidrug Transporter Gene ABCG2 through Estrogen Receptor and NF B Cooperativity at Adjacent Response Elements. Journal of Biological Chemistry. 2010;285(41):31100–31106. doi:10.1074/jbc.M110.155309.

116. Xing D, Feng W, Miller AP, Weathington NM, Chen YF, Novak L, Blalock JE, Oparil S. Estrogen modulates TNF- -induced inflammatory responses in rat aortic smooth muscle cells through estrogen receptor-beta activation. AJP: Heart and Circulatory Physiology. 2007;292(6):H2607–H2612. doi:10.1152/ajpheart.01107.2006.

117. Xing D, Oparil S, Yu H, Gong K, Feng W, Black J, Chen Y-F, Nozell S. Estrogen Modulates NFκB Signaling by Enhancing IκBα Levels and Blocking p65 Binding at the Promoters of Inflammatory Genes via Estrogen Receptor-β. Kovats S, ed. PLoS ONE. 2012;7(6):e36890. doi:10.1371/journal.pone.0036890.s004.

118. BENJAMINI Y, HOCHBERG Y. Controlling the False Discovery Rate - a Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B-Methodological. 1995;57(1):289–300.

119. Frasor J, El-Shennawy L, Stender JD, Kastrati I. ARTICLE IN PRESS. MOLECULAR AND CELLULAR ENDOCRINOLOGY. 2014:1–5. doi:10.1016/j.mce.2014.09.013.

120. Qin B, Dong L, Guo X, Jiang J, He Y, Wang X, Li L, Zhao J. Expression of G protein-coupled estrogen receptor in irritable bowel syndrome and its clinical significance. Int J Clin Exp Pathol. 2014;7(5):2238–2246.

121. Ariazi EA, Brailoiu E, Yerrum S, Shupp HA, Slifker MJ, Cunliffe HE, Black MA, Donato AL, Arterburn JB, Oprea TI, Prossnitz ER, Dun NJ, Jordan VC. The G Protein-Coupled Receptor GPR30 Inhibits Proliferation of Estrogen Receptor-Positive Breast Cancer Cells. Cancer Research. 2010;70(3):1184– 1194. doi:10.1158/0008-5472.CAN-09-3068.

99 122. Scaling AL, Prossnitz ER, Hathaway HJ. GPER Mediates Estrogen-Induced Signaling and Proliferation in Human Breast Epithelial Cells and Normal and Malignant Breast. HORM CANC. 2014;5(3):146–160. doi:10.1007/s12672-014- 0174-1.

123. Frasor J, Weaver AE, Pradhan M, Mehta K. Synergistic Up-Regulation of Prostaglandin E Synthase Expression in Breast Cancer Cells by 17β-Estradiol and Proinflammatory Cytokines. Endocrinology. 2008;149(12):6272–6279. doi:10.1210/en.2008-0352.

124. Pradhan M, Baumgarten SC, Bembinster LA, Frasor J. CBP Mediates NF- B- Dependent Histone Acetylation and Estrogen Receptor Recruitment to an Estrogen Response Element in the BIRC3 Promoter. Molecular and Cellular Biology. 2011;32(2):569–575. doi:10.1128/MCB.05869-11.

125. Perrot-Applanat M, Vacher S, Toullec A, Pelaez I, Velasco G, Cormier F, Saad HES, Lidereau R, Baud V, Bièche I. Similar NF-κB Gene Signatures in TNF-α Treated Human Endothelial Cells and Breast Tumor Biopsies. Federici M, ed. PLoS ONE. 2011;6(7):e21589. doi:10.1371/journal.pone.0021589.t011.

126. Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell. 2003;114(2):181–190.

127. Heijink DM, Jalving M, Oosterhuis D, Sloots IA, Koster R, Hollema H, Kleibeuker JH, Koornstra JJ, de Vries EG, de Jong S. TNF-related apoptosis-inducing ligand cooperates with NSAIDs via activated Wnt signalling in (pre)malignant colon cells. J Pathol. 2010;223(3):378–389. doi:10.1002/path.2797.

128. Hoesel B, Schmid JA. The complexity of NF-κB signaling in inflammation and cancer. Mol Cancer. 2013;12(1):1–1. doi:10.1186/1476-4598-12-86.

129. Daige CL, Wiggins JF, Priddy L, Nelligan-Davis T, Zhao J, Brown D. Systemic delivery of a miR34a mimic as a potential therapeutic for liver cancer. Mol Cancer Ther. 2014;13(10):2352–2360. doi:10.1158/1535-7163.MCT-14- 0209.

130. Shaker O, Maher M, Nassar Y, Morcos G, Gad Z. Role of microRNAs -29b-2, - 155, -197 and -205 as diagnostic biomarkers in serum of breast cancer females. Gene. 2015;560(1):77–82. doi:10.1016/j.gene.2015.01.062.

131. Li J, Li L, Li Z, Gong G, Chen P, Liu H, Wang J, Liu Y, Wu X. The role of miR- 205 in the VEGF-mediated promotion of human ovarian cancer cell invasion. Gynecol Oncol. 2015. doi:10.1016/j.ygyno.2015.01.531.

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