A Dissertation

Entitled

Novel Actions of Receptors that Limit Treatment Response in Breast and Lung

Cancers

by

Mugdha Patki

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Science

Dr. Manohar Ratnam, Committee Chair

Dr. Ivana de la Serna, Committee Member

Dr. Stephan M. Patrick, Committee Member

Dr. Edwin R. Sanchez, Committee Member

Dr. Robert J. Trumbly, Committee Member

Dr. Patricia R. Komuniecki, Dean

College of Graduate Studies

The University of Toledo December 2013

Copyright 2013, Mugdha Patki

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Novel Actions of Steroid Receptors that Limit Treatment Response in Breast and Lung Cancers

by

Mugdha Patki

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Science

The University of Toledo

December 2013

The primary physiological role of is the development of secondary sexual characteristics including development and function of the normal breast, reproductive system, bone homeostasis, cognitive functions and cardiovascular system. has also been implicated as a major player in the progression of normal breast epithelial tissue to a carcinoma. The role of estrogens in has been studied extensively and mainly attributed to the transcriptional activation of growth genes through the . Along with activation of genes, estrogen is also responsible for repression of many genes. Anti-estrogens antagonize both gene activation and gene repression by estrogen. However, the significance and physiological relevance of gene repression by estrogen is poorly understood. mRNA profiling of MCF-7 breast cancer cells combined with a detailed gene ontology analysis revealed that the genes repressed by estrogen are mostly involved in tumor progression including invasion, drug resistance, angiogenesis and immune evasion. This study looks at the mechanism and impact of estrogen in repressing these tumor progression genes in breast cancer cells; we found that

iii estrogen suppresses the invasiveness of breast cancer cells in a manner that is antagonized by anti-estrogens and is independent of HER2 status. These studies have implications in understanding the incidence of invasive breast cancer following hormone replacement therapy and long term anti-estrogen treatment and may also aid in the development of superior drugs for adjuvant treatment in breast cancer.

Variability in response to chemotherapeutic drugs among patients is a longstanding issue in the treatment of several cancers. Likewise, in advanced lung cancer, patient response to the recently introduced drug, pemetrexed is variable. Pemetrexed is an antifolate approved for the first-line treatment of advanced non-squamous non-small lung cancer. Dexamethasone was added to the treatment regimen with pemetrexed to alleviate the serious side effect of severe skin rash observed in many patients. Dexamethasone is administered to patients the day before, the day of and the day after pemetrexed chemotherapy. We show that treatment of non-squamous non-small cell lung cancer cell line models with dexamethasone causes a reduction in the S-phase fraction of cells along with a decrease in the expression of thymidylate synthase and dihydrofolate reductase, which are primary and secondary targets of pemetrexed, respectively. As a consequence of these effects of dexamethasone pemetrexed cytotoxicity is attenuated. The response to dexamethasone is variable among different cell line models. Our correlative and functional studies demonstrate that the variability in pemetrexed sensitivity is causally related to variability in the expression of the receptor isoform alpha, i.e. cells with relatively lower expression of the receptor fail to respond to dexamethasone

iv and hence are sensitive to pemetrexed. These results could help to predict response to pemetrexed therapy leading to the development of individualized treatment strategies.

v

I lovingly dedicate my dissertation to my parents, Minal and Rajiv Patki, for always supporting my dreams and for their endless love and faith in my abilities.

To my maternal grandfather, Ratnakar Mohile and to the memory of my maternal grandmother, Vimal Mohile, for being a source of inspiration and encouragement to me throughout my life.

To my paternal grandmother, Asha Patki and to the memory of my paternal grandfather,

Nilkanth Patki, for teaching me the importance of education and hard work.

vi

Acknowledgements

I would like to thank my advisor Dr. Manohar Ratnam whose eternal wealth of knowledge and ideas have enlightened my life. I deeply acknowledge his guidance and patience in my research, his incessant spirit and love for science inspires me each day. I extend my sincere gratitude for his invaluable support and advice in my career goals and for his endless moral support which has made me a stronger person.

I would like to thank Dr. Stephan Patrick for supporting me in every way and being my advisor during my time away from University of Toledo. I would like to thank Dr. Robert

Trumbly for his advice and help with the analysis of the microarray results and bioinformatics. I would like to thank Dr. Edwin Sanchez for his invaluable suggestions; his experience in the field of nuclear receptors has helped me immensely in developing my research projects. I would also like to thank Dr. Ivana de la Serna for her support and advice.

I am thankful to all the past and present members of the lab, Marcela d’Alincourt Salazar,

Mesfin Gonit, Suneethi Sivakumaran, Venkatesh Chari, Lily Huang, Thomas McFall,

Rayna Rosati and Shoya Yamada for their encouragement and camaraderie. A special thanks to Marcela d’Alincourt Salazar for teaching me everything in the lab and for her advice and friendship. I am thoroughly indebted to Lily Huang for helping me with my

vii experiments in the last few months; my dissertation would not be complete without her assistance.

I thank all the faculty and students of the Department of Biochemistry and Cancer

Biology at University of Toledo for their support and a friendly work environment. I would especially like to acknowledge the efforts of Jenifer Zak in supporting me during my time away from Toledo.

I would also like to thank the members of the labs on the eighth floor of Karmanos

Cancer Institute for supporting me during my transition to Detroit. I am grateful to Dr.

Cecilia Speyer for her advice in troubleshooting my experiments. I would like to extend my sincere gratitude to Dr. Larry Matherly for his support and involving me in the

Wayne State University Cancer Biology program activities. I would also like to thank

Amanda Cook and Jean Guerin for their assistance with everything.

I feel blessed to have my wonderful family and bow to my parents’ for their efforts in encouraging and supporting me. I would like to express my gratitude to my cousin,

Snehal Kharkar and to my boyfriend, Gopal Iyer for their love and moral support.

Lastly, I would like to thank all of my friends for their constant encouragement and appreciation. A special thanks to my roommates Akshada Sawant and Gurpanna Saggu for making my time in Toledo memorable. I would like to thank Thomas McFall and

Rayna Rosati for bringing joy to my life in Detroit.

viii

Table of Contents

Abstract...... iii

Acknowledgments.……………………………………………………………………... vii

Table of contents...………………….……………………………….……………..……. ix

List of tables………………………………………………………………………...…… xi

List of figures…………………………………………………………………...... …… xii

1 – Literature Review...... …………………………………………………………..... 1

1.1 Breast cancer epidemiology, etiology and biologic subtypes...... 1

1.2 Estrogen biology...... 5

1.3 Adjuvant therapy in estrogen receptor positive breast cancer...... 8

1.4 Endocrine therapy...... 13

1.5 Mechanisms of resistance to tamoxifen...... 16

1.6 Estrogen receptor structure and mechanism of action...... 19

1.7 Gene repression by estrogen and de-repression by tamoxifen...... 25

1.8 Carcinoma of lung, epidemiology and etiology...... 28

1.9 Classification and management of lung cancer...... 29

1.10 Pemetrexed in the treatment of NSCLC – single agent and combination

therapy...... 33

1.11 Mechanism of action of pemetrexed...... 36

1.12 Mechanisms of resistance to pemetrexed...... 38

ix

1.13 Therapeutic applications of ...... 40

1.14 Mechanism of action of the glucocorticoid receptor...... 42

1.15 Mechanisms of resistance to glucocorticoids...... 48

2 – Disruption of Estrogen Signaling Enhances Invasiveness of Breast Cancer

Cells by Attenuating a Her2-independent Gene Repression Program..……...... 51

2.1 Abstract ……………………………………………………….………...... …….. 52

2.2 Introduction ……………………..………………………………………….…… 53

2.3 Materials and Methods...... 55

2.4 Results...... 62

2.5 Discussion...... 69

3 – Glucocorticoid Receptor Status is a Principal Determinant of Variability in

the Sensitivity of Non-Small Cell Lung Cancer Cells to Pemetrexed.…...... … 90

3.1 Abstract...... 91

3.2 Introduction...... 92

3.3 Materials and Methods...... 95

3.4 Results...... 98

3.5 Discussion...... 102

3.6 Acknowledgements...... 106

4 – Conclusion...... ……………………………………………………………. 115

References ………………………………………………………….....………………. 119

x

List of Tables

2.1 Functional classification of E2-repressed genes in breast cancer.….…...... … 89

3.1 GR isoform expression and p53 status of model NSCLC cell lines and their

response to Dex...... 114

xi

List of Figures

2-1 Comparison of E2 repressed genes in MCF-7 cells with genes upregulated

in clinical progression of ductal carcinoma in situ (DCIS) to

invasive ductal carcinoma (IDC)...... 74

2-2 Gene ontology of E2 repressed genes in relation to progression of

breast cancer...... 75

2-3 Gene repression by E2 and antagonism by 4-OHT in ER+ breast

cancer cell lines...... 76

2-4 Effect of cycloheximide on gene repression by E2 in MCF-7 cells...... 78

2-5 Co-repressor dependence for gene repression by E2...... 79

2-6 Effect of E2 & 4-OHT on invasiveness of ER+ breast cancer cell lines...... 80

2-7 Lack of an effect of HER2/Neu knockdown on repression of gene

expression or invasiveness by E2...... 82

2-8 ER-dependence for gene repression & inhibition of invasiveness by E2...... 84

2-9 Ectopic overexpression of wild-type ER or ER-L372R in BT474 cells

and the effect on regulation of gene expression and invasiveness by E2...... 86

2-10 Recruitment of ER to binding sites in chromatin associated with genes

repressed or activated by E2...... 88

3-1 Differential effects of Dex on the expression of genes involved in

pemetrexed action in A549 vs. H1299 cells...... 107

3-2 Effect of Dex on cell cycle phase distribution in A549 and H1299 cells...... 108

3-3 Reversibility of Dex effects in A549 cells...... 109

xii

3-4 Effect of restoring GRα on Dex sensitivity in H1299 cells...... 110

3-5 Effect of knocking down p53 on Dex sensitivity in H226 cells...... 111

3-6 Differential effects of Dex on growth inhibition by pemetrexed in

A549 vs. H1299 cells...... 112

3-7 Influence of Dex on inhibition of colony formation by pemetrexed in

relation to GRα status...... 113

xiii

Chapter 1

Literature Review

1.1 Breast cancer epidemiology, etiology and biologic subtypes

Breast cancer continues to be the most common type of cancer diagnosed in women since 1975 and the second leading cause of cancer related deaths in women in

USA (Siegel et al.,2013); less than one percent of breast cancers diagnosed account for male breast cancer (Korde et al.,2010). The dependence of breast cancer on the ovarian hormones has been known since late 19th century. Endocrine ablation by surgical or radiation induced oophorectomy was the main option for patients with advanced breast cancer for the first 60 years of the 20th century, but with a success rate of only 30%. The discovery of the estrogen receptor in 1962 led to development of better targeted treatments (Jensen et al.,1968). The expression of ER in the breast tissue was also established as a predictive marker for ablative surgery (Jensen et al.,1971); and was reported to predict the outcome of breast cancer treatment in 1974 (Jordan,2003). This has provided a molecular basis for classification of breast cancer based on the presence of hormone receptors – estrogen receptor (ER) and progesterone receptor (PR). Hormone receptor positive breast cancer accounts for about 60% of the premenopausal and 80% of the postmenopausal breast cancers (Clark et al.,1984). Tumors expressing ER depend on

1 estrogen signaling for growth and survival; anti-estrogen adjuvant therapy is the mainstay in the treatment of ER positive tumors (discussed in section 1.3). HER2/neu (human epidermal 2), a member of the tyrosine kinase family (Coussens et al.,1985;

Schechter et al.,1985) was shown to be amplified in approximately 30% of breast cancers

(Slamon et al.,1987; Slamon et al.,1989) and to be a negative prognosticator of breast cancer (Ross et al.,1998; Schnitt,2001). Monoclonal antibodies targeting HER2 have been developed – Herceptin/trastuzumab targets the subdomain IV of the HER2 extracellular domain whereas pertuzumab targets subdomain II. Trastuzumab, along with standard chemotherapy, improved the overall survival of metastatic breast cancer patients with HER2 positive tumors from 20.3 months to 25.1 months when compared to chemotherapy alone (Hudis,2007). Triple negative breast cancer (TNBC) is described as a subtype of breast cancer that lacks expression of the hormone receptors ER and PR and does not overexpress HER2 receptor protein and accounts for about 15% of the breast cancers (Hudis et al.,2011). TNBC has poor prognosis as treatment of it solely depends on surgery, chemotherapy and radiation lacking an effective targeted treatment strategy.

Molecular subtypes of breast cancer, classified on the basis of distinct gene expression patterns, include luminal A (ER, PR positive, HER2 negative), luminal B (ER, PR, HER2 positive), basal (ER, PR, HER2 negative) and HER2 (ER, PR negative and HER2 positive) (Perou et al.,2000). The luminal subtype expresses high amounts of luminal cytokeratins and genetic markers of luminal and normal breast epithelial cells, whereas, the basal subtype, arising from the outer basal layer expresses cytokeratins associated with basal types of cancers and is typically high-grade and poorly differentiated (Rakha et al.,2007; Sotiriou et al.,2009).

2 Breast cancer usually develops in the epithelial cells lining the ducts or the lobules required for milk production. The most common histopathological types of breast cancer are invasive ductal carcinoma, invasive lobular carcinoma and ductal carcinoma in situ. Ductal carcinoma is the most common type of cancer diagnosed in women, the two main subtypes – (i) Ductal carcinoma in situ (DCIS) or intraductal carcinoma is a non- invasive tumor where proliferation of malignant cells is confined within the basement membrane of the ducts of the breast which defines early stage of the disease. Treatment options for DCIS include surgical resection – either lumpectomy (discrete removal of lumps) or mastectomy (complete or partial removal of one or both breasts) followed by radiation (Lambert et al.,2012). (ii) Invasive ductal carcinoma (IDC) is the most common form of invasive breast cancer diagnosed and accounts for about 72-80% of invasive breast cancers (Lamovec et al.,1991; Borst et al.,1993; Sastre-Garau et al.,1996; Arpino et al.,2004; Li et al.,2005). Prognosis of IDC depends on histopathological subtype, histologic grade, tumor size, lymph node/distant metastasis, expression of hormone receptors or HER2/neu receptor. Treatment options depend on the size of the tumor which include surgery (lumpectomy or mastectomy) followed by adjuvant radiation, chemo or hormonal therapy. Invasive lobular carcinoma (ILC) is less common and accounts for 5–15 % of all invasive breast cancers (Lamovec et al.,1991; Borst et al.,1993; Sastre-Garau et al.,1996; Arpino et al.,2004). ILC is more likely to occur in older patients and to be estrogen and progesterone receptor positive and HER2 negative as compared to IDC. Treatment of both ILC and IDC improves the 5 year overall survival; it is 85% for ILC whereas it is 84% for IDC (Arpino et al.,2004). The metastatic patterns for ILC and IDC are distinct; metastasis to the peritoneum, ovary and

3 gastrointestinal systems are associated with ILC whereas IDC is more likely to metastasize to the lungs and CNS (Arpino et al.,2004). A few other histopathologic subtypes of invasive breast cancer based on 2003 World Health Organization (WHO) classification include tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, mucinous carcinoma, invasive papillary carcinoma, invasive micropapillary carcinoma and apocrine carcinoma.

Breast cancer etiology is poorly understood, however several risk factors such hereditary factors, genetic mutations, hormonal and reproductive factors (early menarche, late age at first full-term pregnancy, parity and late menopause) which could be based on the exposure of the breast tissue to estrogen as well as exposure to environmental carcinogens have been shown to greatly increase the risk of developing breast cancer

(Russo et al.,1998). Germline mutations in breast cancer predisposition genes BRCA1 and

BRCA2 account for 1% to 4% of all breast cancer cases across different populations

(Kurian,2010); furthermore BRCA1 or BRCA2 mutations carriers have an 80% chance of developing breast cancer (Ford et al.,1994; Easton et al.,1997; Ford et al.,1998). Women with family history of breast cancer related to BRCA1 and BRCA2 mutations have twice the risk of developing sporadic breast tumors than general population (Colditz et al.,1993;

Hedenfalk et al.,2001). Occupational hazards such as exposure to organic solvents, estrogenic chemicals, polyaromatic hydrocarbons, aromatic amines as well as exposure to environmental , active and passive cigarette smoke are risk factors for development of breast cancer (DeBruin et al.,2002).

Several estrogen and progesterone combinations have been prescribed to replace the ceased production of ovarian hormones after menopause which attenuates

4 menopausal symptoms. Randomized, placebo-controlled clinical trials by Women’s

Health Initiative (WHI) were conducted to determine the health outcomes associated with use of conjugated (CEE) and CEE plus medroxyprogesterone acetate

(MPA); a combination of estrogen-progesterone (CEE plus MPA) was directly associated with increase in incidence of breast cancer which are diagnosed at a more advanced stage as compared to placebo (Chlebowski et al.,2003). In contrast, estrogen replacement monotherapy (CEE only) was associated with decreased risk of breast cancer during intervention part of the trial as well as during the follow up period (LaCroix et al.,2011).

1.2 Estrogen biology

Estrogens are steroid hormones primarily responsible for growth and development of female secondary sexual characteristics and reproduction. In premenopausal women, estrogens are produced by the ovaries and their levels vary based on the phase of the menstrual cycle however, during pregnancy the levels are 30-40 times higher than the normal cycle mainly due to estrogen production in the placenta. The granulosa cells in the ovarian developing follicles and the corpus luteum are major sites of estrogen production. The synthesis of estrogens in the ovaries is stimulated by the secretion of the follicle stimulating hormone (FSH) from the . The secretion of FSH is stimulated by the gonadotropin releasing hormone (GnRH) from the . A negative feedback due to high levels of estrogen suppresses hypothalamic release of

GnRH, regulating the level of the hormone (Morrison et al.,2006). In postmenopausal women, extragonadal synthesis of estrogen in adipose tissue, bone, smooth muscle, liver is predominant. There are three main types of estrogens produced in women (E1),

5 (E2), and (E3). E2 is the most active and potent form of estrogens. E2 easily diffuses across the cell membrane due to its hydrophobic nature. In the plasma, a very low percentage of the E2 present in biologically free and active form; it is mainly bound by the protein steroid hormone binding globulin (SHBG). E2 is metabolized to its less active forms and excreted by the kidneys as sulfate conjugates (Zhu et al.,1998).

Development and function of the normal breast is primarily under the influence of ovarian steroid hormones. The spurt of growth that occurs at puberty is dependent on high levels of estrogen and progesterone. Prenatal development of mammary gland does not seem to require estrogen but it is required for pre-pubertal and post-pubertal development of the gland. Normal duct development requires estrogen and progesterone, however, ductal branching and elongation occurring during puberty is regulated by growth hormone secreted by the pituitary (Russo et al.,1998). Apart from its reproductive functions, estrogens are essential for bone homeostasis, cognitive and memory functions in the brain and the cardiovascular system. The first report of a beneficial effect of estrogen on cardiovascular function was published in 1985, which provided evidence that use of postmenopausal estrogen therapy reduces the risk of severe coronary disease

(Stampfer et al.,1985). Observational studies examining the benefits of hormone replacement therapy suggested a 30%-50% reduction in the coronary events (Bush et al.,1987; Stampfer et al.,1991; Grady et al.,1992). WHI’s randomized placebo-controlled trials comparing either CEE alone or CEE plus MPA with placebo provided a large amount of evidence that led to the conclusion that a combination of CEE plus MPA replacement therapy increased coronary heart diseases (Rossouw et al.,2002); whereas

CEE alone neither increased nor decreased risk of coronary heart diseases (Anderson et

6 al.,2004). Estrogen has a favorable effect on the plasma lipid profile by decreasing the level of LDL- and increasing the level of HDL-cholesterol. Both genomic and non-genomic mechanisms are involved in the regulation of vascular function by estrogen.

Estrogen conserves the bone mass by suppressing bone turnover and maintaining balanced rates of bone formation and bone resorption. It maintains the balanced rates of bone formation and bone resorption by decreasing the osteoclast formation, activity and life span by increasing apoptosis; as well as increasing the osteoblast formation, differentiation and proliferation and function. Therefore, estrogen deficiency affects bone mass in several ways leading to higher bone turnover, prolonging the resorption phase and reducing the formation phase (Riggs et al.,2002). Postmenopausal osteoporosis was thus linked to estrogen deficiency; later it was demonstrated that the bone loss could be prevented by estrogen therapy (Lindsay et al.,1976).

The association between hormones and breast cancer growth was established more than 100 years ago, when George Beatson demonstrated that removal of ovaries from a premenopausal woman with advanced breast cancer resulted in dramatic remission of the tumor (Beatson,1899). This was based on the observations by Ashley

Cooper that the size of tumor increased and decreased during the menstrual cycle in premenopausal women with advanced breast cancer (Jensen et al.,2003). Oophorectomy was the standard option of treatment until it was replaced by ovarian radiation, adrenalectomy, hypophysectomy in the 1950s and 1960s. However, this treatment was not completely successful as only one-third of the patients responded. Hence, there was a need for predictive markers to avoid ineffective ablation surgery. The localization of estrogen in the ovaries was discovered in 1923 (Allen et al.,1983) but its target was still

7 unknown. The discovery of ER (Jensen et al.,1968) and its detection in breast carcinoma led to strategies to target the receptor for breast cancer therapy. (Jensen et al.,2003).

The plasma level of estrogens in postmenopausal women is about 20-fold lower than in pre-menopausal women, the mean plasma level of E2 in postmenopausal women is 14.6 pM whereas it is 270.9 pM in pre-menopausal women (Lonning et al.,2009).

Although, there is a dramatic decrease in the plasma estrogen levels in postmenopausal women, the E2 levels in the breast tissue of postmenopausal women are 10-20 fold higher

(Geisler,2003). Increase in the local production of estrogen as well as increase in uptake of estrogen from the circulation could be the reasons for this phenomenon (Lonning et al.,2009). There are two principal pathways involved in the last steps of E2 formation in breast cancer tissue; (i) aromatase pathway which converts androgens and to estrone and estradiol respectively by aromatase; (ii) sulfatase pathway which converts to estrone by steroid sulfatase (STS). The final step converts of estrone to estradiol by 17β-hydroxysteroid dehydrogenase type 1(17β-HSD1).

Among the two pathways, aromatase pathway is considered to be the most important pathway with aromatase being the most important enzyme in estrogen biosynthesis.

Inhibition of aromatase has clinically proven to be very effective in postmenopausal women with breast cancer (Coombes et al.,2004).

1.3 Adjuvant therapy in estrogen receptor positive breast cancer

Breast tumors are generally localized if detected early; however, most patients eventually die of metastatic spread of the disease. The primary treatment (surgery or chemotherapy) is usually followed by adjuvant therapy to minimize the possibility of

8 metastasis. Anti-estrogen adjuvant therapies could be divided into three major categories:

(i) Selective Estrogen Receptor Modulators (SERMs): SERMs are a diverse group of compounds that bind to the ER and manifest variable agonistic or antagonistic properties in the context of estrogen-dependent responses occurring in target tissues. Tamoxifen and are the most widely prescribed SERMs. Tamoxifen is extensively used in adjuvant therapy of estrogen receptor positive breast cancer in both pre- and postmenopausal women (discussed in section 1.4). Raloxifene, a second generation

SERM was initially approved for the treatment of osteoporosis, until the STAR trial

(Study of Tamoxifen and Raloxifene) which directly compared tamoxifen with raloxifene and demonstrated that raloxifene was as effective as tamoxifen in preventing invasive and non-invasive breast cancer, however the toxicity and side effect evaluation favored the raloxifene group (Vogel et al.,2006; Vogel et al.,2010). Raloxifene reduced the incidence of thromboembolic events and the risk of as compared to tamoxifen.

Although, longer administration periods could be required for raloxifene as it is less bioavailable than tamoxifen and has a lower half-life, the far lesser toxicity of raloxifene was appealing (Vogel et al.,2010); hence it was approved by the US Food and Drug

Administration (FDA) as an alternative to tamoxifen in breast cancer prevention.

Toremifene is a tri-phenylethylene derivative, chemically and pharmacologically related to tamoxifen with no significant difference in the survival of post-menopausal women administered either adjuvant tamoxifen or (Lewis et al.,2010). Toremifene has antiestrogenic activity in the breast; its agonist and antagonist profile is similar to tamoxifen and has been approved for postmenopausal women with metastatic breast cancer (Shang,2006). Toremifene varies from tamoxifen in a single

9 chloride ion, resulting in differing metabolism and a potentially more favorable toxicity profile (Lewis et al.,2010). Besides tamoxifen, raloxifene and toremifene, several third and fourth generation SERMs have been developed. , a third generation

SERM and a raloxifene analog with similar profile as raloxifene but higher potency was tested in postmenopausal women in a phase III clinical trial; however the FDA approval was not sought as it did not reach the secondary endpoint of reduction in non-vertebral fractures, cardiovascular events and improvements in cognitive function (Bolognese et al.,2009). Other SERMs such as , bazodoxifene are being developed with a potential for the treatment of postmenopausal osteoporosis (Bolognese,2010).

(ii) Aromatase inhibitors: Aromatase (CYP19) is a microsomal cytochrome P450 enzyme responsible for the synthesis of estrogens (estradiol and estrone) from androgens

(testosterone and androstenedione respectively) in the ovaries and in peripheral tissues including bone, brain, breast and adipose tissue. After menopause, the production of estrogens from the ovaries ceases but aromatase activity and estrogen production persists in peripheral tissues. Aromatase inhibitors selectively inhibit aromatase activity in the whole body. Type I (steroidal) aromatase inhibitors such as exemestane and formestane are analogs of androstenedione and act as competitive substrate inhibitors. Non-steroidal inhibitors (Type II inhibitors) such as anastrozole and letrozole, cause short term inhibition of aromatase activity in a reversible manner by binding to the heme group of the enzyme and competing with androstenedione (Johnston et al.,2003). After aromatase inhibitor treatment, the intratumoral and circulating estrogens are reduced by more than

30 fold to almost undetectable levels (Geisler et al.,2008; Miller et al.,2012). The use of aromatase inhibitors is limited to postmenopausal women due to the intact hypothalamus-

10 pituitary-gonadal feedback loop in premenopausal women, which leads to an increase in gonadotropin secretion and estrogen synthesis in the ovaries (Johnston et al.,2003).

Several multicenter, double-blind clinical trials comparing adjuvant treatment of aromatase inhibitors (anastrozole, letrozole and exemestane) have been conducted

(Brodie,2002; Dixon et al.,2003; Smith et al.,2003; Howell et al.,2005; Cuzick et al.,2010; Miller et al.,2012). These trials demonstrated superior efficacy and safety of aromatase inhibitors over tamoxifen in postmenopausal women. In the 10-year analysis of the ATAC (Arimidex, Tamoxifen, Alone or in Combination) trial, there were significant improvements in the anastrozole group in context of disease-free survival, time of recurrence; however the complete ablation of estrogen caused frequent fractures in the anastrozole group (Cuzick et al.,2010). Interestingly, the incidence of contralateral breast cancer was lower in the anastrozole group. Because of their improved efficacy and tolerability, FDA has approved aromatase inhibitors in the first-line (anastrozole, letrozole) and second-line (exemestane) treatment of breast cancer, in both early and advanced disease in postmenopausal women. When the proliferative indices (Ki67) of tumors before and at 2 and 12 weeks of treatment with aromatase inhibitors were compared, it suggested that virtually all ER positive breast carcinomas had some proliferative dependence on estrogen. (Dowsett et al.,2005; Dowsett et al.,2005). The response rates in the randomized first-line studies of third generation aromatase inhibitors in metastatic disease varied from 21 to 33% (Smith et al.,2003). The response rates after

4 and 3 months of treatment were 55% and 37%, respectively in the neoadjuvant studies of letrozole and anastrozole, respectively. (Eiermann et al.,2001; Dowsett et al.,2005).

These clinical data suggest that a substantial proportion of ER positive tumors are

11 intrinsically resistant to aromatase inhibitor. In vitro studies have helped to understand some of the mechanisms of acquired resistance to aromatase inhibitors. It has been demonstrated that long term estrogen deprivation of MCF-7 cells causes an increase in the expression of ER along with increase in phosphorylation of the receptor at Ser 118 which influences the basal ER mediated transcription as well as estrogen mediated growth of the cells, sensitizing the cells to very low levels of estrogen. (Masamura et al.,1995; Jeng et al.,1998; Chan et al.,2002; Martin et al.,2003). In addition, there is evidence for increased cross-talk between the various growth factor receptor signaling pathways and ER with the function of the receptor as well as its coactivators such as

AIB1 becoming hypersensitive to the action of several different intracellular kinases such as ERK1/ERK2 (Anzick et al.,1997; Font de Mora et al.,2000; Murphy et al.,2000; Shim et al.,2000; List et al.,2001).

(iii) Selective Estrogen Receptor Downregulators (SERDs): Regardless of the type, use of adjuvant therapy and the mechanism of resistance to adjuvants, hormone- sensitive tumors express the estrogen receptor, even after relapse. The major mechanism of resistance to adjuvant therapy is bypassing the classical mechanisms of activation of the ER and its downstream gene targets (Musgrove et al.,2009); the anti-estrogen resistant tumors retain the activity and function of the ER and depend on it for growth and survival. ICI 182, 780 (Faslodex/) is a steroidal pure anti-estrogen that has been approved by the FDA in the second-line therapy of ER positive breast cancer in postmenopausal women who have metastatic disease after anti-estrogen treatment (Bross et al.,2002). Fulvestrant is a competitive inhibitor of ER with no agonistic activity on estrogen target tissues; it binds to the receptor with an affinity 89% that of estradiol and

12 100 times higher than tamoxifen (Howell et al.,2000; Osborne et al.,2004). The long side chain of fulvestrant renders the ER functionally inactive, depletes it by impairing its dimerization, nuclear localization and causing proteolytic degradation of cellular ER

(Robertson,2001). Clinical trials comparing fulvestrant and tamoxifen for the treatment of metastatic/locally advanced breast cancer in postmenopausal women previously untreated with endocrine therapy demonstrate that fulvestrant has similar efficacy and tolerability as compared to tamoxifen (Howell et al.,2004). As a second-line treatment option in postmenopausal women with advanced breast cancer progressing after prior endocrine treatment, fulvestrant was well tolerated and as effective as anastrozole (Howell et al.,2002). However, fulvestrant is not preferred as the first line therapy because it abolishes all the beneficial effects of estrogen. Resistance to pure anti-estrogens is mainly associated with loss of ER expression (Sommer et al.,2003; Massarweh et al.,2006). In vitro studies understanding the mechanisms of fulvestrant resistance have indicated elevated activity of the growth factor signaling such as EGFR expression, ERK1/2,

MAPK activation, activation of HER2/HER3, Src and AKT pathways (Hiscox et al.,2006; Frogne et al.,2009).

1.4 Tamoxifen Endocrine therapy

The first non-steroidal anti-estrogen, ethamoxytriphetol or MER25 was discovered in mid-1950s, it showed anti-estrogenic activity in all species. MER25 was a post-coital contraceptive (morning after pill) in rats but when tested in humans, it showed the opposite effect. MER25 and clomiphene were used for fertility treatment; they were also tested for their ability to treat breast cancer but due to potential side effects of cataract formation led to abandonment of work on breast cancer. Dr. Arthur Walpole

13 discovered ICI46, 474, a non-steroidal compound (which came to be known as tamoxifen) at Alderly Park research laboratories of Imperial Clinical Industries

(ICI) Pharmaceuticals (now AstraZeneca). Tamoxifen was marketed for fertility treatment but never proved successful as a human contraceptive (Jordan,2003;

Jordan,2006). The first preliminary clinical trial of tamoxifen was performed in 1971 showing the similar efficacy to the then accepted treatment of and methylandrostenediol, but an improved toxicity profile in postmenopausal advanced breast cancer patients (Cole et al.,1971). However, ICI Pharmaceuticals was not interested in cancer treatment, hence in 1972, all clinical data on ICI46, 474 was reviewed and it was decided not to develop ICI46, 474. A second clinical study was performed in 1972 by Dr. Harold W.C. Ward (Ward,1973). This study used a higher dose

(20 mg) and the partial, definite response rates added up to 77%. Based on these studies

Dr. Walpole succeeded in convincing ICI to market tamoxifen in UK for breast cancer treatment. He was also involved in supporting and providing funds to Dr. V. Craig

Jordan, who went on to do seminal work with SERMs such as tamoxifen, raloxifene and is considered the ‘father of tamoxifen’. Tamoxifen was approved for clinical use in treatment of advanced breast cancer in the UK in 1973 and in 1977 it was approved for the use in postmenopausal women in the USA (Jordan,2003; Jordan,2006).

Tamoxifen shows partial estrogenic effects on bone, the cardiovascular system and the uterus; however it acts as an antagonist of estrogen in the breast. It binds to the

ER and induces a conformational change in the structure of the receptor leading to either activation or inhibition of transcription based on the cell and promoter context

(Shang,2006). Tamoxifen is administered as tamoxifen citrate and undergoes metabolism

14 in the gastrointestinal tract and liver. In the liver, it is metabolized by hepatic cytochrome

P450 (CYPs) to produce the major metabolites in the plasma, N-desmethyltamoxifen and

4-hydroxytamoxifen which are catalyzed by enzymes CYP3A4 and CYP2D6 respectively. Oxidation of these metabolites results in formation of the most pharmacologically active metabolite 4-hydroxy-N-desmethyltamoxifen ()

(Hoskins et al.,2009). 4-hydroxytamoxifen and endoxifen have a higher affinity for the

ER as compared to the parent compound.

Several randomized trials were performed to assess the effect of 1, 2 or 5 years of adjuvant tamoxifen treatment. It was not until 1998 that the definitive clinical benefit of adjuvant tamoxifen treatment was clear in an overview of trials in women with early breast cancer ("Tamoxifen for early breast cancer: an overview of the randomised trials.

Early Breast Cancer Trialists' Collaborative Group",1998). This undoubtedly proved that adjuvant tamoxifen treatment considerably improved 10-year survival of women with ER positive tumors. The use of adjuvant tamoxifen reduced the mortality of breast cancer patients by 26% and the incidence of contralateral breast cancer by 50% after 5 years of active treatment ("Tamoxifen for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists' Collaborative Group",1998). A course of 5 years treatment is the most effective but one-year course in ineffective but extending the treatment beyond 5 years does not offer any benefits (Jordan,2003). Hence, a 5 year course of treatment was tested for tamoxifen as a chemopreventive agent; tamoxifen prevented invasive breast cancer in women at high risk, hence it was approved as a chemopreventive agent in women with increased risk of breast cancer (Fisher et al.,1998). The benefits of tamoxifen persist for an additional 10 years after cessation of

15 treatment. Typically, a dose of 20 mg or 40 mg per day is recommended for advanced breast cancer; however in the adjuvant setting a dose of 20 mg per day for 5 years following tumor resection is advised. Tamoxifen is well tolerated by most patients; however, some adverse effects experienced more commonly in pre-menopausal than in postmenopausal women are symptoms of menopause, hot flashes, and atrophic vaginits.

Some of the less common symptoms include vascular thrombotic events such as blood clots, stroke, deep vein thrombosis and pulmonary embolism. These side effects continue to build up with increase in duration of therapy (Jordan,2003). Case control studies have also shown that the incidence of endometrial cancer is doubled in patients with tamoxifen treatment for 1 or 2 years and quadrupled in patients with 5 years of treatment

("Tamoxifen for early breast cancer: an overview of the randomised trials. Early Breast

Cancer Trialists' Collaborative Group",1998; Swerdlow et al.,2005). These negative effects of tamoxifen are mainly due to its estrogenic actions on tissues other than breast; nonetheless, agonistic actions of tamoxifen are responsible for the favorable effects on serum cholesterol, protection against osteoporosis and cardiovascular diseases. However, alternative adjuvant treatments such as raloxifene, aromatase inhibitors cannot be administered to pre-menopausal women, whereas, fulvestrant is not preferred as the first line therapy because it abolishes all the beneficial effects of estrogens; thus tamoxifen is the mainstay in hormonal adjuvant therapy for breast cancer.

1.5 Mechanisms of resistance to tamoxifen

Despite the benefits of tamoxifen, after 5 years of adjuvant tamoxifen treatment, the disease recurs in 28.9% of the cases when compared to combined adjuvant polychemotherapy and tamoxifen treatment; moreover, 25% of women die of breast

16 cancer after 15 years ("Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials",2005).

These data suggest that a significant proportion of ER positive breast cancers might be resistant to tamoxifen prior to treatment (de novo), where the tumor is intrinsically refractory to tamoxifen; or acquire resistance, where the tumors initially respond to treatment but subsequently develop resistance resulting in disease recurrence. (i) The primary mechanism for de novo resistance would be the lack of ER expression in the tumors or mutations in the ER genes which may lead to a functionally deficient receptor, without loss in expression. (ii) Several mechanisms altering tamoxifen uptake, retention or metabolism certainly play a role in resistance. Likewise, patients carrying inactive alleles/polymorphisms of the hepatic enzyme CYP2D6 are unable to convert tamoxifen to its active metabolite, which leads to intrinsic resistance (Hoskins et al.,2009; Musgrove et al.,2009). To date, more than 75 CYP2D6 variant alleles have been reported. Individuals carrying the null allele, a gene deletion or polymorphisms that lead to no protein expression are considered poor metabolizers; whereas polymorphisms that reduce enzyme activity are intermediate metabolizers. Poor or intermediate metabolizers have a significantly lower rate of production of endoxifen which compromises their tamoxifen response as compared to individuals with normal metabolizer genotypes (Hoskins et al.,2009). Selective noradrenaline reuptake inhibitors (SNRIs) together with selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed along with tamoxifen to alleviate undesirable symptoms such as hot flashes. SNRIs and SSRIs are potent inhibitors of CYP2D6. Therefore in 2006 FDA recommended an amendment for the package insert of tamoxifen, warning patients with CYP2D6 deficient activity of risk of

17 treatment failure and that anti-depressants such as SSRIs interfere with the bioactivation of tamoxifen (Hoskins et al.,2009). (iii) HER2, a member of the tyrosine kinase family, is amplified in approximately 30% of breast cancers (Slamon et al.,1987; Slamon et al.,1989). Amplification/overexpression of HER2 leads to increase in ligand independent phosphorylation of the ER and results in hormone independent transcription and cell proliferation. Expression of HER2 has been established to be an independent predictor of early metastasis, shorter disease free survival and a major mechanism of intrinsic resistance to tamoxifen (King et al.,1985; Semba et al.,1985; Slamon et al.,1987; Tiwari et al.,1992; Borg et al.,1994). Several studies suggest that patients overexpressing HER2 have a relatively less benefit from adjuvant tamoxifen therapy (Carlomagno et al.,1996;

Knoop et al.,2001; De Placido et al.,2003).

The deregulation of various aspects in estrogen signaling is a common mechanism of acquired tamoxifen resistance. (i) Alteration of the co-regulator complement

(overexpression or downregulation) of the tumor cells converts tamoxifen to an agonist.

Coactivator SRC-3/NCOA3/AIB1 is overexpressed in about 50% of breast tumors

(Anzick et al.,1997); overexpression, increased phosphorylation and activity of coactivators such as AIB1 or SRC1 leads to constitutive transcription through the tamoxifen bound ER (Tzukerman et al.,1994; Smith et al.,1997). Additionally, a reduction in the corepressors such as NCoRI results in increased gene transcription through coactivators and leads to resistance (Lavinsky et al.,1998). AIB1 competes with corepressor PAX2 to bind to ERBB2 (EGF-receptor related protein tyrosine kinase B2); the ratio of AIB1 expression to PAX2 determines whether transcription of the erbB2 gene is repressed or activated (Hurtado et al.,2008). Accordingly, increased expression of

18 AIB1 outcompetes Pax2 leading to increased ERBB2 expression, thus causing tamoxifen resistance. There is evidence indicating that in tamoxifen treated patients high levels of

AIB1 is associated with tamoxifen resistance and shorter disease free survival (Osborne et al.,2003); however in untreated patients, overexpression of AIB1 is associated with better outcome. (ii) ER is phosphorylated at Ser118 by ERK1/2 activated through RAS-

RAF-ERK pathway; or at Ser167 by Akt through the PI3/K pathway (Ali et al.,2002;

Lewis et al.,2005). Increase in activity of the growth factor signaling pathways modulates activity of ER and its co-regulators through increased phosphorylation, thus conferring ligand-independent transcriptional activity. In tamoxifen resistant breast cancer cell lines and clinical tumors, the levels of ERK1/2 have been shown to be elevated (Ring et al.,2004). (iii) The role of ERβ isoform in breast tumor biology is not very well known though a protective role for beta isoform has been suggested. The expression of ERβ is found to be decreased in invasive tumors as compared to normal or benign tissues. ERβ expression has been found to be altered in tamoxifen resistant tumors; however, whether this alteration in expression is causally linked to tamoxifen resistance is not yet known

(Ring et al.,2004). (iv) The reduced intratumoral concentrations of tamoxifen as a result of decreased influx or increased efflux in the tumor could result in tamoxifen resistance.

The mechanism responsible for this reduced efficacy of tamoxifen due to its altered accumulation is still unknown. In some cases, long term tamoxifen treatment, could eventually lead to tamoxifen becoming tumorigenic with the tumor being dependent on tamoxifen for growth. (Osborne,1998; Fisher et al.,2001; Ali et al.,2002; Lewis et al.,2005; Hayashi et al.,2006; Sengupta et al.,2008).

1.6 Estrogen receptor structure and mechanism of action

19 The discovery of estrogenic hormones produced in the ovary in 1923 by Allen and Doisy (Allen et al.,1983) led to the search of a target of estrogen .In the 1950s it was thought that estrogen was interacting with enzymes and participating in metabolic processes. However, Elwood Jensen and colleagues made the seminal discovery that estrogen bound to a specific protein termed as estrophilin, now known as estrogen receptor (Jensen et al.,1968). Later, Bert O’Malley demonstrated that hormones estrogen and progesterone induce messenger RNAs in the nucleus (O'Malley et al.,1974).

ER is a ligand-activated transcription factor which is a member of the steroid/thyroid/retinoid/orphan receptor superfamily (Mangelsdorf et al.,1995). Two isoforms of the ER have been cloned and characterized – ERα and ERβ (Greene et al.,1986; Kuiper et al.,1996). Each isoform is encoded by a different gene – ERα by ESR1 located on chromosome 6q24 and ERβ by ESR2 located on chromosome 14q21. The

ESR1 gene spans nearly 300kb with eight coding exons (about 140kb) and seven introns

(Ponglikitmongkol et al.,1988); the ESR2 gene comprises of eight exons which codes for a 530 amino acids long protein (Zhao et al.,2008). From the N-terminus of the ERprotein, it can be divided into five structurally distinct domains A/B, C, D, E and F which are common to both isoforms. Functionally, it consists of three major domains: (i) N- terminus domain (NTD) which spans the A/B domain and consists of the hormone independent transcriptional activation function 1 (AF-1), it is the most variable domain with the two ER isoforms sharing less than 20% homology (Klinge,2001; Zhao et al.,2008); (ii) DNA-binding domain (DBD) which highly conserved between ERα and

ERβ and shares 95% identity, it consists of the C domain featuring two functionally distinct cys-cys zinc fingers with which the receptor interacts with major groove and

20 phosphate backbone of the DNA; (iii) Ligand binding domain (LBD) (domain E) consists of 12 alpha helices that form the ligand binding pocket and contains the activation function 2 (AF-2), it is responsible for functions activated by ligand binding, such as coregulator binding and receptor dimerization, the two ER isoforms share 55% identity in this region and the ligand binding cavity of ERβ is smaller than the binding pocket of

ERα by about 20% (Klinge,2001; Zhao et al.,2008). ERβ has a weaker AF-1 function hence depends largely on the ligand-dependent AF-2 for its transcriptional activity.

Ligands of ER interact with distinct set of residues in the LBD and induce a unique conformation of helix 12. The precise positioning of helix 12 is different for agonist and antagonist binding – when bound by agonist estrogen, the helix 12 sits snugly over the ligand binding cavity which is a prerequisite for the transcriptional activity of ER, in contrast, when bound by an antagonist such as raloxifene, the alignment of helix 12 over the ligand binding cavity is prevented, instead the helix 12 lies in the groove formed by helices 5 and 3 (Brzozowski et al.,1997). The D domain also known as the hinge region shares only about 30% identity between the two isoforms, it has been implicated in nuclear translocation and reported to contain the nuclear localization signal (Picard et al.,1990; Zhao et al.,2008). The F domain is responsible for modulating transcription in a ligand specific manner, it shares only about 20% homology between the two ER subtypes

(Montano et al.,1995; Koide et al.,2007; Zhao et al.,2008). At least seven promoters have been described which could transcribe the human ERα gene. The transcripts from these promoters vary in their 5’-UTRs. Most of these promoters do not have a TATA box,

CCAAT box or GC box sequences; although auto regulation of some of these promoters by estrogen has been observed. The potential use of multiple promoters could be cell and

21 tissue specific promoter utilization or usage of different promoters during various stages of development or variable alternative splicing of transcripts from different promoters producing various protein isoforms (Kos et al.,2001). The promoters for the ERβ gene have not been characterized completely, although two different promoters ON and OK have been described (Zhao et al.,2008). ERα and ERβ are both expressed in the breast, brain, cardiovascular system, ovaries and bone, however some variation in their expression has been reported – ERα is the main isoform in the liver and ERβ in the colon.

Further, in the ovaries, ERα is largely expressed in the thecal and interstitial cells, in contrast to ERβ which is predominantly present in the granulosa cells (Pearce et al.,2004). In normal breast tissue, only 15-25% of the epithelial cells express ER and this cell population is largely non-dividing (Ali et al.,2002) and act to stimulate the growth of surrounding ER-negative cells in response to estrogen.

ERs primarily act as transcriptional factors regulating expression of several genes in a cell, tissue specific manner. In the absence of any ligand ER is bound by an inhibitory complex of chaperones such as Hsp70 and Hsp90. Various methods have been applied to determine the cellular localization of ER. Ultracentrifugation sucrose gradient studies suggested a two-step mechanism which involved extranuclear localization of ER in the absence of hormone followed by receptor transformation after binding of ligand

(Jensen et al.,1973). Later, monoclonal antibodies were developed against ER; immunoperoxidase staining and cytoplast, nucleoplast fractionation after enucleation showed that the receptor is primarily in the nucleus even in the absence of ligand (apo-

ER) (King et al.,1984; Welshons et al.,1984). A small pool of ER is thought to be

22 localized in the cytoplasm which constantly shuttles between the cytoplasm and nucleus

(Maruvada et al.,2003).

There are several mechanisms by which ER could regulate the expression of genes – (i) Classical ligand-dependent pathway: Upon ligand binding, the complex of chaperones dissociates from ER causing dimerization of the receptor. On dimerization, agonist (such as estradiol) bound ER binds to its estrogen response elements (EREs) in the DNA with high affinity, causing recruitment of coactivators and general transcription machinery leading to activation of downstream genes. However, antagonist (such as tamoxifen) bound ER binds to the EREs in the chromatin and recruits corepressors which cause the repression of genes. ERE is a 13bp palindrome consisting of two half sites separated by a 3bp spacer (GGTCAnnnTGACC). Agonist-bound ER binding to EREs causes recruitment of coactivators such as the p160 family (SRC1, SRC2/TIF2/GRIP1 and SRC3/RAC3/ACTR/AIB1). These p160 family proteins contain LXXLL (NR-box) motifs which interact with AF-2 domain of ER; one of the functions of these proteins is to recruit proteins such as CREB-binding protein (CBP)/p300 and p300/CBP-associated factor (pCAF) that have intrinsic histone acetyltransferase (HAT) activity. The core transcription initiation complex TBP, TFIIB and TFIID interacts with ER (Ing et al.,1992; Jacq et al.,1994; Sadovsky et al.,1995) along with the ATP-dependent chromatin remodeling SWI/SNF (SWItch/Sucrose NonFermentable) complexes altering the local chromatin structure to a more open conformation permitting transcription.

Corepressors such as nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone (SMRT) interact with helices 3 and 5 in the LBD of ER through their CoRNR boxes (LXXI/HIXXXI/L). NCoR and SMRT in turn recruit histone

23 deacetylases (HDAC) and repress the downstream gene activity (Welboren et al.,2009).

(ii) ERE-independent genomic pathway: Almost a third of the genes regulated by ERs lack ERE-like sequences. In such cases, ER is tethered by other transcription factors to their binding sites leading to activation of downstream genes. Activation by estrogen could be mediated through interaction of ER with Fos and Jun at AP-1 (Activator Protein

1) binding sites. These interactions of ER with AP-1 require both AF-1 and AF-2 functions of ER. Furthermore, several genes with GC-rich sequences in their promoters are induced by ER-Sp1 (Specificity protein 1) complexes. Interactions of ER with NF-κB are also well demonstrated. These tethering mechanisms of ER require p160 family of coactivators along with CBP/p300 and SWI/SNF complexes. (iii) Ligand-independent activation of ER: In the absence of estrogen, ER function can be modulated by extracellular signals – polypeptide growth factors such as epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1). Epidermal growth factor receptor (EGFR) or insulin-like growth factor receptor (IGFR) signaling activates MAPK which phosphorylate ER at serine 118. This phosphorylation causes ER activation and enhances its genomic actions in the absence of ligand. Growth factor signaling pathways also phosphorylate coactivators of ER, SRC1 and AIB1 increasing their activity (Hall et al.,2001). (iv) Non-genomic actions of ER: The non-genomic actions of ER include mobilization of intracellular Ca, stimulation of adenylate cyclase and hence an increase in cAMP production, activation of PI3K/Akt and MAPK pathways. These actions of ER are rapid and transient, mediated by plasma membrane associated forms of ER. Splice variants of ERα have been isolated from the plasma membrane – ER46, ER36 but the membrane bound G-protein coupled receptor 30 (GPR30) (Carmeci et al.,1997) has been

24 characterized as the major form of membrane ER. GPR30 is a G-protein coupled receptor with 7 transmembrane domains which binds to ligands in addition to estrogens. The membrane bound ER is now officially called G-protein coupled ER (GPER). The expression of GPER is not only found in ER-positive cells lines but also in ER-negative cell lines. The role of GPR30 in breast cancer pathophysiology is not clear; however, the

GPR30 signaling pathway is activated by both estrogen and tamoxifen in vitro. Thus, tamoxifen is an agonist of the non-genomic actions of ER (Maggiolini et al.,2010).

1.7 Gene repression by estrogen and de-repression by tamoxifen

Gene expression profiling of MCF-7 ER-positive breast cancer cells in the presence of estrogen showed that out of all the genes regulated by estrogen, 70% of the genes were down-regulated (Frasor et al.,2003). This repression of genes by estrogen was largely reversed by tamoxifen, raloxifene and fulvestrant (Frasor et al.,2004). The general mechanisms of this estrogen-mediated gene repression are poorly understood.

There have been various studies elucidating the mechanism of estrogen/ER-mediated gene repression in context of specific genes. The repression of some genes is mediated by

ER through direct interaction with other transcription factors such as GATA-1 (Blobel et al.,1995), C/EBPβ, NF-κB (Stein et al.,1995), Sp1 (Stossi et al.,2006; Higgins et al.,2008) or AP-1 (Jones et al.,2002). ER inhibited the transcriptional activation of GATA-1 in response to estrogen, inhibiting GATA-1 target genes and thus regulating erythropoiesis

(Blobel et al.,1995). Transcription factors C/EBPβ and NF-κB also interact with estrogen bound ER causing transcriptional repression of the IL-6 in human osteoblasts

(Stein et al.,1995). This transcriptional repression of by estrogen is an important mechanism of preventing inflammatory diseases (Cvoro et al.,2006). In

25 postmenopausal women, the drop in the estrogen levels leads to production of cytokines giving rise to inflammatory conditions like osteoporosis. Repression of the hepatic lipase gene by estrogen was attributed to the recruitment of ER by AP-1 and has been associated with the increase in HDL-cholesterol during estrogen replacement therapy

(Jones et al.,2002). Additionally, ER mediates gene repression through recruitment of co- repressors such as NCoR (Stossi et al.,2006; Higgins et al.,2008), SMRT (Higgins et al.,2008), NRIP1 (Teyssier et al.,2003), CtBP1 (Stossi et al.,2009) or REA (Karmakar et al.,2009). ER is recruited to the GC-rich region in the cyclin G2 promoter by transcription factor Sp1 causing co-repressor NCoR and histone deacetylase (HDAC) binding, thus repression of cyclin G2 (Stossi et al.,2006). Another study illustrated the recruitment of co-repressors NCoR and SMRT to the VEGFR2 promoter by ERα/Sp1 and

ERα/Sp3 interactions leading to estrogen dependent down-regulation of tyrosine kinase

VEGFR2 gene (Higgins et al.,2008). Alternatively, co-repressor NRIP1/RIP140 mediates repression of estrogen-induced AP-1 dependent transcription by competing with co- activator GRIP1 (Teyssier et al.,2003). Whereas, the p300-interacting partner, co- repressor CtBP1 is recruited to the ER binding sites of early estrogen-repressed genes by p300/ER complex eliciting chromatin modifications causing gene repression (Stossi et al.,2009). Estrogen also represses the tumor necrosis factor α (TNF-α) promoter which requires the AF-2 surface of the ER along with the recruitment of coregulators. After menopause, the increase in TNF-α production is associated with pathogenesis of osteoporosis (An et al.,1999). Low levels of plasma protein S (PROS1) in pregnant women are associated with a risk of deep venous thrombosis. In hepatocytes and other tissues of the body, estrogen represses the expression of protein S by associating with

26 Sp1, Sp3 at the GC-rich motifs in the promoter of the PROS1 gene. Furthermore, it was demonstrated that this was followed by recruitment of RIP140 and NCoR/SMRT/HDAC complexes (Suzuki et al.,2010). When the repression of the erbB2 gene by ER was first demonstrated (Dati et al.,1990; Read et al.,1990; Warri et al.,1991; Antoniotti et al.,1994), the mechanism was unknown. This mechanism was elucidated in a step-by step manner; the suppression of ERBB2 mRNA and protein was found to be a transcriptional effect of ER and a 409bp region within the erbB2 gene was recognized as an estrogen suppressible enhancer (Bates et al.,1997). Later, it became clear that this repression by

ER was ligand-dependent requiring the AF-2 domain and involved ER cofactors

(Newman et al.,2000). The ER cofactor required for the repression of erbB2 gene was demonstrated to be PAX2. In the presence of estrogen, co-repressor PAX2 inhibits expression of ERBB2, while AIB1 competes with PAX2 for binding to ER; overexpression of AIB1 leads to upregulation of ERBB2 expression (Hurtado et al.,2008). Among all the instances of gene repression by estrogen, the mechanism of repression has been shown to be non-classical, ERE-independent and in all cases tamoxifen antagonizes estrogen-mediated repression. Studies published by our laboratory have previously discovered a non-classical mechanism of gene repression by estrogen where estrogen bound-ER was recruited by TAFII30 to the core promoter of folate receptor α (FRα) gene. ER also recruits corepressors NCoR and SMRT leading to repression of the core promoter (Kelley et al.,2003; Hao et al.,2007). Tamoxifen blocks the recruitment of ER and the corepressor complex resulting in de-repression of the promoter. However, there is a gap in our understanding about the significance of this gene repression by estrogen and its antagonism by tamoxifen.

27 1.8 Carcinoma of lung, epidemiology and etiology

An estimated 230,000 new cases of lung cancer are expected to be diagnosed in

United States in 2013. Lung cancer is the leading cause of cancer related deaths worldwide and is expected to account for 26% of all female cancer deaths and 28% of all male cancer deaths in United States (Siegel et al.,2013).

A single etiologic agent, exposure to tobacco smoke or use of cigarettes is the leading cause of nearly 90% of lung cancers (Alberg et al.,2007). The first evidence establishing a scientific, causal link between tobacco smoking and lung cancer were two case-control studies in 1950s (Doll et al.,1950; Wynder et al.,1950). The duration of cigarette smoking and the number cigarettes smoked per day are determinants of the risk of lung cancer, which increases by about 70-fold in a person smoking 40 cigarettes a day for 20 years compared to a lifelong non-smoker (Keshamouni et al.,2009). The lung cancer risks associated with cigar and pipe smoking are substantial but less than the risks observed for cigarette smoking. The risk decreases steadily over a period of time in those who quit smoking. However, the risk among former smokers remains elevated compared to non-smokers (Alberg et al.,2007). Lung cancer risk is increased by up to 25% due to passive or secondhand smoking or exposure to cigarette smoke (Taylor et al.,2007).

About 10% of lung cancer patients are non-smokers (Govindan et al.,2006). The biology of the disease differs from smokers to non-smokers; adenocarcinomas with EGFR mutations are more frequent in non-smokers. Various other environmental exposures, occupational and non-occupational have been associated with increased risk of lung cancer; they include asbestos, air pollution, radon, polycyclic aromatic hydrocarbons, nickel, chromium and arsenic (Alberg et al.,2007; Keshamouni et al.,2009).

28 1.9 Classification and management of lung cancer

Lung cancer is mainly classified into two types based on the histology: Small cell lung carcinoma (SCLC) and Non-small cell lung carcinoma (NSCLC).

Small cell lung cancer: The overall incidence rate of SCLC in the Unites States has decreased in the past few decades and accounts for about 15% of lung cancers diagnosed. Nearly all cases of SCLC are attributed to smoking cigarettes (Govindan et al.,2006). This is the most aggressive of lung cancers with a rapid doubling time, earlier development of metastases and median survival from diagnosis of only 2 to 4 months without treatment. Regardless of the stage, patients presenting with SCLC have very poor prognosis despite the improvements in diagnosis and treatment. Extent of disease is an important prognostic factor for SCLC. Patients with limited stage disease have a better prognosis with a 5-year survival of 12.1% compared to patients with extensive disease

(Janne et al.,2002). At the time of diagnosis, most patients usually present with metastatic

SCLC.

Chemotherapy and radiation therapy has been shown to improve the overall survival of patients with SCLC. However, chemotherapy is presently the mainstay in the treatment of SCLC. A combination of platinum-based drugs, cisplatin or carboplatin, with the topoisomerase inhibitor etoposide is the standard regimen for patients with small cell lung cancer (Evans et al.,1985; Johnson et al.,2004; Jackman et al.,2005). The combination of carboplatin and the topoisomerase inhibitor irinotecan is an option for patients with extensive-stage disease (Lima et al.,2010). Patients with SCLC tend to develop distant metastases and localized treatments such as surgical resection rarely

29 produce long-term survival (Prasad et al.,1989). SCLC is highly sensitive to initial chemotherapy and radiotherapy but patients usually die of recurrent disease.

Non-small cell lung cancer: NSCLC is the most common type of lung cancer and accounts for 80% of the lung cancer cases diagnosed. NSCLC may be classified into three main subtypes based on histology – adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Adenocarcinoma is the most common subtype of lung cancer diagnosed in lifelong non-smokers (Subramanian et al.,2007) and accounts for 40% of lung cancers (Travis et al.,1995). EGFR mutations are predominantly found in adenocarcinomas of non-smokers (Pao et al.,2011). Approximately 90% of mutations are exon 19 deletions and exon 21 L858R point mutations which activate downstream signal transduction independent of ligand. EGFR mutations are often accompanied by gene amplification (Ladanyi et al.,2008). Amplification of MET occurs in 20% of EGFR mutant tumors with acquired resistance to EGFR inhibitors (Engelman et al.,2007).

ERBB2 mutations also occur in about 1–2% of adenocarcinomas. Mutations in KRAS appear to be smoking-related and are predictors of poor response or failure to benefit from EGFR TKI therapy. Younger patients and never-smokers with adenocarcinomas could also harbor ALK fusions (Ladanyi et al.,2008). Squamous cell carcinomas are the second most common type of lung cancers diagnosed also associated with smoking, often centrally located derived from bronchial epithelial cells. The incidence of this subtype of

NSCLC has decreased as a result of reduction in yields of tar and nicotine in cigarette smoke due to the introduction of cigarette filter (Stellman et al.,1997). Large-cell carcinomas constitute approximately 5% of lung cancers (Ginsberg et al.,2007). These tumors show no evidence of squamous differentiation. Large-cell neuroendocrine

30 carcinomas reveal histologic features suggestive of neuroendocrine differentiation and express neuronal markers (Langer et al.,2010).

The choice of therapy for patients with NSCLC is based on the stage of the disease on diagnosis. The confinement of primary tumor or its metastasis to lymph node or distant organs is considered. Surgical resection of the tumor by lobectomy or pneumonectomy (removal of entire affected lung) is the preferred treatment for an early stage (I/II) NSCLC, followed by adjuvant chemo or radiation therapies. Nearly 40% of newly diagnosed NSCLC are in advanced stages (III/IV). In advanced disease, if complete surgical resection of tumor and lymph nodes is not possible, treatment options include cytotoxic chemotherapy and targeted agents to prolong survival and control disease-related symptoms (Keshamouni et al.,2009).

Platinum- based combination therapy has been the standard first line treatment for patients with advanced NSCLC since the 1990s (Non-small Cell Lung Cancer

Collaborative Group,1995). Toxicity reasons prevented the use of combinations of platinum therapy with targeted agents like bevacizumab and cetuximab in most patients even though it showed improved survival rates. Different schedules of cisplatin in combination with vinorelbine or paclitaxel or docetaxel or irinotecan showed advantages in terms of response rate, toxicity and quality of life, but little improvement in terms of survival (Bunn,2002). Until recently, the combination of gemcitabine and cisplatin was most commonly used as the first-line therapy as this combination improved the progression-free survival of advanced NSCLC patients when compared to other platinum- combination therapies (Le Chevalier et al.,2005). In 2008, FDA approved the combination of pemetrexed (an antifolate which targets key enzymes in the de novo

31 purine and pyrimidine synthesis) and cisplatin as the first-line treatment for advanced non-squamous NSCLC. (Scagliotti et al.,2008; Ciuleanu et al.,2009). Addition of third drug to the standard chemotherapy doublet offered no therapeutic benefit but increased toxicity (Alberola et al.,2003). A better understanding of the molecular oncology and genetics of NSCLC has led to the development of various alternative molecular targeted agents discussed below.

Approximately 10% of NSCLC patients have EGFR mutations (Lynch et al.,2004). Tyrosine Kinase Inhibitors (TKI) erlotinib and gefitinib are small molecules that block the EGFR pathway and have been approved for second-line treatment of patients who are refractory to platinum based therapy. The expression of EGFR in lung cancer patients predicts sensitivity to these inhibitors (Paez et al.,2004; Pao et al.,2004).

Cetuximab is a recombinant, chimeric murine-human monoclonal antibody directed against the ligand binding site of EGFR which competes with endogenous ligands EGF and TGFα. Phase III FLEX trial showed modest improvement in overall survival rates in combination with platinum-based chemotherapy in EGFR expressing advanced NSCLC patients (Pirker et al.,2009).

Crizotinib, an ALK (anaplastic lymphoma kinase, receptor tyrosine kinase) inhibitor is shown to be effective in ALK fusion-positive lung cancers, which are resistant to EGFR inhibitors. A phase II clinical trial with crizotinib reported at the 46th Annual

Meeting of the American Society of Clinical Oncology in 2010, demonstrated a high response rate in patients selected for ALK fusion (Pao et al.,2011).

32 The angiogenesis inhibitor, bevacizumab is a recombinant human monoclonal antibody that targets the vascular endothelial growth factor (VEGF). The addition of bevacizumab to the combination of paclitaxel-cisplatin prolonged the progression-free survival and improved the response rate in advanced non-squamous NSCLC patients but there was in increase in toxicity due to bevacizumab (Sandler et al.,2006).

A small molecule inhibitor of proteasome, bortezomib has yielded promising results in combination with gemcitabine and carboplatin in phase II studies of patients with advanced NSCLC (Davies et al.,2007).

1.10 Pemetrexed in the treatment of NSCLC – single agent and combination therapy

Pemetrexed is a newer antifolate approved by the FDA for first-line treatment of advanced non-squamous NSCLC in combination with cisplatin (Scagliotti et al.,2008;

Ciuleanu et al.,2009), as a single agent in second-line treatment of advanced NSCLC

(Hanna et al.,2004) and in combination with cisplatin as a first-line treatment for malignant pleural mesothelioma (Vogelzang et al.,2003).

Pharmacokinetic single-agent dose-escalating studies were conducted in three phase I trials (Rinaldi et al.,1995; McDonald et al.,1998; Rinaldi et al.,1999) and a maximum tolerated dose (MTD) of 600 mg/m2 once every 21 days was defined. The major toxicities included neutropenia, thrombocytopenia, fatigue, anorexia, nausea, diarrhea and rash. Pemetrexed was shown to eliminate rapidly from plasma with a terminal elimination half-life of between 2 and 5 hours. The MTD was modified to

500mg/m2 based on the evaluation of safety of pemetrexed in patients with moderate renal function as it is mainly metabolized by the kidneys undergoing largely urinary

33 excretion as unchanged parent compound within 24 hours (Rinaldi,1999). Single-agent phase II studies (Rusthoven et al.,1999; Clarke et al.,2002; Smit et al.,2003) including two studies (Rusthoven et al.,1999; Clarke et al.,2002) in patients with previously untreated NSCLC were conducted using the dose recommended by the phase I studies which demonstrated a moderate single-agent activity of pemetrexed in chemo-naïve patients or first-line chemotherapy treated patients with advanced NSCLC. Toxicities were neutropenia and grade 3 or 4 rash in more than a third of the patients. Toxicity was positively correlated with increasing plasma homocysteine levels which is an indirect measure of folate and vitamin B12 status in the patients (Niyikiza et al.,2002). Based on this finding folate/B12 supplementation was added to the regimen. Pretreatment of patients with dexamethasone prevented the occurrence of rash. Combination studies of pemetrexed with platinum-based chemotherapy (Manegold et al.,2000; Shepherd et al.,2001; Scagliotti et al.,2005; Zinner et al.,2005) were executed in phase II studies with encouraging response rates and survival times. On the basis of the above trials a 500 mg/m2 dose of pemetrexed given on day 1 of a 21-day cycle as 10-minute infusion supplemented with vitamin B12, folic acid and dexamethasone was tested as a single agent or in combination with platinum in various phase III trials (Hanna et al.,2004;

Scagliotti et al.,2008; Ciuleanu et al.,2009; Gronberg et al.,2009). A randomized phase III trial was performed to compare the efficacy and toxicity of pemetrexed vs. docetaxel in advanced NSCLC patients, previously treated with chemotherapy which revealed similar response rates and median survival times in both arms but pemetrexed was better tolerated with significantly fewer side effects in the second-line treatment of advanced

NSCLC (Hanna et al.,2004). Pemetrexed was then approved by the FDA for the

34 treatment of relapsed NSCLC in August 2004; until this study docetaxel was the only

FDA-approved chemotherapy for second-line treatment of NSCLC. A pivotal randomized, noninferiority, phase III trial was conducted to evaluate the overall survival of cisplatin/ pemetrexed combination as a first-line treatment for advanced NSCLC compared to an effective widely used regimen of cisplatin/gemcitabine in chemotherapy- naive NSCLC patients (Scagliotti et al.,2008). The combination of cisplatin/pemetrexed provided similar efficacy but better tolerability with significant reduction in the drug- related hematologic toxicities than cisplatin/gemcitabine. Additionally, this study identified a significant therapeutic advantage for pemetrexed by classifying the effect of cisplatin/pemetrexed on survival, based on tumor histology. The overall survival of patients with non-squamous NSCLC was significantly improved with combination of cisplatin/pemetrexed versus cisplatin/gemcitabine – large-cell carcinoma 10.4 months vs.

6.7 months respectively and adenocarcinoma 12.6 months vs. 10.9 months respectively.

(Scagliotti et al.,2008). However, in patients with squamous cell NSCLC cisplatin/pemetrexed combination was inferior to cisplatin/gemcitabine combination with overall survival of 9.4 months with cisplatin/pemetrexed and 10.8 months with cisplatin/gemcitaine combination. This led to the FDA approval of pemetrexed in combination with cisplatin for the first-line treatment of patients with advanced non- squamous NSCLC in September 2008. In July 2009, FDA approved pemetrexed for maintenance therapy for patients with locally advanced or metastatic non-squamous

NSCLC who have not progressed after platinum therapy based on the study that demonstrated significantly improved progression-free survival (4.3 months vs. 2.6 months) and overall survival (13.4 months vs. 10.6 months) of patients with advanced

35 non-squamous NSCLC on pemetrexed maintenance therapy as compared to placebo.

(Ciuleanu et al.,2009).

1.11 Mechanism of action of pemetrexed

Pemetrexed or LY231514, marketed by Eli Lilly as Alimta (N-[4-[2-(2-amino-

3,4-dihydro-4-oxo-7H-pyrrolo[2,3,-d]pyrimidin-5-yl)-ethyl]-benzoyl]-L-glutamic acid) is a multitargeted antifolate which possess a unique 6-5 fused pyrrolo[2,3-d]pyrimidine nucleus unlike previously discovered antifolates (Shih et al.,1998). Pemetrexed was developed from an inhibitor of de novo purine synthesis, lometrexol, which targeted glycinamide ribonucleotide formyltransferase (GARFT). The substitution of 5- deazapteridine ring of lomotrexol with pyrrolopyrimidine ring led to synthesis of pemetrexed (Taylor et al.,1992). The development of pemetrexed was a collaborative effort between Edward C. Taylor at Princeton University and a team of chemists at Eli

Lilly led by Chuan Shih (Chattopadhyay et al.,2007). Pemetrexed is an inhibitor of several tetrahydrofolate (THF)-cofactor requiring enzymes involved in the synthesis of purines and thymidine – thymidylate synthase (TS), dihydrofolate reductase (DHFR),

GARFT and to a lesser extent 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT) and C1-tetrahydrofolate synthase (Shih et al.,1998). TS catalyzes the rate-limiting step in the synthesis of deoxythymidine monophosphate

(dTMP) from deoxyuridine monophosphate (dUMP). This is the sole de novo thymidine synthesis reaction which requires the folate cosubstrate 5,10-methylenetetrahydrofolate

(CH2-THF) which is oxidized to dihydrofolate (Touroutoglou et al.,1996). By inhibiting

TS, pemetrexed decreases the de novo thymidine synthesis required for DNA synthesis.

Inhibition of DHFR by pemetrexed blocks the replenishment of CH2-THF enhancing the

36 inhibitory effects of TS. Pemetrexed has an antiproliferative effect on CCRF-CEM leukemia cells which can be rescued by addition of thymidine at low concentrations of the drug, indicating TS is the major target. However, at higher concentrations of pemetrexed, protection from the antiproliferative effect requires both thymidine and hypoxanthine, suggesting that inhibition of TS and GARFT is responsible for secondary cytotoxic effects of the drug. The inhibition of TS is 50 times more potent than inhibition of GARFT (Shih et al.,1998; Chattopadhyay et al.,2007). Pemetrexed is transported into the cells principally via the reduced folate carrier (RFC); the drug has high affinity for folate receptor-α but it is not dependent on it for intracellular transport (Shih et al.,1997).

Efflux transporters ATP-binding cassette (ABC) transporters including multidrug resistance proteins (MRPs) which hydrolyze ATP to transport their substrates across the cell membrane are implicated in the efflux of pemetrexed (Assaraf,2006). Pemetrexed is administered as a prodrug; once inside the cell it is poly-γ-glutamated by folylpolyglutamate synthase (FPGS). The polyglutamated derivatives (tri-, tetra- and penta-glutamates) have a longer half-life and are more negatively charged (polyanions) than the parent compound which increases their solubility, resulting in reduction in the cellular efflux and increase in accumulation within the cells allowing them to attain significantly higher levels of cellular retention than could be achieved by the parent compound. These derivatives cause more potent and prolonged inhibition of the target enzymes (e.g.: the pentaglutamated form is100 times more potent inhibitor of TS than the monoglutamed form); this confers a pharmacologic advantage to pemetrexed (Taylor et al.,1992; Shih et al.,1997; Shih et al.,1998). The cytotoxic effects of pemetrexed are enhanced due to its high affinity to multiple folate-requiring enzymes in combination

37 with its increased cellular retention. The inhibition of TS leads to a ‘thymine-less’ state and also increases the misincorporation of dUTP in the DNA resulting in DNA fragmentation and apoptosis (Touroutoglou et al.,1996). Accordingly, the accumulation of deoxyuridine (dUrd) in the circulation is exploited as a marker for the inhibition of TS by antifolates in vivo (Joerger et al.,2010).

1.12 Mechanisms of resistance to pemetrexed

The various mechanisms that have been attributed to antifolate resistance are impaired drug transport into the cell, increase in expression or amplification of target enzymes or decrease in polyglutamation due to mutations or alterations in the expression of FPGS. Intracellular folate pools also affect the efficacy of antifolates by competing with FPGS; high folate pools suppress the formation of active polyglumated antifolates.

Mechanisms of acquired resistance to various antifolates could be similar despite a few differences depending on the drug. The polyglutamation of pemetrexed by FPGS is a prerequisite to its anticancer activity and also determines its selectivity. The entry of pemetrexed into the cell is facilitated by RFC. A small decrease in mRNA and protein expression of both RFC and FPGS has been observed in pemetrexed-resistant L1210 murine leukemia cells (Wang et al.,2003). However, mutations in FPGS or downregulation of FPGS activity are recognized as one of the predominant mechanisms of resistance to pemetrexed (Liani et al.,2003). A decrease in or defective polyglutamation due to mutations in FPGS results in altered binding affinity for either the glutamic acid or the antifolate substrate (Mauritz et al.,2002). The polyglutamated derivatives of pemetrexed are substrates for γ-glutamyl hydrolase (GGH) which catalyzes the hydrolysis of these derivatives by removal of the γ-linked polyglutamates. The

38 pharmacological effectiveness of the drug has been inversely correlated to the expression and cellular activity of GGH. (Rhee et al.,1999). Increase in the expression of the target enzyme of pemetrexed, TS has been proven to be the major mechanism of resistance to pemetrexed in vitro (Sigmond et al.,2003; Ozasa et al.,2010; Takezawa et al.,2011).

Indeed, the poor response to pemetrexed in squamous cell carcinoma is thought to be a result of higher expression of TS mRNA and protein in squamous cell carcinoma compared to other NSCLC histotypes (Ceppi et al.,2006; Scagliotti et al.,2008; Monica et al.,2009) and level of TS expression has been negatively correlated to response to pemetrexed in NSCLC (Christoph et al.,2013) and to TS-targeted drugs in various cancers (Johnston et al.,1994; Johnston et al.,1997; Pestalozzi et al.,1997; Ferguson et al.,1999). Increase in the expression of the efflux transporters, MRPs, can also mediate resistance to pemetrexed (Assaraf,2006; Uemura et al.,2010).

Pemetrexed is widely used in the front-line therapy of non-squamous NSCLC; the median progression free survival in patients with non-squamous NSCLC on a combination of cisplatin/pemetrexed was 5.3 months (Scagliotti et al.,2008; Scagliotti et al.,2009) whereas it was only 3.1 months for pemetrexed as a single agent (Hanna et al.,2004; Scagliotti et al.,2009). However, this modest efficacy of pemetrexed suggests that benefit of pemetrexed in patients with advanced NSCLC is variable. Alternative treatment options such as gemcitabine/cisplatin could be beneficial in patients that might not respond to pemetrexed. Hence, there is a pressing need for predictors of pemetrexed response and benefit. Clinical studies have been conducted implicating TS expression as a predictor of pemetrexed response (Nakagawa et al.,2002; Christoph et al.,2013). The unique histologic specificity of pemetrexed was also linked to higher expression of TS in

39 squamous cell carcinoma (Ceppi et al.,2006; Scagliotti et al.,2008; Monica et al.,2009).

Prospective clinical trials evaluating predictive biomarkers of pemetrexed response is the need of the hour as this would lead to individualized treatment to overcome poor outcome of patients with NSCLC.

1.13 Therapeutic applications of glucocorticoids

Endogenous glucocorticoids are secreted by the adrenal cortex under the control of the hypothalamus-pituitary-adrenal (HPA) axis. These hormones are responsible for regulation of homeostasis; and are secreted in response to stress factors that signal the hypothalamus to regulate the secretion of adrenocorticotropic hormone (ACTH) from the pituitary gland. ACTH interacts with specific receptors in the adrenal gland leading to production and release of cortisol from the adrenal cortex. The lipophilic cortisol readily diffuses into the cell and binds to its receptor, the glucocorticoid receptor which is virtually expressed in all cells. Biologically active free cortisol is converted to cortisone

(inactive) by type 2 11β-hydroxysteroid dehydrogenase, whereas type 1 11β- hydroxysteroid dehydrogenase catalyzes the conversion of cortisone to cortisol (Rhen et al.,2005; Duma et al.,2006).

The Nobel Prize for Medicine in 1950 was awarded jointly to Kendall, Reichstein and Hench for their discoveries relating to the biological and therapeutic effects of pituitary adrenocorticotropic hormone and adrenal cortex secreted hormone, cortisone.

Synthetic glucocorticoids such as prednisone and dexamethasone have since been used effectively in controlling symptoms of a variety of acute and chronic inflammatory and autoimmune diseases including asthma, rheumatoid arthritis, ulcerative colitis and

40 allergic rhinitis. The role of glucocorticoids in induction of apoptosis has been exploited in the treatment of Hodgkin’s lymphoma, acute lymphoblastic leukemia and multiple myelomas (Duma et al.,2006).

Glucocorticoids impair glucose uptake in insulin sensitive cells elevating serum glucose levels, hence chronic use of these leads to glucose intolerance and steroid-induced diabetes. The main side effect of glucocorticoid therapy is osteoporosis and increased risk of fractures due to a reduction in the number and activity of osteoblasts, whereas an increase in the number of osteoclasts. In addition, glucocorticoids have immunosuppressive effect and increase skin fragility and thinning due to an augmentation of muscle catabolism. These effects are a result of pleiotropic effects of the glucocorticoid receptor on multiple signaling pathways (Buckbinder et al.,2002; Rhen et al.,2005).

Chemotherapy induced nausea and vomiting (CINV) are the most common side effects of cancer chemotherapy. Various neurotransmitters such as dopamine, serotonin and their receptors play an important role in this process by influencing areas of the brain

(vomiting center) and areas of the gastrointestinal tract. Corticosteroids - dexamethasone or methylprednisolone are commonly administered with type 3 serotonin receptor antagonists to prevent chemotherapy-induced emesis. (Markman,2002). Randomized trials have shown that corticosteroids can potentiate the anti-emetic effects of serotonin receptor antagonists caused by high emetogenic potential chemotherapy like carboplatin or cisplatin regimens (Roila et al.,1991; Hesketh et al.,1994; Italian Group Antiemetic

Research,1995; Ioannidis et al.,2000). The anti-inflammatory properties of corticosteroids may prevent the release of serotonin in the gut (Fredrikson et al.,1992). Dexamethasone

41 is effective in the prevention of CINV in both acute and delayed phases. A single dose of

8mg before chemotherapy is recommended during the use of a moderate emetogenic chemotherapeutic agent (e.g: oral etoposide, carboplatin) whereas a dose of 12mg or

20mg is recommended prechemotherapy for high emetogenic chemotherapy (e.g: cisplatin) (Lohr,2008).

Another common side effect of chemotherapy is extensive skin rashes.

Dexamethasone is used as prophylactic measure to prevent skin rashes, with certain chemotherapeutic agents like gemcitabine (Chen et al.,1996), docetaxel (Chouhan et al.,2011) or pemetrexed (Hanna et al.,2004). Radiation recall dermatitis is a cutaneous toxicity induced by chemotherapeutic drugs in patients with advanced disease previously treated radiation therapy. It is described as localized erythema, edema, dry desquamation, blistering, or ulceration that occurs in an area of a previously quiescent field of irradiation, usually occurring a few days after the injection of an antitumor antibiotic

(Donaldson et al.,1974). This phenomenon has been reported after intravenous administration of doxorubicin, methotrexate, gemcitabine and paclitaxel. Administration of oral dexamethasone completely resolves these cutaneous side effects and allows resumption of chemotherapy (Castellano et al.,2000).

1.14 Mechanism of action of the glucocorticoid receptor

Glucocorticoid receptor (GR) was first cloned in 1985 as two different isoforms

GRα (777 amino acids) and GRβ (742 amino acids) (Hollenberg et al.,1985). The hGR gene is located on chromosome 5q31 and spans over 140kb, although less than 2% (

2.5kb) of it comprises of exons. Promoters of the hGR gene do not contain a consensus

42 TATA or CAAT box, but all of them contain multiple CpG islands and binding sites for various transcription factors – AP1, AP2, Sp1, NF-κB, Yin Yang 1 (YY1) and CREB.

The presence of wide range of transcription factor binding sites could account for the constitutive expression of hGR under a variety of physiological conditions. The hGR gene comprises of 9 exons, the coding sequence constitutes exon 2-9. Exon 1 is a leader sequence which is not a component of the coding sequence due to presence of an in- frame stop codon at the beginning of exon 2. Alternative promoter usage and alternative mRNA splicing can yield at least 5 exon 1 variants producing hGR transcripts with variable leader sequences. Alternative splicing in the coding sequence of the GR mRNA produces different isoforms. Exons 2 to 8 are a common component of both GRα and

GRβ mRNA, splicing of exon 9α of GR produces GRα mRNA and splicing of exon 9β produces GRβ mRNA. Thus, the GR isoforms are identical from the amino terminus to amino acid 727, with GRα having an additional 50 amino acids and GRβ an additional non-homologous 15 amino acids beyond amino acid 727. GRα is ubiquitously expressed but GRβ has varying levels of expression and a more tissue-selective distribution (Lu et al.,2004). GRβ is expressed abundantly in the epithelial cells that line the terminal bronchiole of the lung, form the outer layer of the Hassall’s corpuscle in the thymus and in cells lining the bile duct in the liver. GRβ has been largely found in the nucleus, independent of glucocorticoids; it does not bind to glucocorticoids owing to an altered ligand-binding domain hence fails to initiate transcription of downstream gene targets.

However, GRβ can bind to glucocorticoid response elements (GRE) in promoters of target genes in the absence of glucocorticoids. GRβ represses transactivation activity of

GRα as GRβ:GRα heterodimers are inactive and interfere with the formation of

43 transcriptionally active GRα homodimers demonstrating dominant-negative inhibitory activity of GRβ (Oakley et al.,1997). The relative levels of GRα and GRβ in cells may cause a differential sensitivity to glucocorticoids, with higher expression of GRβ causing glucocorticoid resistance (Pujols et al.,2001). Translation of the GRα mRNA starts after recognition of the first start codon by ribosomes; however translation reinitiation can occur at alternate translation start sites. There are seven internal translation start sites apart from the first AUG codon in the GRα mRNA, generating eight receptor isoforms with varying lengths of N-terminus termed GRα- A to D (A, B, C1, C2, C3, D1, D2, D3).

The GRα-A isoform is the full length 777 amino acid GRα protein. All the GRα isoforms are transcriptionally active due to an intact ligand-binding domain; however they have variable subcellular distribution and tissue specificities and also differ in their abilities to activate GRE-driven promoters. These isoforms of GRα are responsible for the transcriptional regulation of unique sets of genes; hence the ratios of their expression can alter the transcriptional potential, in the context of single cell type. The N-terminal sequences of the two GR isoforms GRα and GRβ are identical; hence eight GRβ isoforms can also be generated as a result of the alternative translation initiation sites (Lu et al.,2006). In addition to GRα and GRβ, another variant, GRγ, has been detected in childhood acute lymphoblastic leukemia. This isoforms differs from the other isoforms due to insertion of an additional arginine between exons 3 and 4 that is retained after splicing (Duma et al.,2006). GR-P (also known as GRδ) isoform is encoded by exons 2-7 plus several basepairs from the subsequent intronic region as a result of which, it lacks the ligand binding domain (LBD) and does not bind to glucocorticoids. GR-P has been shown to be upregulated in ALL, non-Hodgkin lymphoma and multiple myeloma patients

44 (Tissing et al.,2003). Another isoform, GR-A has been described in multiple myeloma cells, which lacks exons 5-7 (Moalli et al.,1993).

GR is a member of the steroid/thyroid/retinoid/orphan receptor superfamily of nuclear transactivating factors (Mangelsdorf et al.,1995). Members of nuclear receptor superfamily share a common structure with a variable length amino terminus, a centrally located zinc finger DNA-binding domain (DBD), hinge region and a carboxyl terminal ligand-binding domain (LBD). GR has two transcription-activating regions, one in the amino terminal domain which can be activated independent of ligand (AF-1 or τ1) and second transactivation domain, dependent on ligand in the LBD (AF-2 or τ2) (Lewis-

Tuffin et al.,2006). GR that is not bound by any ligand primarily resides in the cytoplasm is complex of molecular chaperones including heat shock proteins Hsp90, 70, 23 and immunophilins FKBP51, FKBP52, Cyp40, PP5 (Pratt et al.,2003). The interaction with specific immunophilins determines the subcellular localization of GR with recruitment of

FKBP52 correlating with nuclear localization whereas binding to FKBP51 correlates with cytoplasmic localization (Banerjee et al.,2008). The ligand-bound GR dimerizes and binds to DNA sequences termed glucocorticoid response elements (GRE) in the promoters of target genes. The sequence of GRE is highly conserved across mammalian species, which consists of two conserved six-nucleotide halves which are separated by three nonconserved bases (5’-GGTACAnnnTGTTCT). The ligand-bound receptor bound to GREs recruits coactivator or corepressor complexes thereby facilitating or inhibiting the recruitment of the basal transcription machinery followed by activation or repression of target genes. Two types of GREs exist – positive GREs that activate transcription and negative GREs that repress transcription of downstream targets (Sakai et al.,1988; Surjit

45 et al.,2011). GR could also exert genomic effects without directly binding to DNA, in such cases it interacts with other transcription factors like NF-κB, AP-1 (Buckbinder et al.,2002). Glucocorticoids exert rapid anti-inflammatory and immunosuppressive effects that are mediated through non-genomic, transcription independent mechanisms. These non-genomic effects of glucocorticoids on cellular processes including actin structures, neuronal membranes, intracellular Ca+2 mobilization and signal transduction have been reported. At high concentrations, glucocorticoids may integrate into the plasma membrane changing their physiochemical properties and activities of membrane proteins which results in reduced calcium and sodium cycling contributing to the rapid immunosuppressive and subsequent anti-inflammatory effects. A plasma membrane bound form of GR has been reported in B cells and peripheral blood mononuclear cells

(PBMNCs). There is accumulating evidence for a role of membrane bound form of GR; however their functional importance is not yet well understood (Lowenberg et al.,2007).

Glucocorticoids mediate various physiological processes including glucose metabolism, immune responses and electrolyte homeostasis. The anti-inflammatory effects of glucocorticoids are exerted by various mechanisms, a few of which are explained further. Inflammatory signals like cytokines, bacterial and viral infections activate MAPK cascades. MAPK cascade phosphorylates and activates Jun N-terminal kinase (JNK) which in turn phosphorylates transcription factor c-Jun. Phosphorylated c-

Jun forms homodimers or heterodimers with c-Fos and bind to the activator protein 1

(AP-1) response or TPA response elements (TRE) in the DNA. AP-1 induces the transcription of genes regulating inflammation and immune responses. Glucocorticoids induce MAPK phosphatase 1 which dephosphorylates and inactivates JNK inhibiting AP-

46 1 mediated transcription. In addition to inhibition of JNK phosphorylation, GR can directly interfere with AP-1 mediated transcription. This transcriptional interference between GR and AP-1 due to protein-protein interactions is a major mechanism of anti- inflammatory actions of glucocorticoids (Buckbinder et al.,2002; Rhen et al.,2005).

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor which plays a central role in regulating both innate and adaptive immunity. In its inactive state, NF-κB is present in the cytoplasm in association with inhibitory protein of NF-κB (IκB) (Li et al.,2002). In response to various stimuli including microbial pathogens, viral infections, stress signals, pro-inflammatory cytokines like tumor necrosis factor α (TNFα) and interleukin 1 (IL-1), IκB kinases

(IKK) are activated that phosphorylate IκB. This leads to ubiquination of IκB and degradation by proteasome, exposing the nuclear localization signal of NF-κB. In the nucleus, NF-κB binds to NF-κB elements and stimulates the transcription of cytokines, chemokines, cell adhesion molecules, complement factors. The ligand-bound GR physically interacts with the p65 (Rel A) subunit of NF-κB to block its transcriptional activity. This direct interaction with NF-κB accounts for most of the inhibitory effects of glucocorticoids. A secondary mechanism of inhibition of NF-κB is thought to be an increase in the synthesis of IκB by GR (Buckbinder et al.,2002; Rhen et al.,2005).

Ligand-bound GR also suppresses inflammatory genes such as IL-1 and IL-2 via negative

GREs (Dostert et al.,2004).

Glucocorticoids can both transcriptionally induce annexin-1 or lipocortin 1 and induce rapid serine phosphorylation and membrane translocation of annexin-1, through a non-genomic GR-dependent mechanism involving phosphotidylinisitol-3-kinase (PI3K)

47 and MAPK-dependent pathways (Solito et al.,2003; Lowenberg et al.,2007). Annexin-1 in turn inhibits the synthesis of major inflammatory mediators including prostaglandins and leukotrienes. However, the non-genomic anti-inflammatory mechanisms of glucocorticoids are less well understood than the genomic mechanisms. Both genomic and non-genomic mechanisms may be required for the multiple effects of glucocorticoids exerted on major immune cells including dendritic cells, , neutrophils, B- and T-lymphocytes (Baschant et al.,2010).

1.15 Mechanisms of resistance to glucocorticoids

Glucocorticoid resistance is characterized by attenuation (partial or generalized) of response to glucocorticoids (van Rossum et al.,2006). Familial glucocorticoid resistance or Chrousos syndrome is a rare genetic condition, inherited as an autosomal recessive or dominant disease with mutations in the GR gene affecting the ligand binding domain (LBD) or the DNA binding domain (DBD) with clinical symptoms of generalized cortisol resistance. These mutations reduce the ligand binding affinity, transactivation potential of GR and alter its interactions with other proteins. Due to reduction in the activity of GR, the negative feedback of glucocorticoids is reduced, leading to hypercortisolism – a compensatory increase in the hypothalamic-pituitary-adrenal axis causing increased concentrations of adrenocorticotropic hormone (ACTH) followed by elevation in circulating pituitary corticotropin, cortisol and excess secretion of non- corticoid adrenal steroids such as and androgens (van Rossum et al.,2006).

Two commonly occurring mutations causing generalized glucocorticoid resistance in patients have been characterized by genetic analysis (Ruiz et al.,2001; Charmandari et al.,2006). The R477H mutation affects the DBD of GR whereas the G679S alters the

48 LBD of GR. Both these mutants affect function of GR with R447H mutant having no transcriptional activity whereas G679S mutant displaying 55% reduced transcriptional activity as compared to the wild type GRα (Ruiz et al.,2001; Charmandari et al.,2006).

Various other mutations affecting the DBD or the LBD of GR associated with generalized glucocorticoid resistance have been reported in patients. Mutations in exons

6-9 of GR affect the LBD whereas mutations in exon 5 affect the DBD; both LBD and

DBD mutant GRα proteins exhibit reduced transcriptional activity (van Rossum et al.,2006). The molecular mechanism underlying glucocorticoid resistance has been revealed in some patients, however, a large number of patients suffer from unexplained glucocorticoid resistance, without any mutations in the GR gene. Two-linked single nucleotide polymorphisms in codons 22 and 23 (exon 2) in the GR gene convert GAG

AGG to GAA AAG causing ER22/23EK alteration. This polymorphism is present in normal population at a frequency of 5-12% and is associated with mild loss of sensitivity to glucocorticoids resistance in healthy individuals. The relative expression of the isoforms of GR – GRα, GRβ, GRγ, GRδ is an important determinant of GR sensitivity.

As GRβ is a dominant-negative inhibitor of the active isoform GRα; increased expression of the GRβ splice variant can cause glucocorticoid resistance (familial and acquired).

GRγ was originally defined to have a reduced transcriptional activity (Rivers et al.,1999) and its expression in childhood leukemia results in glucocorticoid resistance (Beger et al.,2003; Yang et al.,2012). Expression of GR-A and GRδ are increased in myeloma and leukemia (Yang et al.,2012). Moreover, increase in expression of GR chaperones hsp90

(Bronnegard et al.,1995) or FKBP51 (Scammell et al.,2001) may be responsible for some cases of glucocorticoid resistance. Furthermore, insufficient ligand i.e. insufficient

49 glucocorticoids in the plasma due to impaired uptake, increased steroid-binding protein, overexpression of ABC transporters like multidrug resistance protein (MRP) or increased expression of glucocorticoid metabolizing enzymes such as 11β-hydroxysteroid dehydrogenase type 2 (catalyzes synthesis of inactive cortisone from cortisol) might cause glucocorticoid resistance (Schmidt et al.,2004). Besides the familial glucocorticoid resistance, acquired resistance occurs in pituitary tumors and hematological malignancies. Patients suffering from several other diseases like inflammatory bowel disease, Cushing’s disease, asthma, rheumatoid arthritis have also demonstrated glucocorticoid resistance (van Rossum et al.,2006). In hematological malignancies glucocorticoids induce apoptosis; however, chronic treatment promotes development of resistance and the cells of the lymphoid lineage fail to undergo apoptosis and cell-cycle arrest in response to glucocorticoids (Schmidt et al.,2004).

50

Chapter 2

Disruption of Estrogen Signaling Enhances Invasiveness of Breast Cancer Cells by Attenuating a Her2-independent Gene Repression Program

Mugdha Patki1, 2, Marcela d’alincourt Salazar2, 3, Robert Trumbly2 and Manohar Ratnam1

1Department of Oncology, Barbara Ann Karmanos Cancer Institute, 4100 John R., Detroit, MI 48201. 2Department of Biochemistry and Cancer Biology, University Medical Center, Toledo, OH 43614. 3Division of Translational Research, Beckman Research Institute, City of Hope, Duarte, CA 91010.

To whom correspondence should be addressed: Manohar Ratnam, Ph.D., Barbara Ann Karmanos Cancer Institute, 4100 John R., Detroit, MI, USA 48201-2013, Tel.: 313-576- 8612, Fax: 313-579-8928 Email: [email protected]

Keywords: Estrogen receptor; Tamoxifen; Anti-estrogens; Gene repression; Cell invasion; Breast cancer

This work was supported by NIH grant 5R01CA140690 and The Harold & Helen McMaster endowment to M.R.

M.P. and M.D.S. are equal contributors

51 2.1 Abstract

In breast cancer, anti-estrogens inhibit proliferation by antagonizing activation of growth supporting genes by estrogen (E2). However, the overall physiological and therapeutic consequence of gene repression by E2 and its antagonism by anti-estrogens is unclear. In ER+ breast cancer cells, genes directly and indirectly repressed by E2, but not

E2-activated genes, overlapped the gene overexpression signature of clinical progression of ductal carcinoma in situ to invasive ductal carcinoma and showed a strong collective functional bias toward tumor progression. In MCF-7 (ER+/PR+/Her2-), ZR-75-1

(ER+/PR+/Her2-) and BT474 (ER+/PR+/Her2+) cells hormone depletion or tamoxifen treatment restored gene expression and invasiveness that were inhibited by E2. Gene repression by E2 was causally linked to inhibition of invasiveness as a co-repressor binding site mutation in ER that selectively disrupted the ability of E2 to repress genes also prevented inhibition of invasiveness. Although E2 inhibits expression of Her2, and

Her2 is known to support tumor progression, neither the general gene repression program of E2 nor the effect of E2 on invasiveness was affected by depletion of Her2. Gene repression by E2 involved ER binding at non-classical chromatin sites that was prevented by tamoxifen. Therefore, E2 depletion and tamoxifen treatment are permissive to cell invasion due to loss of a clinically relevant and Her2-independent gene repression program of E2. The findings suggest that selective antagonism of gene activation vs. gene repression by E2 may offer better treatment outcomes in breast cancer.

52 2.2 Introduction

Estrogen receptor type α (ER)-positive breast cancer accounts for up to 75% of all breast cancer cases (Dunnwald et al.,2007). Estrogen, predominantly in the form of estradiol

(E2), binds to ER to drive tumor cell growth through activation of genes that support mitosis and cell cycle progression (Feigelson et al.,1996). Tamoxifen and raloxifene inhibit breast tumor growth primarily by antagonizing E2-induced gene activation whereas aromatase inhibitors, given post-menopause, act by blocking peripheral and intratumoral E2 synthesis (Wong et al.,2004). Tamoxifen is also used as a chemopreventive agent in women at risk for breast cancer (Hershman et al.,2002). In

ER+ breast cancer, a 5-year adjuvant treatment with tamoxifen given to inhibit residual tumor growth decreases recurrence by about 50 percent ("Tamoxifen for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists' Collaborative

Group",1998). Breast tumors however can become growth adapted to attenuated E2 signaling through dysregulated alternative signaling pathways (Ring et al.,2004).

Intrinsic or acquired resistance derives from subpopulations of cells within the tumor with an aggressive growth phenotype and migratory capacity (Husemann et al.,2008) ultimately leading to tumor recurrence, drug resistance and metastasis (Tredan et al.,2007). In up to 90 percent of the cases however, when the primary tumor is ER+, lymph node and distant metastases retain ER expression even though their growth may have escaped hormonal control (Gomez-Fernandez et al.,2008).

Studies of the actions of E2 in breast cancer have overwhelmingly focused on the mechanisms by which E2 promotes and supports breast cancer cell proliferation (Clemons

53 et al.,2001). Nevertheless, E2 also plays physiological roles in normal and malignant breast epithelial cells that are unrelated to growth. In normal breast development E2 acts on a subset of ER+ ductal cells to induce ductal morphogenesis and differentiation

(Shyamala,1997; LaMarca et al.,2007). In ER-expressing breast cancer cells, E2 also inhibits invasiveness and metastasis, in vitro and in vivo (Garcia et al.,1992; Long et al.,1996; Garcia et al.,1997). Indeed, recent studies have revealed that, in postmenopausal women with prior hysterectomy, hormone replacement with estrogen as a monotherapy is actually associated with a persistent decrease in the onset of invasive breast cancer (LaCroix et al.,2011). Understanding mechanisms underlying the effects of

E2 and its anti-estrogens on breast tumor cell invasiveness is potentially highly significant in understanding tumor progression within subpopulations of cells that survive anti- estrogens. This is a particularly significant issue because breast tumor cells could migrate at a relatively early stage from the primary tumor to distal sites (Husemann et al.,2008).

At the level of gene regulation, E2 represses as many or more of its target genes than it activates, in a manner that is antagonized by tamoxifen or raloxifene (Frasor et al.,2003).

Except for ERBB2, much of the focus in detailed studies of gene repression by E2 has been on selected individual genes encoding negative regulators of the cell cycle and tumor suppressors (Varshochi et al.,2005; Stossi et al.,2006; Karmakar et al.,2009). The choice of genes for those studies is apparently based on the a priori assumption that gene repression by E2 must also serve to support cell growth. However, the reported studies are limited in scope, lacking a broader picture of the physiological and therapeutic relevance of gene repression by E2. The ERBB2 gene is pro-invasion and pro-metastatic (Tan et

54 al.,1997; Palmieri et al.,2007) and is known to be repressed by E2 in ER+ cell lines

(Hurtado et al.,2008). It has therefore been suggested that down-regulation of Her2 by E2 might explain the relatively low invasiveness of luminal breast tumors (Beauchemin et al.,2011). However, this possibility has not been proven. Moreover, about 15 % of invasive and ER+ breast tumors also overexpress Her2 (Clarke et al.,2012) which may confer estrogen-independent growth; in those cases, the role of estrogen signaling in tumor biology is unclear.

For the foregoing reasons, it was undertaken to more systematically examine gene repression by E2 and its antagonism by anti-estrogens with respect to breast tumor related functions of the target genes. We chose as models, breast cancer cells representing different clinical subtypes of ER+ tumors including ER+/PR+/Her2- (MCF-7 cells),

ER+/PR+/Her2- (ZR-75-1 cells) and ER+/PR+/Her2+ (BT474 cells); the BT474 cells are capable of robust growth without dependence on E2. The studies are meant to enable a better understanding of the long-term clinical consequences of loss of gene repression by

E2 during anti-estrogen therapies and chemoprevention or restoration of gene repression during estrogen replacement therapy.

2.3 Materials and Methods

Chemicals and reagents. Dulbecco’s minimum essential medium (DMEM), glutamine and penicillin/streptomycin/glutamine stock mix were purchased from Life

Technologies Corporation (Carlsbad, CA). Fetal bovine serum (FBS) and charcoal- stripped FBS were from Life Technologies Corporation (Carlsbad, CA). Reagents for real

55 time PCR, primers and TaqMan probes for human ABCG2, ANXA1, BAG1, CCL5,

CD24, CD55, CEACAM6, CITED2, CTGF, CTSF, CTSH, CTSK, CTSS, CXCL2,

DDR1, EGR3, ERBB2, ERBB3, ERBB4, ESR1, FSCN1, FZD7, GREB1, ICAM1, ID1,

ID3, IGFBP3, IL-6, IL-8, KLF5, KRT7, LIMK2, LYN, MAPK10, MMP14, MSX2,

PGR, PKCD, PKCZ, PSAP, pS2, RND3, SDC4, S100P, TACSTD2, TM4SF1,

TSPAN31, USP25 and GAPDH were purchased from the Life Technologies Corporation inventory (Carlsbad, CA). Primers and probes used to amplify the ABCG2, CEACAM6,

GREB1 and PS2 promoter regions were purchased as a set from Integrated DNA

Technologies (IDT, Coralville, IA). 17β-estradiol (E2) and 4-hydroxytamoxifen (4-OHT) were purchased from Sigma Aldrich (Saint Louis, MO). Phenol-red free growth factor reduced Matrigel (356231) and Calcein AM Fluorescent Dye (354216) were purchased from BD Biosciences (San Jose, CA).

Antibodies and siRNA. Affinity purified anti-human ER (sc-543), anti-human RIP140

(sc-8997) rabbit polyclonal antibodies and anti-human Neu (sc-33684) and glyceraldehyde-3-phosphate dehydrogenase mouse monoclonal antibodies (GAPDH; sc-

44724) were from Santa Cruz Biotechnologies (Santa Cruz, CA). Anti-human NCoR

(ab24552) and PAX2 (ab23799) rabbit polyclonal antibodies were from Abcam

(Cambridge, MA). Affinity purified mouse anti-human monoclonal SMRT (611386) was purchased from BD Biosciences (San Jose, CA). The small interfering RNAs (siRNA) for

ERBB2 (#J-003126-20; GCUCAUCGCUCACAACCAA), ESR1 (#J-003401-12;

GAAUGUGCCUGGCUAGAGA), NCoRI (AAGAAGGAUCCAGCAUUCGGAUU),

PAX2 (GAAGUCAAGUCGAGUCUAUUUUU), RIP140

56 (GGAGGAAGCUUUGCUAGCUUU), SMRT (#J-020145-10;

CAGCCAGGGAAGACGCAAA) and Non-targeting siRNA (#D-001810-02) were from

Thermo Fisher Scientific Dharmacon Research, Inc (Waltham, MA).

Cell culture and transfection. MCF-7, T47D, ZR-75-1, BT474 breast cancer cells

(American Type Culture Collection) were cultured in DMEM supplemented with FBS

(10%), penicillin (100unit/ml), streptomycin (100μg/ml) and L-glutamine (2mM). Breast cancer cells were cultured in phenol-red free DMEM medium supplemented with 10%

○ charcoal-stripped FBS (v/v) and incubated at 37 C with 5% CO2 for 48 h to 72 h to achieve complete hormone depletion prior to treatments.

Cells were plated to 20% confluence in phenol-red free DMEM medium supplemented with 10% charcoal-stripped FBS and transfected with the appropriate siRNA using Dharmafect 1(Thermo Fisher Scientific Dharmacon Research, Inc.,

Waltham, MA) according to the vendor’s protocol.

Mutagenesis and lentiviral transduction. The ER-L372R mutant was generated by creating a point mutation in ERα cDNA at nucleotide position 1116 by converting the codon CTC to CGC using the QuickChange II Site-Directed Mutagenesis kit (Agilent

Technologies, Santa Clara, CA). The wild type ER cDNA and ER- L372R mutant cDNA were inserted in the pCDH lentiviral vector at the SalI site in the polylinker.

293FT cells were used to generate the lentiviral particles by transfection using

Lipofectamine 2000 (Life Technologies Corporation, Carlsbad, CA). Packaging plasmids pMD2G, pMDLg/RRE and pRSV/Rev were cotransfected along with the appropriate

57 pCDH plasmid. The virus containing supernatant was harvested 48 h and 72 h after transfection, filtered and stored at -80°C until the time of transduction. 48 h before transduction, BT474 cells were plated in phenol-red free DMEM medium supplemented with 10% heat inactivated charcoal-stripped FBS at 25% confluence. Cells were transduced with either pCDH-ER lentivirus or pCDH-ER-L372R lentivirus with polybrene (8μg/mL) for 5 h followed by a similar second lentiviral transduction for an additional 5 h. After the second transduction, the virus was replaced with fresh phenol red free medium containing 10% charcoal stripped FBS.

Chromatin immunoprecipitation. MCF-7 cells (4x106 cells/10cm plate) were plated in hormone-depleted media for 48 h and treated with vehicle (ethanol), E2 (100 nM) or E2

(100 nM) plus 4-OHT (10 M) for 45 min and then subjected to ChIP using anti-ER antibody (sc-543x) or rabbit anti-IgG control. The ChIP assay was performed using the

EZ ChIP chromatin immunoprecipitation kit (17-371) per the vendor’s protocol

(Millipore, Temecula, CA) and ChIP signals were measured by real-time PCR analysis of chromatin-immunoprecipitated products. Each sample was tested in triplicate. The optimal target sequence chosen to amplify the promoter regions were as follows:

ABCG2:

Forward primer-

5’-TACGAGAATCACCAGGCGCTCATT-3’

Reverse primer-

5’-TCCCATTCACCAGAAACCACCCAT-3’

Probe- 5’-/56-FAM/TACCTCGTCTGACCTAGCTGGGTTT/36-TAMSp/-3’

58 CEACAM6:

Forward primer-

5’-ACACACCCAGAGTATGGCCTTTGA-3’

Reverse primer-

5’-TGACGACTCAGTGCTATGTGCTCT-3’

Probe- 5’-/56-FAM/TTTCCATGCCTCGTGGCCTCTATCTT/36-TAMSp/-3’

GREB1:

Forward primer-

5’-AGGAGCCCTTCATCAGTCAACACT-3’

Reverse primer-

5’-TTTCATGAACCTCCCTCGCTCCAA-3’

Probe- 5’-/56-FAM/CCGGGAAACAGATGGGAAAGACAACT/36-TAMSp/-3’

PS2:

Forward primer-

5’-CGTGAAAGACAGAATTGTGGTTTT-3’

Reverse primer-

5’- CGTCGAAACAGCAGCCCTTA-3’

Probe- -5’/56-FAM/TGTCACGCCCTCCCAGTGTGCA/36-TAMSp/-3’

Transwell invasion assay. MCF-7, ZR-75-1 and BT474 cells were seeded in 6-well plates in phenol-red free DMEM medium supplemented with 10% charcoal stripped FBS and treated with vehicle (ethanol), E2 (1nM) or 4-OHT (500 nM) for 72 h. In experiments involving tranfection of siRNA or lentiviral transduction, the ligand treatments were

59 initiated 24 h and 72 h after transfection and transduction respectively. 1x105 cells were re-suspended in serum free/phenol-red free DMEM medium and added in the top chamber of fluoroblok cell culture inserts (#351152; 8µM pore membrane; BD

Biosciences, Bedford, MA) coated with matrigel (0.2 mg/mL). 20% FBS in phenol-red free DMEM media was used as the chemoattractant. The appropriate treatments were included in both the top and the bottom chambers. Cells were allowed to invade for 24 h.

Invaded cells on the bottom surface of the insert were labeled with Calcein AM (2μg/ml -

4µg/ml) for 1 h at 37°C in the dark and imaged using a fluorescent microscope. The invasion assay for each treatment was performed in triplicate wells. Images were captured from each well from five fields of view using a 4x objective. The images were processed using the Image J software (Schneider et al.,2012) and the number of cells invaded was quantified on the basis of brightness and pixel size.

Western blot analysis. Cell lysates were generated using RIPA buffer (150mM NaCl,

1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris of pH 8.0) containing protease inhibitor cocktail (Pierce Biotechnology, Thermo Fisher Scientific, Rockford, IL

) and incubated on ice for 1 h. Total protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA). The blots were performed as previously described (Salazar et al.,2011). Protein samples (10-20μg) were resolved by electrophoresis on 6% (NCoRI and SMRT) or 8% (ER, Her2, PAX2 and RIP140) SDS- polyacrylamide gels and electrophoretically transferred to PVDF membranes (Millipore

Corporation, Bedford, MA) for 1 h at 100V (ER, Her2, PAX2 and RIP140) or overnight at 40V (NCoRI and SMRT). The blots were probed with the appropriate primary

60 antibody and the appropriate horse radish peroxidase conjugated secondary antibody. The protein bands were visualized using enhanced chemiluminescence reagents (Hyglo Quick

Spray, Denville Scientific) following the vendor’s instructions.

RNA isolation, reverse transcription PCR and Real time PCR. Total RNA from

MCF-7, T47D, ZR-75-1 and BT474 breast cancer cells were isolated using the RNeasy mini kit (Qiagen, Maryland, MD). Reverse transcription PCR reactions were performed using 500ng of total RNA and the high capacity complementary DNA Archive kit (Life

Technologies Corporation, Carlsbad, CA) according to the vendor’s protocol. cDNA was measured by quantitative real time PCR using the StepOne Plus Real time PCR System

(Life Technologies Corporation, Carlsbad, CA). All samples were measured in triplicate and normalized to GAPDH Ct values.

mRNA profiling. The Affymetrix DNA microarray analysis was performed as a full service global gene expression study at the transcriptional profiling core facility of the

Cancer Institute of New Jersey. Total RNA samples were used to generate labeled cRNAs, which were hybridized to human U133 Plus2.0 Affymetrix microarrays. Scanned image files were analyzed using the Gene Chip Operating System version 1.4 software, and standard thresholding and filtering operations were used. The data were normalized using housekeeping genes. Normalization assumes that for a subset of genes (i.e. housekeeping genes), the ratio of measured expression averaged over the set should be one.

Differentially expressed genes were identified by comparing E2 treatment with vehicle treatment or E2 plus 4-OHT treatment using the indicated fold-cutoff values. The adjusted

61 p values for the comparisons, calculated using the Bioconductor limma program were <

0.05.

Statistical analyses. Experimental values are presented as mean ± s.d. The statistical significance of differences (P value) between values being compared was determined using ANOVA. The P values are noted in the figures.

2.4 Results

The estrogen repressed gene set is enriched for genes overexpressed in clinical progression of ductal carcinoma in situ to invasive ductal carcinoma. In MCF-7 cells, a total of 2,373 unique annotated genes in the Affymetrix platform were repressed by E2 by at least 30 percent and 667 genes were repressed by at least 50 percent (Supplemental

Table 1). Whereas, hormone depletion by definition completely prevented gene repression by E2, tamoxifen antagonized gene repression by E2 by at least 50 percent in the vast majority (>80 percent) of its target genes (Supplemental Table 2). Of a total of

557 unique annotated genes activated by E2 by at least 2-fold (Supplemental Table 3), tamoxifen antagonized gene activation by E2 by at least 50 percent in 302 of the genes

(Supplemental Table 4). Ontology analysis using DAVID bioinformatics Resources 2008

(Dennis et al.,2003; Huang da et al.,2009) showed that the E2 activated genes predominantly supported cell cycle and mitosis, with an enrichment score of 46.60 and a

Benjamini score of 4.5E-49. The tamoxifen-sensitive E2 activated genes also predominantly supported cell cycle and mitosis with an enrichment score of 55.28 and a

62 Benjamini score of 3.0E-63. In contrast, the gene set that was repressed by E2 failed to show significant enrichment in favor of genes known to negatively impact cell growth.

The data is consistent with previous findings that E2-dependent breast cancer cell growth and its antagonism by tamoxifen or hormone depletion occur principally through the modulation of E2-activated genes.

In an elegant study, Schuetz et al. (Schuetz et al.,2006) have established the gene overexpression profile associated with progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) using laser dissected epithelial cells obtained from matched DCIS/IDC samples from 9 patients. Comparison of the E2 repressed genes identified in MCF-7 cells in our study with the top 10 percent of the invasiveness associated genes from Schuetz et al. (Schuetz et al.,2006) showed a highly significant

-3 overlap with a P value of 8.7 x 10 for genes repressed by E2 by at least 30 percent

-8 (Figure 2-1A) and the much lower P value of 4.7 x 10 for genes repressed by E2 by at least 50 percent (Figure 2-1B). In contrast, there was not a significant overlap with genes activated by E2 (P = 0.9). Thus, in MCF-7 cells, E2 represses a significant number of genes associated with clinical progression of DCIS to an invasive phenotype.

Estrogen repressed genes are enriched for functions known to collectively support breast cancer progression. In order to obtain a clearer understanding of the potential functional impact of E2 repressed genes in breast cancer, all of the genes currently known to be associated with breast cancer were first comprehensively identified from the larger pool of E2 repressed genes. Complementary approaches were used to identify the breast

63 cancer associated genes. In one approach, the genes occurring in a previously published list of 1,000 breast cancer genes (the BC1000 list) (Witt et al.,2006) were identified. In a second approach, MILANO (Microarray Literature-based Annotation) (Rubinstein et al.,2005) was used to thoroughly scan the literature database (GeneRIF and Medline through PubMed) to identify relevant research articles in which individual genes repressed by E2 on the microarray list were associated with breast cancer. The program was used to generate an annotation table containing the number of publications in which the term ‘breast cancer’ as well as the name of each gene on the microarray list occurs in the text with a cut off at 4 publications per gene. A list of 470 genes was obtained in this manner, representing > 20 percent of the E2 repressed genes. The literature was then manually scanned to obtain updated information on the specific functions of each gene in breast cancer; in this manner, 199 genes (Table 2.1) were found to have well established functions related to breast cancer as indicated in Figure 2-2. As some of the 199 genes are known to belong to more than one functional category indicated in Figure 2-2, the numbers of genes placed in the individual groups in Figure 2-2 add up to 232.

As seen in Figure 2-2, among genes functionally associated with breast tumor invasion and metastasis, the E2 repressed genes overwhelmingly supported this phenotype. Among genes influencing breast tumor cell survival and growth, a greater number of the E2 repressed genes supported the phenotype. Additionally, E2 repressed genes included those known to support drug resistance, angiogenesis and immune evasion in breast tumors and none that opposed these phenotypes. The results demonstrate a strong functional bias of

E2 repressed genes towards the general phenotype associated with breast cancer

64 progression. The repression of these genes is fully prevented by hormone depletion and either partially or fully prevented by tamoxifen.

Repression of breast tumor progression genes occurs in a variety of hormone- sensitive and tamoxifen resistant models and is antagonized by tamoxifen. Among the breast tumor progression genes in Figure 2-2, 44 genes were selected for further quantitative studies, based on the ready commercial availability of optimized TaqMan probes for quantitative RT-PCR analysis. The breast cancer cell lines used represented different subtypes of ER+ tumors: MCF-7 cells (ER+/PR+/HER2-), ZR75-1 cells

(ER+/PR+/HER2-) and BT474 cells (ER+/PR+/HER2+) (Figure 2-3A). Tamoxifen, at a pharmacological concentration (0.5 µM), antagonized the action of E2 in virtually all of these genes in the MCF-7 cells (Figure 2-3B). The results obtained with MCF-7 cells extended to ZR75-1 cells (Figure 2-3C).

The rescue of expression of tumor progression genes in breast cancer cells by anti- estrogens could potentially support tumor progression in sub-populations of breast tumor cells that are intrinsically refractory to growth inhibition by the anti-estrogens. It was therefore of interest to test whether these genes continued to be under E2 regulation in

HER2/ErbB2 amplified ER+ cells (BT474 cells). Most members of the set of 44 tumor progression genes tested were also repressed by E2 in BT474 cells (Figure 2-3D) in which growth is not inhibited by tamoxifen. Thus E2 retains the ability to repress breast tumor progression genes even in hormone-independent ER+ cells.

65 Repression of tumor progression genes by estrogen occurs by both direct and indirect mechanisms. E2 target genes that required de novo protein synthesis for repression by E2 were identified using cycloheximide (Chx) to block protein synthesis during 24 hours of treatment with E2. Affymetrix DNA microarray analysis of the E2 repressed genes in MCF-7 cells showed that the repression was prevented by Chx treatment in 73 percent of the genes (2,421 of 3,069 probe sets). The data was validated by real time RT-PCR analysis for a representative subset of tumor progression genes from Figure 2-3 that showed a substantial decrease in mRNA levels within the 24 hour duration of the experiment (Figure 2-4A); Chx treatment prevented repression of many of the genes (Figure 2-4B). Therefore, among the genes functionally associated with an aggressive phenotype in breast cancer, a little over a fourth of the genes are direct target genes of E2 repression.

Several estrogen receptor co-repressors support repression of breast tumor progression genes by estrogen. The co-repressor dependence of gene repression by E2 was tested for breast tumor progression genes by individually knocking down NCoRI,

PAX2, RIP140 or SMRT (Figure 2-5A). The co-repressor knockdown partially (> 30 percent) decreased repression by E2 in 30 of the 44 genes tested in the order of frequency:

NCoRI>PAX2>RIP140>SMRT with NCoRI and PAX2 supporting E2 repression in approximately 57 percent and 27 percent of the genes, respectively (Figure 2-5B). The partial and overlapping effects of co-repressor depletion also indicate functional redundancy of the co-repressors.

66 Inhibition of invasiveness by estrogen is tamoxifen-sensitive and estrogen dose dependent. MCF-7 cells are weakly invasive in matrigel, whereas ZR-75-1 and BT474 cells are relatively strongly invasive. In all three cell lines, E2 inhibited invasiveness and tamoxifen blocked the effect of E2 (Figure 2-6 A-C). To relate the effect of E2 on invasiveness to physiological E2 levels, the E2 dose response for inhibition of invasiveness was determined. E2 was able to inhibit invasiveness of BT474 cells in the subnanomolar range with virtually complete inhibition observed at 1 nM (Figure 2-6D).

Repression of breast tumor progression genes and suppression of invasiveness by estrogen are both independent of repression of the ERBB2 gene. As increased expression of HER2/ErbB2 is a causal factor in the anti-estrogen resistant growth as well as progression of breast cancer and as HER2/ErbB2 is a target gene for repression by E2, it was of interest to test whether the repression of other tumor progression genes by E2 was mediated by the repression of the ERBB2 gene. Knocking down the basal expression of HER2/ErbB2 in MCF-7 cells (Figure 2-7A, inset) did not affect the repression of the other genes by E2 (Figure 2-7A) demonstrating that the various tumor progression genes tested and HER2/ErbB2 are independent targets of repression by E2. Likewise, knocking down HER2/ErbB2 in BT474 cells (Figure 2-7B, inset) did not impact the repression of the breast tumor progression genes by E2 (Figure 2-7B) demonstrating that the various tumor progression genes tested and HER2/ErbB2 are independent targets of repression by

E2.

67 Knocking down HER2/ErbB2 also failed to show any effect on the ability of E2 to suppress invasiveness in BT474 cells (Figure 2-7C) indicating that amplified

HER2/ErbB2 does not play a role in the effect of E2 on invasiveness of ER+ cells.

Gene repression and inhibition of invasiveness by estrogen is dependent on ER. In

BT474 cells, depletion of ER (Figure 2-8A inset) prevented E2 mediated gene repression

(Figure 2-8A) as well as the activation of classical E2 target genes (Figure 2-8B). The ability of E2 to inhibit invasiveness was also abrogated by knocking down ER (Figure 2-

8C) confirming ER-dependence of this effect.

A co-repressor binding site mutant of ER selectively disrupts the ability of E2 to repress genes as well as its ability to inhibit invasiveness. Mutation of L372 in ER to

R372 is known to attenuate the ability of ER to interact with the CoRNR box motifs of

ER co-repressors without affecting interaction with the LXXLL motifs of ER co- activators (Huang et al.,2002). We overexpressed wt ER and the ER L372R mutant in

BT474 cells using lentiviral transduction to achieve a 6-8 fold increase in the expression of ER mRNA (Figure 2-9A, inset). The ectopic mutant ER showed the expected dominant-negative effect on gene repression by E2 (Figure 2-9B) without affecting gene activation (Figure 2-9C). The control ectopic wt ER did not affect either gene repression or gene activation by E2 (Figures 2-9B and 2-9C). The transduced mutant ER but not wt

ER abrogated inhibition of invasiveness by E2 (Figure 2-9D). The results demonstrate a causal link between gene repression by E2 and the role of the hormone in suppression of invasiveness.

68

Tamoxifen binding dissociates the ER from both promoter-proximal and -distal sites of estrogen-repressed genes. The classical mechanism by which tamoxifen antagonizes gene activation by E2 is through inducing a conformational change in ER that favors recruitment of co-repressors rather than co-activators to the receptor bound to E2 response elements (ERE). However, in the case of E2-repressed genes, studies of global genome associations of ER in MCF-7 cells (Bourdeau et al.,2008), have commonly found functional ER binding sites lacking ERE at both promoter-proximal and -distal sites of

E2-repressed genes. Consistent with these observations chromatin immunoprecipitation

(ChIP) analysis showed that E2 induced ER recruitment at putative sites of repressive ER- binding associated with the ABCG2 gene (-495 to -308nt) and CEACAM6 gene (+18999 to +19167 nt); however 4-OHT blocked ER recruitment at these sites (Figure 2-10). As controls, ChIP analysis of ER binding at the known classical EREs of genes activated by

E2, GREB1 and pS2, demonstrated that as expected, ER recruitment was not significantly affected by 4-OHT treatment (Figure 2-10).

2.5 Discussion

Although many studies have demonstrated a strong link between gene activation by E2 and breast cancer cell proliferation (Frasor et al.,2003; Frasor et al.,2004), the functional significance of gene repression by E2 on the physiology of breast cancer cells is not at once apparent from standard gene ontology analyses. In the present study, the E2 repressed gene pool (including both direct and indirect targets of E2) was examined by an initial identification of breast cancer associated genes using complementary approaches

69 followed by an updated literature survey of the known functions of the individual genes in this subset. This method of analysis revealed that gene repression by E2 could mitigate an aggressive phenotype in breast cancer cells by directly or indirectly inhibiting the expression of many genes known to support breast tumor progression, compared to a relatively small number known to oppose this phenotype. The functions of most of the E2 repressed genes associated with breast cancer included invasiveness and metastasis, drug resistance, angiogenesis and immune evasion, all characteristics associated with progression of breast cancer. The clinical relevance of E2 repressed genes to breast tumor progression is also clear from the strong overlap between this set of genes and those found to be consistently elevated in the clinical progression of DCIS to IDC. Among E2- repressed genes, although a greater number encoded positive rather than negative regulators of cell growth and survival, there was not a strong functional bias towards either group of genes. It appears rather that the influence of E2 on cell proliferation is overwhelmingly mediated by target genes that are activated by the hormone. In contrast to E2-repressed genes, there was not a significant overlap of E2-activated genes with those consistently elevated in the progression of DCIS to IDC. The present study supports a clear functional distinction between genes activated by E2 and those repressed by it; the former set of genes is related to growth whereas the latter is related to important aspects of tumor progression.

A causative link between gene repression by E2 and the inhibition of invasiveness by

E2 was established by taking advantage of the co-repressor dependence of ER for repression of tumor progression genes. A mutation in the co-repressor binding site of ER

70 abrogated both gene repression and inhibition of invasiveness by E2 but not E2-dependent gene activation. This result indicates that the ability of E2 to repress genes is critical for suppression of invasiveness by the hormone.

E2 is known to repress the ERRB2 gene in breast cancer cells (Hurtado et al.,2008); this was a significant issue in the present study as overexpression of Her2 is known not only to enable hormone-independent growth in breast cancer but also to support progression of several types of tumors (Tan et al.,1997; Palmieri et al.,2007). Using both

Her2-amplified and -unamplified cell line models of ER+ breast cancer, it was also demonstrated that neither the expression level nor regulation of Her2 by E2 was linked to the general gene repression program of E2 or the ability of E2 to inhibit invasiveness.

As anti-estrogens generally antagonize both gene activation and gene repression by

E2, the phenotypic impact of antagonizing gene repression by E2 during anti-estrogen therapy may be manifest in only the tumor cells that are able to survive or resist the growth inhibitory effects of the anti-estrogens. The ability of E2 to suppress the invasive potential of ER+ cells that are hormone-refractory was evident from the response of

BT474 cells to E2; the hormone-independent growth acquired by these cells through overexpression of Her2 represents a clinically observed event in breast tumor progression. Therefore in ER+ tumor cells harboring Her2 amplification or other mechanisms that confer hormone-independence, it is likely that the effect of anti- estrogens is to support tumor progression by attenuating estrogen signaling.

71 The E2 dose required for substantial or optimal suppression of invasiveness in the hormone-independent HER2 overexpressing ER+ cells (0.2 nM – 1nM) corresponds to the literature consensus (Geisler,2003) for circulating and tumor levels of E2 in pre- menopausal women and tumor levels of E2 in post-menopausal women. Whereas pharmacological concentrations of tamoxifen (0.1 – 0.5 µM) should optimally antagonize the effects of E2 on tumor invasiveness, aromatase inhibitors may be expected to have a similar effect by decreasing tumor E2 levels well below its physiological range (Geisler et al.,2008).

We have previously demonstrated a mechanistically distinct aspect of gene repression by E2 wherein, the hormone caused ER and co-repressor recruitment to the target gene and tamoxifen dissociated the proteins from the gene; moreover, placing a classical estrogen response element (ERE) upstream of the promoter caused a switch to gene activation by E2 (Hao et al.,2007). The studies suggested fundamental mechanistic differences between gene repression and the classical model of gene activation, both with respect to E2 action and the manner in which tamoxifen antagonized it. This view is also supported by the observation from global chromatin binding studies that, in contrast to E2 activated genes, E2 repressed genes are not enriched for associated EREs (Bourdeau et al.,2008). A similar non-classical mechanism at least partly underlies the repression of tumor progression genes by estrogen as demonstrated in this study at both promoter proximal and distal sites of repressive binding of ER. Mechanistic differences between gene activation and gene repression by estrogen could potentially be exploited to develop agents that would selectively antagonize gene activation or gene repression by E2.

72 In conclusion, this study addresses the need to better understand molecular mechanisms involved in the frequent transition from a hormone-responsive phenotype towards a more aggressive hormone-refractory phenotype in the course of anti-estrogen therapy of ER+ breast cancer. The role of E2 in repressing genes associated with breast tumor progression offers a mechanistic basis for the emergence of invasive breast tumors during long-term treatment with conventional anti-estrogen drugs. Whereas the antiproliferative effects of the anti-estrogen treatments are overwhelmingly mediated by blocking gene activation by E2, the consequence of antagonizing gene repression by E2 by the same treatments would predictably be detrimental by encouraging a more invasive phenotype in tumor cells that have acquired hormone-independence for growth. By this reasoning, a superior anti-estrogen for ER+ breast cancer would antagonize activation of critical growth genes by E2 without affecting its ability to repress critical genes involved in tumor progression.

73 Figure 2-1

A

E2 repressed Top 10 percent up- (≥30 percent) 2020 261 1646 regulated genes in genes IDC vs DCIS

P=8.7x10-3

B

E2 repressed 434 92 1815 Top 10 percent up- (≥50 percent) regulated genes in genes IDC vs DCIS

P=4.7x10-8

Figure 2-1. Comparison of E2 repressed genes in MCF-7 cells with genes upregulated in clinical progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Affymetrix DNA microarray analysis was used to determine genes repressed by E2 (1 nM) in MCF-7 cells at the end of 48 h of treatment. The gene sets that were repressed by ≥ 30 percent (Panel A) or ≥ 50 percent (Panel B) were compared for overlap with the top 10 percent genes upregulated in association with progression of DCIS to IDC in 9 paired clinical tumor specimens as reported by Schuetz et al. 2006 (Schuetz et al.,2006). P values for the overlap were calculated using Fisher’s exact test.

74

Figure 2-2

Functional categories Invasion/metastasis* Inhibition of invasion and metastasis

Support cell/tumor growth/survival Negative regulation of cell growth or survival

Drug resistance Associated with: Good prognosis Angiogenesis Poor prognosis Immune evasion 0 10 20 30 40 50 60 70 80 Number of genes * Includes migration, EMT, adhesion and ECM degradation

Figure 2-2. Gene ontology of E2 repressed genes in relation to progression of breast cancer. Affymetrix DNA microarray analysis was used to determine genes repressed by

E2 (1 nM) in MCF-7 cells at the end of 48 h of treatment. Breast cancer associated genes were identified within this group of genes by comparison with the BC1000 list (Witt et al.,2006) and also by scanning the literature database (GeneRIF and Medline through PubMed) using the MILANO (Microarray Literature-Based Annotation) (Rubinstein et al.,2005) to identify publications in which the text contained the term “breast cancer” and the name of each gene; the genes were selected on the basis of a cut off at 4 publications for each gene. The literature for each selected gene was then scanned to assign one or more functions. The figure indicates the numbers of genes assigned to functional categories covering all aspects of breast cancer progression.

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Figure 2-3

76

Figure 2-3. Gene repression by E2 and antagonism by 4-OHT in ER+ breast cancer cell lines. Whole cell lysates from MCF-7 cells, ZR-75-1 cells and BT-474 cells were extracted using RIPA buffer. 10μg of protein was loaded on SDS-PAGE gels and transferred to a PVDF membrane. Blots were probed for ER, HER2/Neu and GAPDH (loading control) (Panel A). MCF-7 cells (Panel B), ZR-75-1 cells (Panel C) and BT-474 cells (Panel D) were plated at 20% confluence in media containing charcoal-stripped FBS for hormone depletion for 48 h – 72 h. Cells were treated with either vehicle (ethanol), E2 (1 nM) or E2 (1 nM) plus 4-OHT (500 nM) for 48 h. Cells were harvested to measure mRNA expression by real time RT-PCR for the indicated genes. All Ct values were normalized to the Ct values of GAPDH.

77

Figure 2-4

A

) 150 Vehicle ol MCF-7 cells E2 * P < 0.05 on tr ec

cl 100

vehi * * *

of * * ** * * * nt * * * * * * * ce 50 * *

er *

(P *

el * * ev l 0 5 3 1 5 R P3 G1 RNA F2 MP m PK10 CD5 PSAP CTSS CTSF KLF5 CCL BA KRT7 RND3 CTSH CTGF PKCZ PKCD TI ERBB2 ERBB4 ANXA1 NDRG IGFB MA TSPAN31 CEACAM6 B

l) 300

ro * P < 0.05 Vehicle + Chx cont E2 + Chx icle 200 eh tofv en

rc 100

Pe * * * * * * l( * ** ve le 0 5 1 3 5 Z R P3 G1 RNA F2 MP m PK10 CD5 PSAP CTSS CTSF KLF5 CCL BA KRT7 RND3 CTSH CTGF TI ERBB2 ERBB4 ANXA1 NDRG PRKC PRKCD IGFB MA TSPAN31 CEACAM6

E2 repression BLOCKED by Chx E2 repression NOT blocked by Chx

Figure 2-4. Effect of cycloheximide on gene repression by E2 in MCF-7 cells. Hormone depleted MCF-7 cells were treated with either vehicle (ethanol) or E2 (1 nM) for 24 h. In each case, the cells were pre-treated with either cycloheximide (Chx) (10 μM) or vehicle for 2 h followed by the continued presence of Chx or vehicle for the next 24 h. Total RNA was then purified. Affymetrix DNA microarray analysis was used to identify genes repressed by E2 in the absence or in the presence of Chx. The Figure shows validation of the results for representative genes by real time RT-PCR. Panel A shows repression of all of the genes by E2 in the absence of Chx; Panel B shows the effect of E2 on the expression of the same genes in the presence of Chx.

78

Figure 2-5

A B Ctrl Co-repressor siRNA siRNA nd nd ga ga li li (1nM) (1nM) NCoRI PAX2 2 2

No E No E 3 NCoRI 13 8 PAX2 1 3 RIP140 RIP140 SMRT GAPDH SMRT 2

Figure 2-5. Co-repressor dependence for gene repression by E2. MCF-7 cells were plated in charcoal-stripped media for hormone depletion. Cells were transfected with control siRNA or siRNA against each of the co-repressors using Dharmafect 1. 24 h after transfection, cells were treated with vehicle (ethanol) or E2 (1 nM) for 48 h. Whole cell lysates were extracted to measure knockdown of each of the co-repressors by western blot analysis, using GAPDH as the loading control (Panel A).Cells were harvested for RNA extraction and mRNA levels of the genes indicated in figure 3B were measured using real time RT-PCR (Panel B). Panel B only includes the tested genes for which repression by E2 was significantly reduced (P < 0.05 from at least 3 separate experiments) by knocking down the indicated co-repressor.

79

Figure 2-6

A B ZR-75-1 cells MCF-7 cells * P < 0.0001 (Veh vs.E2) * P = 0.007 (Veh vs.E2) ** P = 0.0018 (E2 vs. E+4-OHT) ** P = 0.062 (E2 vs. E+4-OHT) *** P = 0.0034 (E2 vs. 4-OHT) *** P = 0.029 (E2 vs. 4-OHT) 1500 80 ed

ed *** 60 1000 ** invad

invad ** *** ls cel

cel ls 40 of

of * er 500 er * mb

mb 20 Nu Nu 0 0 e T T e T T trol cl E2 H cl E2 H hi O OH trol hi on - on -O OH Ve 4 4- Ve 4 4- gc E+ gc E+ Ne Ne C D BT474 cells

* P = 0.001 (Veh vs.E2) ** P = 0.0004 (E2 vs. E+4-OHT) BT474 cells *** P < 0.0001 (E vs. 4-OHT) 2 150 400

*** sion ed m) va

300 in ** mu 100 ll xi invad ce ls ma cel

200 of of nt

induced 50 er * ce er m-

mb 100 (p ru Nu Se 0 0 0.001 0.01 0.1110 e T T cl E2 H trol hi on -O OH E2 (nM) Ve 4 4- gc E+ Ne

80

Figure 2-6. Effect of E2 and 4-OHT on invasiveness of ER+ breast cancer cell lines. MCF-7 cells (Panel A), ZR-75-1 cells (Panel B) and BT-474 cells (Panel C) were plated in media containing charcoal-stripped FBS for hormone depletion. 48 h later, cells were treated with either vehicle (ethanol), E2 (1 nM), 4-OHT (500 nM) or E2 (1 nM) plus 4- OHT (500 nM) for72 h. Cells were trypsinized and 1x105 cells were plated on matrigel coated fluroblok inserts for the invasion assay with the appropriate treatments (vehicle,

E2, 4-OHT or E2 plus 4-OHT) included in both the top and the bottom chambers. 20% FBS was used as chemoattractant. Cells were allowed to invade for 24 h and stained with calcein AM for visualization. In Panel D, BT474 cells were plated in media containing charcoal stripped serum for 48 h and treated with vehicle (ethanol) or different 5 concentrations of E2; 72 h after treatment, cells were trypsinized and 1x10 cells were plated on matrigel coated fluroblok inserts and the invasion assay was performed as described above.

81

Figure 2-7

Ctrl ERBB2 A siRNA siRNA

l) 150 MCF-7 cells Ctrl si + E2 HER2/NEU ro ERBB2 si + E 2 GAPDH cont

le E2 (1nM) - + - + ic 100 eh tofv

rcen 50 Pe l( ve le 0 4 5 1 1 3 2 5 2 N P3 P3 D7 X2 G1 RNA ID ID BB3 BB4 IL-6 IL-8 LY 4SF1 MK m PK10 CD2 CD5 FZ CTSS PSAP SDC4 CTSF KLF5 CCL BA DDR1 KRT7 RND3 S100P CTSH CTSK MS CTGF USP25 FSCN1 ER ER ICAM1 LI CXCL ABCG2 ANXA1 NDRG SPINT1 PRKCZ PRKCD IGFB CITED2 TM TGFB MA TSPAN31 TACSTD2 CEACAM6

Ctrl ERBB2 B siRNA siRNA HER2/NEU ) 150 ol BT474 cells GAPDH Ctrl si+E2 E2 (1nM)

contr - + - + ERBB2 si+E2

icle 100 eh tofv en

rc 50 Pe l( ve le 0 4 2 2 14 P3 D7 G1 RNA IL-8 MK m PK10 CD2 FZ CTSS PSAP SDC4 CTSF KLF5 KRT7 BA DDR1 S100P CTSH CTSK CTGF USP25 ERBB3 ERBB4 LI CXCL PRKCZ IGFB MMP PRKCD CITED2 MA TSPAN31 TACSTD2 CEACAM6 C BT474 cells * P = 0.0034 (Ctrl si + Veh vs. Ctrl si + E2) 2000 ** P = 0.0002 (ERBB2 si + Veh vs. ERBB2 si + E2) d

de 1500 inva ls

cel 1000

of **

er *

mb 500 Nu

0 h E2 eh E2 trol Ve V + i+ + on i+ s si gc s rl si rl Ct Ne Ct ERBB2 ERBB2

82

Figure 2-7. Lack of an effect of HER2/Neu knockdown on repression of gene expression or invasiveness by E2. MCF-7 cells (Panel A) and BT474 cells (Panel B) were plated in media containing charcoal-stripped FBS 48 h prior to transfection with either control siRNA or ErbB2 siRNA using Dharmafect 1. 24 h after the transfection, cells were treated with either vehicle (ethanol) or E2 (1 nM) for a period of 48 h. Cells were harvested for RNA extraction and mRNA levels of the indicated genes were measured using real time RT-PCR. Whole cell lysates were also extracted to ensure knockdown of HER2; GAPDH was used as a loading control (Inset in panels A and B). In Panel C, BT474 cells were trypsinized 72 h after treatment and 1x105 cells were plated on matrigel coated fluroblok inserts for invasion assay; media with 20% FBS was used as chemoattractant. Cells were allowed to invade for 24 h and stained with calcein AM to visualize under the microscope (Panel C).

83

Figure 2-8

A Ctrl ER Ctrl si + Veh siRNA siRNA Ctrl si + E2 ER si + Veh ER ER si + E 2 GAPDH

) 150 ol * P < 0.001 E2 (1nM) - + - + tr on ec

cl 100 f vehi

to * en * 50 * ** rc * Pe

l( * ve le 0 6 1 5 0

RNA 1 SF R 00P AM LF 1 m CT DD FBP3 K S IG MAPK CEAC B C

Ctrl si + Veh * P < 0.005 (Ctrl si+Veh vs. Ctrl si+E2) Ctrl si + E2 ** P < 0.001 (Ctrl si+Veh vs. ER si+Veh) ER si + Veh *** P < 0.001 (Ctrl si+E2 vs. ER si+E2) ER si + E 2000 2 *** 50

* P < 0.0001 ed ** 1500 *

40 invad ls

cel 1000 30 of er 20 mb 500 * Nu 10 * 0 h

mRNA level (Relative to control) E2 eh E2 0 trol Ve V i+ i+ GREB1 EGR3 on i+ s i+ s gc s rl s rl Ct ER Ne Ct ER

84

Figure 2-8. ER-dependence for gene repression and inhibition of invasiveness by E2. BT474 cells were plated in media containing charcoal-stripped FBS 48 h prior to transfection with control siRNA or ER siRNA using Dharmafect 1. 24 h after transfection, cells were treated with either vehicle (ethanol) or E2 (1 nM). RNA was extracted 48 h after treatment to measure mRNA levels for the indicated genes by real time RT-PCR (Panels A and B); whole cell lysates were also extracted to measure ER levels by western blot using GAPDH as the loading control (Panel A, inset) . In Panel C, cells were trypsinized 72 h after treatment and 1x105 cells were plated on matrigel coated fluroblok inserts for the invasion assay using 20% FBS as the chemoattractant. Cells were allowed to invade for 24 h and visualized by staining with calcein AM (Panel C).

85

Figure 2-9

A B

Controlwt ER ER-L372R

ER wt ER+Veh GAPDH wt ER+E2 ER-L372R+Veh E2 (1nM) - + - + - + ER-L372R+E2

l) 250 10 * P < 0.0001 * P < 0.0001 ro cont control) 200 8 to icle ve eh

ti 150 6 la Re tofv

l( 100 4 en rc leve

Pe * 50 l(

NA 2 * * ve le mR 0 0 ER

l h h RNA 00P o E2 E2 1 tr m FBP3 n Ve + Ve + S + + IG ER Co ER wt 372R wt 372R -L -L ER ER C D

wt ER+Veh 1500 * P < 0.05 wt ER+E2

ER-L372R+Veh ed ER-L372R+E2 1000 invad l) 20 *, ** P < 0.0001 cel ls ntro ** * of co 15 ** ** er to * 500 * ve * mb lati 10 Nu Re

l( 0 ve h h le 5 e E2 E2 + Ve + +V + ER ER 372R

RNA wt 372R -L wt -L m 0 ER ER 2 R S EB1 p PG R G

86

Figure 2-9. Ectopic overexpression of wild-type ER or ER-L372R in BT474 cells and the effect on regulation of gene expression and invasiveness by E2. BT474 cells were plated in media containing heat-inactivated charcoal-stripped FBS 48 h prior to lentiviral transduction with wt ER or ER-L372R. 72 h after transduction, cells were treated with either vehicle (ethanol) or E2 (1 nM). 48 h after treatment, cells were harvested for RNA extraction and the mRNA expression levels of ER (Panel A) and other indicated genes (Panels B and C) were measured using real time RT-PCR. Whole cell lysates were also extracted to measure ER levels by western blot using GAPDH as the loading control (Panel A, inset). In Panel D, cells were trypsinized 72 h after treatment and 1x105 cells were plated on matrigel coated fluroblok inserts for invasion assay; media with 20% FBS was used as chemoattractant. Cells were allowed to invade for 24 h and visualized by staining with calcein AM (Panel D).

87

Figure 2-10

* P < 0.001 Vehicle E 11 * 2 10 E+4-OHT 9 d) 8 ol

(F 7 6

gnal 5 Si 4 * 3 ChIP 2 * 1 * 0 ABCG2CEACAM6GREB1 PS2 E2 repressed genes E2 activated genes

Figure 2-10. Recruitment of ER to binding sites in chromatin associated with genes repressed or activated by E2. MCF-7 cells were plated in media containing charcoal- stripped serum and treated with either vehicle or E2 (100 nM) or tamoxifen (10 µM) for 45 min. The cells were then subjected to ChIP analysis using anti-ER antibody. ChIP signals were measured by real time RT-PCR analysis of the chromatin- immunoprecipitated products as described under Materials and Methods.

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Table 2.1: Functional classification of E2-repressed genes in breast cancer

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Chapter 3

Glucocorticoid Receptor Status is a Principal Determinant of Variability in the Sensitivity of Non-Small Cell Lung Cancer Cells to Pemetrexed

Mugdha Patki1, 2, Shirish Gadgeel2, Yanfang Huang2, Thomas McFall2, Anthony F. Shields2, Larry H. Matherly2, Gerold Bepler2 and Manohar Ratnam2

1 Department of Biochemistry and Cancer Biology, The University of Toledo Medical Center, 3000 Arlington Ave., Toledo, OH 43614 2Barbara Ann Karmanos Cancer Institute and Department of Oncology, Wayne State University, 4100 John R, Detroit, MI 48201

Address for correspondence: Manohar Ratnam, Ph.D., Room 840.1, Hudson Weber Research Center, Barbara Ann Karmanos Cancer Institute, 4100 John R, Detroit, MI 48201. Phone: 313-576-8612. E-mail: [email protected]

Key Words: Non-small cell lung cancer, Pemetrexed, p53, Glucocorticoid Receptor

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3.1 Abstract

Introduction: Pemetrexed is an S-phase targeted drug in front-line or maintenance therapy of advanced non-squamous non-small cell lung cancer (NSCLC) but methods are needed for predicting the drug response. Dexamethasone (Dex) is typically administered the day before, the day of and the day after pemetrexed. As Dex strongly regulates many genes including p53 through the glucocorticoid receptor (GR), we hypothesized that Dex influences tumor response to pemetrexed. Methods: Eight non-squamous NSCLC cell line models with varied p53 and GRα/GRβ status were used for gene expression and cell cycle analyses and for loss/gain-of-function experiments. Results: In three cell lines Dex profoundly, but reversibly, suppressed the fraction of S-phase cells. Dex also reversibly repressed expression of thymidylate synthase and dihydrofolate reductase which are primary targets of pemetrexed but are also quintessential S-phase enzymes as well as the

S-phase dependent expression of thymidine kinase 1. Dex also decreased expression of the major pemetrexed transporters, the reduced folate carrier and the proton coupled folate transporter. Only cells expressing relatively high GRα showed these Dex effects, regardless of p53 status. In cells expressing low GRα, the Dex response was rescued by ectopic GRα. Further, depletion of p53 did not attenuate the Dex effects. The presence of

Dex during pemetrexed treatment protected against pemetrexed cytotoxicity, in only the

Dex responsive cells. Conclusions: The results predict that in non-squamous NSCLC tumors, reversible S-phase suppression by Dex, possibly combined with a reduction in the drug transporters, attenuates responsiveness to pemetrexed and that GR status is a principal determinant of tumor variability of this response.

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3.2 Introduction

Non-small cell lung cancer (NSCLC) comprises 85% of all lung cancer cases and the majority of those patients have regional or systemic metastases. Therefore systemic chemotherapy forms an import ant component of the management of NSCLC. The efficacy of various chemotherapeutic agents utilized for the treatment of NSCLC was, for many years, similar among the different histologic subtypes. Randomized clinical trials have now shown that the efficacy of pemetrexed is superior to other chemotherapy drugs in non-squamous NSCLC and inferior to other drugs in squamous cell lung cancer when given as mono-therapy or in combination with a platinum compound (Hanna et al.,2004; Scagliotti et al.,2008; Ciuleanu et al.,2009;

Scagliotti et al.,2009). Pemetrexed has now received approval by regulatory agencies in the U.S. and in Europe for non-squamous NSCLC patients for front- line therapy in advanced stage disease in combination with cisplatin, as maintenance therapy following front-line therapy, and in patients with recurrent disease. Based on these data and in consideration of its limited toxicity profile, pemetrexed has become the preferred chemotherapy drug in the management of non-squamous NSCLC.

Despite its widespread use, the efficacy of pemetrexed in advanced stage NSCLC is modest, with a median progression-free survival of 5.5 month s in the front-line setting when combined with cisplatin and only 3.5 month s as single agent in patients with recurrent non-squamous NSCLC. These data suggest that the clinical benefit from pemetrexed is quite variable even among patients with non-squamous

NSCLC highlighting the need to identify predictors of clinical benefit from

92 pemetrexed. Such predictors would enable treatment decisions that would greatly benefit individual patients, while avoiding extended chemotherapies that are ineffective.

Pemetrexed is transported into the cell through two principal membrane transporters, the reduced folate carrier (RFC) and the proton coupled folate transporter (PCFT) (Chattopadhyay et al.,2007). Polyglutamation of pemetrexed by folyl-polyglutamate synthase (FPGS) results in increased cellular retention o f t h e d r u g and increases its affinity for its target enzymes

(Mendelsohn et al.,1999). Pemetrexed inhibits nucleotide biosynthesis principally by inhibiting thymidylate synthase (TS) butm at phar acologic doses it is also an inhibitor of dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT) and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT) (Shih et al.,1998). It is generally accepted that TS inhibition causes cytotoxicity via deprivation of dTMP needed for DNA synthesis and the associated ‘thymineless death’(Touroutoglou et al.,1996) and also via elevation of dUMP and its misincorporan tio into DNA (Wilson et al.,2012). Clearance of pemetrexed through the kidneys is rapid, resulting in a systemic half-life of only 3.5 hours, with up to 90 percent of the drug being eliminated in the urine within 24 hours (Rinaldi,1999). The multi-targeted action of pemetrexed, together with its prolonged cellular retention due to its relatively higher affinity for FPGS results in greater anti-tumor activity compared with older generation antifolate drugs such as methotrexate.

93

The synthetic glucocorticoid dexamethasone (Dex) is used in standard practice as a concomitant medication during treatment with pemetrexed. Dex is typically dosed at

4 mg twice daily the day before, the day of and the day after therapy with pemetrexed. An important role of Dex is to reduce the possibility of severe skin rash caused by pemetrexed. Dex is also included for its anti-emetic properties particularly when pemetrexed is combined with cisplatin or carboplatin. The pharmacological actions of Dex are mediated by the glucocorticoid receptor (GR), which principally acts as a transcription factor (Mangelsdorf et al.,1995).

Glucocorticoids regulate cell proliferation and apoptosis as well as inflammation and immune response (Vilasco et al.,2011). The nature of the physiological response as well as sensitivity to synthetic glucocorticoids such as Dex is tissue-dependent and variable.

This variability occurs among different individuals as well as among different tissues in the same individual (Lu et al.,2006).

A single GR gene generates two major splice variants, GRα and GRβ; each isoform has variants that result from multiple translation start sites within their mRNAs (Lu et al.,2006). Although GRβ only diverges from GRα by substitution of the carboxyl- terminal 50 amino acids in GRα by a non-homologous 15 amino acid sequence, GRβ is unable to bind GR ligands (Oakley et al.,1997). GRβ heterodimerizes with GRα to exert a dominant-negative effect on the transcriptional activity of GRα (Oakley et al.,1997).

Therefore the degree of Dex sensitivity of a tissue may be related to the functional GR status, determined by the level of GR expression as well as the ratio of GRα to GRβ.

94

Whereas GRα is expressed ubiquitously, GRβ expression is potentially also significant in the present study of lung cancer because the limited types of tissues expressing this isoform include epithelial cells of the terminal bronchioles (Oakley et al.,1997).

Given the profound tissue-specific effects of Dex, its ability to regulate many genes and the reported ability of Dex to modulate cellular senescence in lung cancer cells (Ge et al.,2012), it was of interest to examine the possibility that Dex may act on NSCLC cells to influence their responsiveness to pemetrexed. Immunohistochemical analysis of clinical NSCLC tumors shows considerable heterogeneity in total GR expression with relatively high total GR observed in approximately half of the tumors (Lu et al.,2006).

Further, approximately 33% of lung adenocarcinomas harbor p53 mutations (Toyooka et al.,2003) and the p53 gene is a known target for activation by Dex (Crochemore et al.,2002). It was therefore of interest to explore both the GR status and the p53 status

(wild type p53, p53 deletions or p53 mutations) of NSCLC cells as possible determinants of responsiveness to Dex, particularly molecular and cellular effects of

Dex that could influence the action of pemetrexed.

3.3 Materials and Methods

Cell Culture and Reagents. The non-squamous NSCLC cell lines A549, H1299,

H358, H226, H460, H1650, ADLC-5M2 and H292 were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies), 100 units/ml penicillin, 100 μg/ml streptomycin and 2mM glutamine. The MTT assay reagents were from MP Biomedicals (Solon, OH). Charcoal-stripped fetal bovine serum

95 was from Life Technologies (Carlsbad, CA). Dexamethasone was purchased from EMD

Millipore (Billerica, MA). Dimethyl sulfoxide (DMSO) and crystal violet were purchased from Fisher Scientific (Pittsburgh, PA). Propidium iodide (PI)/RNase solution was purchased from BD Biosciences (San Jose, CA). PCR primers and TaqMan probes were either purchased from the Life Technologies inventory or custom synthesized by

Integrated DNA Technologies (Coralville, IA). The GRα-expressing lentivirus was from

GenTarget Inc. (San Diego, CA). p53shRNA expressing lentivirus was a kind gift from

Dr. Yubin Ge at Karmanos Cancer Institute. Blasticidin and puromycin were from Life

Technogies, Carlsbad, CA and Sigma-Aldrich, St. Louis, MO.

Measurement of Gene Expression. To measure gene expression, mRNA was quantified by real time reverse transcription PCR. Total RNA from cells was isolated using RNeasy minikit (Qiagen, Georgetown, MD) according to the manufacturer’s protocol. Reverse transcription was performed using 500ng of total RNA and High-Capacity cDNA Archive kit (Life Technologies, Carlsbad, CA) according to the vendor’s protocol. cDNA was measured by quantitative real-time PCR using the

StepOnePlus Real-Time PCR System (Life Technologies, Carlsbad, CA) and TaqMan

Fast Universal PCR Master Mix (Life Technologies, Carlsbad, CA). All samples were measured in triplicate and CT values normalized to those of GAPDH.

Cell Proliferation Assay. Cells (2000 per well) were seeded in 96-well plates in phenol-red-free medium supplemented with charcoal-stripped FBS. Treatments were initiated after the cells were attached. At the appropriate time points, cell viability was

96 determined by MTT assay. 10μl of MTT (5mg/ml in phosphate buffered saline) was added to each well followed by incubation at 37°C for 2 h. The formazan crystal sediments were dissolved in 100μl DMSO and absorbance was measured at 570nm using the BioTek Synergy 2 Microplate Reader (BioTek, Winooski, VT). Each treatment was performed in six replicate wells.

Colony Formation Assay. Cells were trypsinized and 2000 cells per well were plated in duplicate 6-well plates in phenol-red-free medium supplemented with charcoal- stripped FBS. Colonies were formed after 6 days. The cells were fixed with ice cold methanol and stained with crystal violet. Images were obtained using the Oxford

Optronix GelCount colony counter (Oxford Optronix Ltd., Abingdon, UK).

Cell Cycle Analysis. Cells were trypsinized and harvested in phenol-red-free medium supplemented with charcoal stripped FBS. Cells (1 x 106) were washed and suspended in

500μl phosphate-buffered saline. The cells were fixed with ice cold 100% ethanol added drop wise with constant agitation and incubated on ice for 20 min. The cell pellet was obtained by centrifugation and suspended in 500μl of PI/RNase solution and incubated in the dark at room temperature for 20 min. Cell cycle distribution was determined using the

BD LSR II analyzer (BD Biosciences, San Jose, CA) at the Microscopy, Imaging and

Cytometry Resources Core at Karmanos Cancer Institute. The data was analyzed using

ModFit software.

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Gene Transduction and Selection of Recombinant Cells. To generate recombinant cells overexpressing GRα, cells were plated in a 24-well plate to 75% confluence and transduced with 50µl lentivirus. 72 h later, blasticidin (4µg/ml or 6µg/ml) was added to the cells. The cells were treated with blasticidin for 72 h and then subcultured in complete media. Blasticidin was added to the media at every third passage of the subculture. The recombinant cells expressing p53 shRNA were generated by an identical procedure except that the antibiotic used for selection was puromycin.

Statistical Analyses. Statistical significance was determined using one way

ANOVA. The error bars in all graphs represent standard deviation of the mean. The P values are indicated in the figure legends.

3.4 Results

Effect of Dex on the Expression of Genes Involved in Pemetrexed Action. As

Dex is known to regulate the transcription of many genes through GR, we tested its effect on the expression of genes whose products have direct roles in the action of pemetrexed. For this purpose, we first chose two commonly used lung adenocarcinoma cell li ne models, A549 and H1299. The cells were plated in hormone depleted media and then treated with Dex (100 nM) for 48 h. Gene expression was quantified by real time RT-PCR. As seen in Figure 3 - 1A, in A549 cells, Dex suppressed the expression of TS and DHFR by 75 percent and also the expression of

RFC (by 45 percent) and PCFT (by 60 percent). The expression levels of GARFT,

AICARFT and FPGS were unaltered by Dex. In contrast, Dex did not significantly

98 influence the expression of any of the genes tested in H1299 cells (Figure 3-1B).

The experiments were extended to additional lung cancer cell lines and they exhibited both sensitivity (H292 and H226 cells) and insensitivity (H460, H358, ADLC-5M2 and H1650 cells) to Dex (Table 3.1). The Dex sensiti vity of lung cancer cell line models were thus variable with respect to the regulan tio of the genes whose products are directly involved in mediating the cytotoxicity of pemetrexed.

Effect of Dex on Cell Cycle Progression. As TS and DHFR are essential S-phase enzymes, we also tested the effect of Dex on thymidine kinase 1 (TK1), another S-phase dependent enzyme. TK1 was also decreased by Dex in A549 cells but not in H1299 cells

(Figure 3-1A and 3-1B). Hence, it was of interest to test the effect of Dex on cell cycle phase distribution in the lung cancer cell lines. Treatment with Dex caused a striking suppression of S-phase in A549 cells (Figure 3-2A) but did not significantly alter the cell cycle phase distribution in H1299 cells (Figure 3-2B). Thus, the differential sensitivity of A549 and H1299 cells to transcriptional regulation by Dex noted above is associated with the differential ability of Dex to suppress the S-phase fraction of cells.

This association also extended to the other NSCLC cell line models (Table 3.1).

Reversibility of the Effects of Dex. Next, we tested the duration of the transcriptional and cellular effects of Dex in A549 cells. Withdrawal of Dex restored the

S-phase fraction to the original level (Figure 3-3A); the reversal occurred within 24 hours of Dex withdrawal. Further, the S-phase recovery was accompanied by full

99 restoration of TS and DHFR expression (Figure 3-3B). These results demonstrate that the effects of Dex on the lung cancer cells are fully reversible.

Relationship of the Dex Effects to GR Status. The cell line models chosen for this study showed variable levels of GRα and GRβ determined by quantitative RT-PCR

(Table 3.1). Despite the fact that epithelial cells of the terminal bronchioles express

GRβ, the expression of GRβ in the NSCLC cell lines was low compared to GRα (Table

3.1). The down-regulation of TS and DHFR or the suppression of S-phase by Dex occurred in A549 and H292 cells that expressed relatively the highest GRα and in

H226 cells that expressed a moderate level of GRα. In contrast, the remaining cell lines that expressed lower levels of GRα were insensitive to Dex (Table 3.1).

However, when GRα was transduced in the low GRα-expressing H1299 cells (H1299-

GRα cells in Figure 3-4A), the Dex effect was restored, both in terms of down- regulation of TS and DHFR (Figure 3-4B compared with Figure 3-1B) and suppression of S-phase (Figure 3-4C compared with Figure 3-2B). Therefore, variability in GRα expression level alone could be an important factor underlying the differences among

NSCLC cells in terms of their ability to respond to Dex.

Relationship of the Dex Effects to p53 Status. The cell line models chosen also have variable p53 status, including wt p53, mutant p53 and p53 deletions (Table 3.1).

Ectopic GRα rescued the Dex effects in the p53-null H1299 cells, indicating that in the presence of optimal GRα expression the cells may be responsive to Dex regardless of their p53 status (Figure 3-4). However H226 cells, which expressed the P72R mutant

100 p53 were responsive to Dex although they expressed a moderate level of GRα in contrast to ADLC-5M2 cells that also harbored the same p53 mutation but expressed much less GRα (Table 3.1). As the P72R mutant is known to share functional similarities with wt p53 (Whibley et al.,2009), it was of interest to test for a possible role of the mutant p53 in mediating the Dex effects in H226 cells. In H226 cells depleted of p53 by lentiviral transduction of p53shRNA (H226-p53KD cells, Figure 3-

5A), Dex still inhibited the expression of TS, DHFR and the additional S-phase marker thymidine kinase 1 (TK1) (Figure 3-5B); the effects of Dex in the transduced cells were comparable in degree to its effects in the parental H226 cells (Figure 3-5C). In

H226 cells, inhibition of the S-phase enzymes (Figure 3-5C) as well as suppression of the S-phase fraction of cells (data not shown) was moderate compared with A549 cells, likely reflecting the moderate expression level of GRα in H226 cells (Table 3.1).

Taken together, the above results demonstrate that in non-squamous NSCLC cells, an optimal expression level of GRα sensitizes cells to Dex independent of p53 status.

Effect of Dex on Cytotoxicity of Pemetrexed. As previously noted, the standard clinical regimen for pemetrexed treatment includes administration of Dex prior to, during and following pemetrexed. The reversible Dex effects observed above implies that in Dex-sensitive non-squamous NSCLC tumors, Dex could protect the tumor cells against the actions of pemetrexed for the duration of the drug treatment by temporarily suppressing transition through S-phase. To test the effect of Dex on sensitivity to pemetrexed, A549 and H1299 cells were pre-treated with Dex and a pharmacologically relevant dose (5 µM) of pemetrexed was introduced for a 24 hour

101 period prior to withdrawing Dex. Following the treatments, the effect of Dex on susceptibility of the cells to pemetrexed was examined both by the standard MTT growth assay and by monitoring clonogenic survival. In the absence of Dex treatment pemetrexed profoundly inhibited the growth (Figure 3-6) and clonogenicity (Figure 3-

7) of both A549 and H1299 cells, although when the cells were treated with Dex, there was a striking difference in the response of A549 cells versus H1299 cells. Dex had a protective effect on A549 cells against inhibition of growth and clonogenicity by pemetrexed (Figures 3-6A and 3-7A); in contrast, Dex treatment did not affect the sensitivity of H1299 cells to pemetrexed (Figures 3-6B and 3-7B). However, Dex did protect against pemetrexed in H1299 cells transduced with GRα (Figure 3-7C).

Therefore, in a clinically relevant in vitro context, Dex-sensitive non-squamous

NSCLC cells are protected by Dex against growth inhibition by pemetrexed and this effect is dependent on the expression of an optimal level of GRα.

3.5 Discussion

Our studies of diverse non-squamous NSCLC cell line models indicate that NSCLC cells are variable in their ability to respond to a pharmacological concentration of Dex. In the responsive cells, Dex slowed cell cycle progression, specifically by suppressing progression into S-phase. Concomitantly, Dex suppressed the expression of TS and

DHFR, both of which are principal target enzymes of pemetrexed as well as critical S- phase enzymes. Among other determinants of pemetrexed cytotoxicity, Dex also decreased the expression of RFC and PCFT, the principal membrane transporters of pemetrexed. The effects of Dex were reversible such that, following withdrawal of Dex,

102 both the S-phase fraction of the cells as well as the levels of TS and DHFR were restored within 24 hours. As a predictable consequence of these reversible effects of Dex, the cytotoxicity of pemetrexed was attenuated by Dex when the cells were exposed to Dex prior to, concomitant with and following pemetrexed treatment. Similar treatment with

Dex did not influence pemetrexed cytotoxicity in cells in which it was unable to regulate the cell cycle or the expression of gene products involved in pemetrexed action.

Among the eight non-squamous NSCLC model cell lines examined, the cells expressing a relatively high level of GRα were sensitive to Dex regardless of their p53 status. Further the Dex effects were restored upon ectopic expression of GRα, in cells expressing relatively low endogenous GRα, even if they were p53 null. Cells that expressed a moderate level of GRα were moderately sensitive to Dex. In these cells, the studies also ruled out any contribution to Dex sensitivity in NSCLC cells attributable to the P72R mutant of p53 which reportedly (Whibley et al.,2009) could be more effective in inducing stress response and apoptosis than wt p53 in different cell types. Although the p53 gene is a known target of glucocorticoids, p53 status is unlikely to have a major influence on response to Dex in non-squamous NSCLC cells. Remarkably, despite the fact that epithelial cells of the terminal bronchioles which may be progenitors of lung adenocarcinoma express GRβ (Oakley et al.,1997), expression of GRβ was low in all of the NSCLC cell line models.

Patient response to pemetrexed has previously been shown to correlate negatively with tumor expression of TS and positively with tumor expression of folate receptor type α

103

(FRα) (Bepler et al.,2008; Christoph et al.,2013) but their relative expression is not a powerful independent predictor of clinical response. Moreover, this association cannot be attributed to a direct mechanistic link between drug sensitivity and the tumor levels of TS or FRα due to the following reasons: (i) the range of variability in tumor levels of TS determined by immunohistochemistry and related to pemetrexed treatment outcome is considerably narrower than that known to cause significant differences in cellular drug sensitivity at pharmacologic doses. For example, in the study of Christoph et al

(Christoph et al.,2013) patient response correlated with a mean H-score for TS expression of 187±5 and a median score of 180 whereas the non-responders had a mean score of

201±4 with a median of 210. In contrast, in vitro studies of lung cancer cell lines (Ozasa et al.,2010) have shown that variability in IC50 values for pemetrexed occurs over a much larger dynamic range of TS levels. Among various cell lines, the variability in TS levels that correlated with the variability in IC50 for pemetrexed occurred over a TS expression range of two orders of magnitude. Further, within a single cell line, TS induction partially or fully contributing to pemetrexed resistance ranged from 2.5-fold to

20-fold (Ozasa et al.,2010). (ii) FRα is not the pharmacologically relevant transporter of pemetrexed except in unusual situations when its expression level may be very high, combined with defects in RFC- or PCFT- mediated transport (Chattopadhyay et al.,2007).

On the other hand, as lower TS and higher FRα are known to be associated with reduced cell growth, they could reflect a smaller proportion of proliferating tumor cells.

Therefore, it is quite likely that relative total TS and FRα values in the treatment-naive tumors may simply reflect the relative intrinsic growth rates of the tumors and hence relate to duration of survival following drug treatment. The present study advances a

104 mechanism-based approach to understanding tumor responsiveness to pemetrexed, based on the effects of Dex and offers a rational basis for predicting individual responses.

It is believed that despite the rapid systemic clearance of pemetrexed (Rinaldi,1999), cellular retention of pemetrexed by polyglutamation confers a special advantage with effective outcomes through less frequent treatment cycles compared to other chemotherapy drugs. Indeed, both reduced FPGS expression and elevated γ-glutamyl hydrolase have been associated with pemetrexed resistance (Schneider et al.,2006).

However, clearance of Dex is relatively slow, with physiologically effective concentrations remaining in circulation for several days following Dex withdrawal

(Weijtens et al.,1998). This consideration, combined with the protective effects of Dex observed under the in vitro conditions used in the present study, strongly suggests that

Dex sensitive non-squamous NSCLC tumors will be less responsive to standard pemetrexed treatment regimens. The findings in this study of the effects of Dex on

NSCLC cell susceptibility to pemetrexed would likely extend to other S-phase acting chemotherapy drugs as well. However, in the clinical setting, Dex may have a lower impact on the action of platinum drugs that can form DNA adducts at any stage of the cell cycle, with the downstream cellular effects manifesting after the clearance of Dex.

Our findings warrant clinical studies of non-squamous NSCLC tumor responsiveness to pemetrexed in relation to Dex sensitivity and GR status of the tumors.

105

3.6 Acknowledgements

The authors acknowledge Jessica Back, Ph.D. for assistance with flow cytometry and the

Microscopy, Imaging and Cytometry Resources Core at the Karmanos Cancer Institute,

Wayne State University. The Microscopy, Imaging and Cytometry Resources Core is supported, in part, by NIH Center grant P30CA22453 to The Karmanos Cancer Institute,

Wayne State University and the Perinatology Research Branch of the National Institutes of Child Health and Development, Wayne State University. The authors thank Dr. Yubin

Ge for sharing p53 shRNA lentivirus.

106

Figure 3-1

A B

Vehicle Vehicle 1.4 A549 cells 1.6 H1299 cells Dex Dex 1.2 1.4 1.2 1 1 0.8 0.8 * 0.6 0.6 * * 0.4 * * 0.4 mRNA Level (Fold) mRNA mRNA Level (Fold) Level mRNA 0.2 0.2 0 0

Figure 3-1: Differential effects of Dex on the expression of genes involved in pemetrexed action in A549 vs. H1299 cells. A549 cells (Panel A) and H1299 cells (Panel B) were plated at 20 percent confluency in media containing charcoal stripped serum for 24 h for hormone depletion. Cells were then treated with either vehicle (ethanol) or Dex (100nM) for 48 h. Cells were harvested, and the total RNA extracted to measure mRNA levels by real time RT-PCR. * P < 0.01.

107

Figure 3-2

A B

G2/M G2/M A549 cells H1299 cells S S 120 G1 120 G1

100 100

80 80

60 60

40 40 Distribution in cell cycle cell in Distribution Distribution in cell cycle cell in Distribution 20 20

0 0 Vehicle Dex Vehicle Dex

Figure 3-2: Effect of Dex on cell cycle phase distribution in A549 and H1299 cells. A549 cells (Panel A) and H1299 cells (Panel B) were plated at 20 percent confluency in media containing charcoal stripped serum for 24 h for hormone depletion. Cells were then treated with either vehicle (ethanol) or Dex (100nM) for 48 h and harvested for flow cytometry analysis.

108

Figure 3-3

A B

G2/M A549 cells Vehicle (48h) A549 cells S Dex (48h) 120 G1 24h post Dex withdrawal 100 48h post Dex withdrawal 80 1.4 60 1.2 40 20 1

Distribution in cell cycle cell in Distribution 0 0.8

(Fold) mRNA 0.6 * 0.4 * 0.2 0 TS DHFR

Figure 3-3: Reversibility of Dex effects in A549 cells. A549 cells were plated at 20 percent confluency in media containing charcoal stripped serum for 24 h for hormone depletion. Cells were then treated with either vehicle (ethanol) or Dex (100nM). After 48 h, Dex was removed by washing the cells twice and replacing with fresh media. Cells were cultured for additional periods of 24 h and 48 h. Cells were harvested for flow cytometry analysis (Panel A) or for mRNA measurement by real time RT-PCR (Panel B). * P < 0.01.

109

Figure 3-4

A B C Vehicle H1299-GRα Dex G2/M H1299-GRα 1.6 1.2 120 S 1.4 G1 1.2 1 100 d) d)

1 cycle

0.8 ll 80 (Fol (Fol 0.8 * 0.6 0.6 ce in 60 RNA * RNA on m ti m 0.4

α 0.4 * bu 0.2 40 GR 0.2 0 Distri 20 0 DHFR TS 0 Vehicle Dex

Figure 3-4: Effect of restoring GRα on Dex sensitivity in H1299 cells. H1299-GRα cells were generated as described under Materials and Methods. RNA extracted from A549 cells, H1299 cells and H1299-GRα cells was used to measure the relative mRNA levels for GRα (Panel A). H1299-GRα cells were plated at 20 percent confluency in media containing charcoal stripped serum for 24 h for hormone depletion. Cells were then treated with either vehicle (ethanol) or Dex (100nM) for 48 h. Cells were harvested for mRNA measurement by real time RT-PCR (Panel B) or for cell cycle analysis (Panel C). * P < 0.01.

110

Figure 3-5

A B C

Vehicle 1.2 Dex H226-p53KD Vehicle H226 1.2 Dex 1 1.4 1 1.2 0.8 * 1 0.6 0.8 * * 0.8 0.4 p53 mRNA (Fold) mRNA p53 0.6 * * 0.6 * 0.2 * mRNA (Fold)mRNA 0.4 mRNA (Fold)mRNA 0.4 0 0.2 0.2 0 0 TS DHFR TK1 TS DHFR TK1

Figure 3-5: Effect of knocking down p53 on Dex sensitivity in H226 cells. The relative p53 expression levels in the parental H226 cells vs. H226-p53KD cells were determined by real time RT-PCR (Panel A). H226-p53KD cells (Panel B) or the parental H226 cells (Panel C) were plated at 20 percent confluency in media containing charcoal stripped serum for 24 h for hormone depletion. Cells were then treated with either vehicle (ethanol) or Dex (100nM) for 48 h and then harvested for mRNA measurement by real time RT-PCR. * P < 0.01.

111

Figure 3-6

A B

A549 cells Pemetrexed H1299 cells Pemetrexed 140 120 Dex + Pemetrexed Dex + Pemetrexed 120 100 100 80 80 60 60 40 40

Viable cells (Percent control) (Percent cells Viable 20 20 control) (Percent cells Viable 0 0 0 4 5 6 0 4 5 6 Days after pemetrexed treatment Days after pemetrexed treatment

Figure 3-6: Differential effects of Dex on growth inhibition by pemetrexed in A549 vs. H1299 cells. A549 cells (Panel A) and H1299 cells (Panel B) were plated in sextuplicate wells in 96 well plates in media containing charcoal stripped serum as described under Materials and Methods for MTT assays. 24 h after plating, the cells were treated with vehicle (ethanol) or Dex (100 nM) for 72 h – 96 h. The cells were exposed to pemetrexed (5 µM) or vehicle (water) for a 24 hour window in the midst of the Dex (or vehicle) treatment. MTT assays were performed on the indicated days, beginning with the day of pemetrexed treatment (Day 0). At each time point, the ratio of the absorbance for pemetrexed treatment to its vehicle control was used to determine the percentage of viable cells.

112

Figure 3-7

C A B H1299-GRα cells A549 cells H1299 cells

Vehicle

Pemetrexed

Dexamethasone

Figure 3-7: Influence of Dex on inhibition of colony formation by pemetrexed in relation to GRα status. A549 cells (Panel A), H1299 cells (Panel B) and H1299-GRα cells (Panel C) were first plated at 20 percent confluency in 10 cm dishes in media containing charcoal stripped serum for hormone depletion. 24 h later cells were treated with vehicle (ethanol) or Dex (100 nM) for 72 h – 96 h. The cells were exposed to pemetrexed (5 µM) or vehicle (water) for a 24 hour window in the midst of the Dex (or vehicle) treatment. The cells were then plated for the colony formation assay as described under Materials and Methods. After the colonies were formed, they were stained with crystal violet.

113

Table 3.1: GR isoform expression and p53 status of model NSCLC cell lines and their response to Dex

p53 GRα GRβ Effect of Dex on Cell line a a status level level TSb DHFRb S-phase A549 wt 1 0.008 Decrease Decrease Suppressed H292 wt 1.2 0.005 Decrease Decrease Suppressed H226 P72R 0.4 0.003 Decrease Decrease Suppressed H460 wt 0.2 0.002 No effect No effect No effect H358 null 0.2 0.001 No effect No effect No effect H1299 null 0.2 0.001 No effect No effect No effect ADLC-5M2 P72R 0.01 0.002 No effect No effect No effect H1650 wt 0.003 0.0002 No effect No effect No effect

a Levels of GRα and GRβ mRNAs are normalized values relative to the level of GRα in A549 cells. b TS and DHFR gene expression levels were determined by measuring mRNA by real time RT-PCR.

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Chapter 4

Conclusion

Breast cancer is the most common type of cancer diagnosed in women. The tumor is usually localized at the early stages. The initial treatments include surgery, chemotherapy and radiation therapy generally followed by adjuvant treatments to prevent tumor recurrence and metastasis. The expression of the estrogen receptor in a breast tumor is generally an indicator of dependence of the tumor on estrogen for growth and survival. This offers a strategy for targeting estrogen signaling and the estrogen receptor for therapeutic applications. Anti-estrogens adjuvant therapy has been a mainstay in the treatment of estrogen receptor-positive breast cancer. Despite the widespread success of anti-estrogen therapy (including aromatase inhibitors and tamoxifen) the major obstacle in improving the overall disease-free survival of patients is the development of resistance to long-term anti-estrogen therapy. The resistance could be either intrinsic (de novo) or acquired during the treatment. Several mechanisms have been established for the development of resistance to anti-estrogens. However, the recurring tumor is generally not dependent on estrogen signaling and is more aggressive than the primary tumor. The mechanism of this transition from a hormone-responsive to a more aggressive hormone- refractory state is unclear. The results from studies in Chapter 2 suggest that a gene

115 repression program of estrogen decreases the aggressiveness of the tumor as the target genes are responsible for invasion, metastasis, angiogenesis, immune evasion and drug resistance. The repressed subset of estrogen target genes significantly overlapped with the subset of genes upregulated in invasive ductal carcinoma compared to matched samples of ductal carcinoma in situ. Our studies were consistent with previous reports in the literature that the genes activated by estrogen are responsible for growth and survival of the breast tumor and that inhibition of growth by anti-estrogens is mediated by antagonism of gene activation. Gene repression by estrogen is also antagonized by anti- estrogens including hormone depletion and tamoxifen. Our studies suggest that antagonism of genes repressed by estrogen supports breast tumor progression and may have significant implications in understanding the effects of long-term anti-estrogen adjuvant therapy. Our studies show that, estrogen suppresses the invasiveness of estrogen receptor positive breast cancer cells in a manner that is antagonized by tamoxifen and hormone depletion. The gene repression and suppression of invasiveness by estrogen was dependent on but was independent of the Her2 status of the cells.

A causal link between gene repression by estrogen and its inhibition of invasiveness was established as a co-repressor binding site mutation in the estrogen receptor that selectively disrupted the ability of estrogen to repress genes also prevented inhibition of invasiveness. Gene repression by estrogen involved recruitment of the estrogen receptor to non-classical chromatin sites that was prevented by tamoxifen. These studies suggest that selective antagonism of gene activation vs. gene repression by estrogen may offer better long-term treatment outcomes in breast cancer.

116

Variability in response to chemotherapeutic drugs among patients is a major obstacle in the treatment of several cancers. The studies in chapter 3 were aimed at predicting response to pemetrexed chemotherapy in advanced non-small cell lung cancer.

Pemetrexed is approved by regulatory agencies for the first-line treatment of advanced non-squamous non-small cell lung cancer in combination with cisplatin and as maintenance therapy following front-line therapy. The response to pemetrexed is variable among patients with different histologic subtypes of non-small cell lung cancer with pemetrexed/cisplatin combination being superior to gemcitabine/cisplatin combination in advanced non-squamous non-small cell lung cancer but inferior to gemcitabine/cisplatin in advanced squamous non-small cell lung cancer. However, the efficacy and benefit of pemetrexed is variable even in patients with advanced non-squamous non-small cell lung cancer, as seen from the median overall survival rates of patients treated with the pemetrexed/cisplatin combination. Pemetrexed is an antifolate which blocks nucleotide biosynthesis primarily by inhibiting thymidylate synthase and dihydrofolate reductase; its secondary targets include glycinamide ribonucleotide formyltransferase and 5- aminoimidazole-4-carboxamide ribonucleotide formyltransferase which are key enzymes in purine and pyrimidine synthesis. The phase II studies of pemetrexed revealed major toxicity issues with neutropenia and grade 3 or 4 rash in more than a third of the patients.

Toxicity was positively correlated with increasing plasma homocysteine levels which is an indirect measure of folate and vitamin B12 status in the patients. Based on this finding folate/B12 supplementation was added to the regimen along with pretreatment of patients with dexamethasone to prevent skin rash. Dexamethasone is administered to patients the day before, the day of and the day after pemetrexed chemotherapy. The studies in chapter

117

3 show that treatment of non-squamous non-small cell lung cancer cell line models with dexamethasone caused a profound but reversible reduction in the S-phase fraction of cells in three out of the eight cell lines tested. Additionally, there was a decrease in the expression of thymidylate synthase and dihydrofolate reductase, which are not only primary targets of pemetrexed but are also quintessential S-phase enzymes. The expression of the S-phase dependent enzyme, thymidine kinase 1 was also decreased in response to dexamethasone. This decrease in expression of S-phase enzymes by dexamethasone was reversible. Dexamethasone also decreased expression of the major transporters of pemetrexed, the reduced folate carrier and the proton coupled folate transporter. As a consequence of these effects of dexamethasone pemetrexed cytotoxicity was attenuated in the cell lines that were responsive to dexamethasone. Our studies further demonstrated that only the three cell lines expressing relatively high glucocorticoid receptor isoform alpha were responsive to dexamethasone, regardless of the p53 status. The variability in pemetrexed sensitivity was causally related to variability in the expression of the glucocorticoid receptor isoform alpha, as the response to dexamethasone was rescued in cells expressing low glucocorticoid receptor alpha, by ectopic expression of glucocorticoid receptor alpha. These results suggest that glucocorticoid receptor status is a principal determinant of tumor variability in response to pemetrexed. The studies predict that the glucocorticoid receptor status could be used as a marker to predict response to pemetrexed therapy enabling to the development of more effective and individualized treatment strategies.

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