Nghiên cứu vai trò của PSAT1 trong tiến triển ung thư vú - Stephanie Metcalf

University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 12-2019 Investigation of phosphoserine aminotransferase 1(PSAT1) in breast cancer progression. Stephanie Metcalf University of Louisville Follow this and additional works at: https://ir.edu/etd Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Metcalf, Stephanie, "Investigation of phosphoserine aminotransferase 1(PSAT1) in breast cancer progression. Electronic Theses and Dissertations.18297/etd/3345 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository.

This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact thinkir@louisville. INVESTIGATION OF PHOSPHOSERINE AMINOTRANSFERASE 1(PSAT1) IN BREAST CANCER PROGRESSION By Stephanie Metcalf B. Indiana University Purdue University Indianapolis 2014 M.

University of Louisville 2017 A Dissertation Submitted to the Faculty of the School of Medicine at the University of Louisville In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biochemistry and Molecular Genetics Department of Biochemistry and Molecular Genetics University of Louisville Louisville, Kentucky December 2019 INVESTIGATION OF PHOSPHOSERINE AMINOTRANSFERASE 1(PSAT1) IN BREAST CANCER PROGRESSION By Stephanie Metcalf B. Indiana University Purdue University Indianapolis 2014 M. University of Louisville 2017 A Dissertation Approved on August 23, 2019 by the Following Dissertation Committee: ____________________________________ Brian Clem, Ph. ____________________________________ Carolyn Klinge, Ph.

____________________________________ David Samuelson, Ph. ____________________________________ Paula Bates, Ph. ____________________________________ Steve Ellis, Ph. ii DEDICATION This dissertation is dedicated to my parents Mr.

Elbert Dee Metcalf and Mrs. Luann Michelle Metcalf who have supported me throughout my educational career. iii ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Brian Clem, for his guidance and assistance at the beginning of my scientific career.

I would also like to thank my committee members Drs. Klinge, Samuelson, Bates, and Ellis for their insights, expertise, and contribution to my scientific education. I would also like to thank all the former and current members of the Clem laboratory as well as the collaborators that have contributed to the work presented in this dissertation. Lastly, I would like to thank my family and friends for their support during the last couple of years and their understanding of the demands of obtaining a Ph.

iv ABSTRACT INVESTIGATION OF PHOSPHOSERINE AMINOTRANSFERASE 1(PSAT1) IN BREAST CANCER PROGRESSION Stephanie Metcalf August 23, 2019 This dissertation describes my research into the involvement of phosphoserine aminotransferase 1 (PSAT1) in breast cancer progression; specifically, in triple negative breast cancer (TNBC) metastasis and endocrine resistance in estrogen receptor positive breast cancer (ER+BC). Breast cancer is the most common tumor diagnosis among women. While the overall 5-year survival for breast cancer is reaching 90%, the 5-year survival for metastatic disease is only 22%. Metastasis and endocrine resistance combined can affect over 50% of patients.

One of the proteins and pathways implicated in both metastasis and endocrine resistance in breast cancer is phosphoserine aminotransferase 1 (PSAT1) and the serine synthetic pathway (SSP). From prior reports and preliminary studies within the lab, I hypothesized that PSAT1 may play a role in metastasis within TNBC and contribute to endocrine v within ER+BC. The role of PSAT1 in TNBC metastasis was evaluated via examination of the effects of altered PSAT1 expression on metastatic potential in TNBC cell lines that were “serine synthesis-independent”. Functional relevance of PSAT1 on sensitivity to endocrine therapy was tested in matched endocrine sensitive and endocrine resistant cell lines upon alteration of PSAT1 expression.

Through this work, I found that suppression of PSAT1 significantly inhibited the in vitro motility and invasiveness of “serine synthesis-independent” TNBC which was not recapitulated upon suppression of PHGDH, which is the first enzyme within the SSP. I also found that suppression of PSAT1 reduced the number of micro-metastases within the lungs in an experimental metastasis model. In addition, I found that both PSAT1 and PHGDH correlated to poorer progression free survival in multiple patient cohorts, manipulation of PSAT1 or PHGDH in both sensitive and resistant ER+BC cell lines altered sensitivity to 4-hydroxytamoxifen treatment. This body of work has demonstrated that PSAT1 selectively promotes metastasis in “serine synthesis-independent” TNBC via a function unrelated to de novo serine synthesis.

It also has shown that both PSAT1 and PHGDH contribute to tamoxifen resistance in ER+BC and thereby implicating a role for the SSP in this context. Taken together, this dissertation demonstrates that PSAT1 contributes to breast cancer progression through promotion of TNBC metastasis and ER+BC endocrine resistance. vi TABLE OF CONTENTS Page Acknowledgements…………………………………………………………………….v List of Figures…………………………………………………………………………. ix Chapter 1: Introduction…………………………………………………………………1 Chapter 2: Selective Loss of Phosphoserine Aminotransferase 1 (PSAT1) Suppresses Triple Negative Breast Cancer Metastasis………………………….30 Methods and Materials……………………………………………………….

41 Discussion………………………………………………………………………62 Extended Results and Discussion…………………………………………. 67 Chapter 3: Phosphoserine Aminotransferase 1 (PSAT1) and Serine Synthesis Pathway Alters Sensitivity to 4-Hydroxytamoxifen Treatment in Estrogen – Receptor Positive Breast Cancer……………………………………………………. 73 Methods and Materials………………………………………………………. 81 Discussion……………………………………………………………………… 93 Chapter 4: Conclusion……………………………………………………………….

114 viii LIST OF FIGURES Figure Page 1. General Subtypes of Breast Cancer…. Endocrine Therapy Mechanisms of Action…………………………………. The Metastatic Cascade………………………………………………………….

Schematic of the Serine Synthesis Pathway……. PSAT1 Expression Increases with TNBC Grade……………………………. PSAT1 and PHGDH Transcript and Protein Expression in MDA-MB-231 and HCC1806 TNBC Cell Lines……………………………………………. Loss of PSAT1 Does Not Affect Proliferation but alters Actin-Cytoskeleton.

SiRNA suppression of PSAT1 Has No Proliferative Effect…………………. Loss of PSAT1 Suppresses MDA-MB-231 Motility and Invasion In Vitro…. SiRNA suppression of PSAT1 Decreases Metastatic Potential of MDA-MB- 231 Cells In Vitro…………………………………………………………………. Suppression of PSAT1 in HCC1806 Cells Does Not Have an Anti- Proliferative Effect………………………………….

Decreased Metastatic Potential of HCC1806 Cells Upon Suppression of PSAT1……………………………………………. PHGDH is Dispensable for MDA-MB-231 Proliferation In Vitro……………. SiRNA Suppression of PHGDH in MDA-MB-231 Cells Has No Effect On In Vitro Proliferation…………………………………………………………………. PHGDH Is Dispensable for MDA-MB-231 Metastasis In Vitro……………….

SiRNA Suppression of PHGDH in MDA-MB-231 Cells Has No Effect On Motility or Migration……………. Loss of PHGDH Does Not Affect HCC1806 Proliferation……………………. Loss of PHGDH Does Not Suppress HCC1806 Motility or Migration Potential…………………………………………………………………………… 58 19. PSAT1 Silencing Inhibits MDA-MB-231 Experimental Metastasis….

Suppression of PSAT1 Does Not Affect Anchorage-Independent Growth of MDA-MB-231 Cells………………………………………………………………. Exogenous Serine Does Not Rescue PSAT1 Suppression on Cell Motility………………………………………………………………………. Exogenous Serine Does Not Rescue Effect of PSAT1 Suppression on Cell Migration…………………………………………………………………. PSAT1 and TIMP2 are Binding Partners……………………………………….

Clinical Relevance of PSAT1 in Tamoxifen treated ER+BC. Differential SSP Expression Between Sensitive and Resistant Cell Types. Metabolomic Differences Between Endocrine Sensitive and Endocrine Resistant Cell Types……………………………………………………………. Overexpression of PSAT1 Reduces Sensitivity to 4-Hydroxytamoxifen…… 86 28.

Suppression of PSAT1 Increases Sensitivity to 4-Hydroxytamoxifen……… 87 x 29. Clinical Relevance of PHGDH in ER+ Patients Treated with Tamoxifen…. Suppression of PHGDH Increases to 4-Hydroxytamoxifen…………………. Correlation Between SSP Enzymes and Relapse-Free Survival and In Vitro Sensitivity to Endocrine Therapy is Selective for Tamoxifen…………….

Model of SSP Contribution to Endocrine Resistance………………………… 96 xi CHAPTER 1 INTRODUCTION Breast cancer is the most common malignancy in U. women, constituting 30% of all new cancer diagnoses. As the second leading cause of cancer-related deaths in women [1], it comprised 6.8% of all cancer related mortalities in 2016 [2]. The lifetime risk for a woman developing breast cancer is currently 1 in 8 or about 12% [2].

It is estimated that 268,600 women will be diagnosed with invasive breast cancer while another 62,930 will be diagnosed with non-invasive breast cancer this year [1]. At the beginning of 2019 there were in excess of 3.1 million women in the U. with a history of breast cancer [1]. These women though all diagnosed with breast cancer, do not all have the same disease.

Breast cancer has been classified into 21 different histological subtypes that can be categorized into four main categories based on their hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status [3]. The four main subtypes are luminal A (HR+/HER2-), luminal B (HR+/HER2+), triple negative (HR-/HER2-), and HER2 enriched (HR-/HER2+) [3]. These all have different clinical presentations, oncogenic drivers, treatment options, affected patient populations, and confer different patient prognoses [3]. This dissertation will focus on two of these subtypes; triple negative breast cancer (TNBC) and luminal A, which will be referred to as estrogen receptor positive breast cancer (ER+BC).

1 TNBC is characterized by lack of estrogen receptor-α (ERα), progesterone receptor (PR), and HER2 expression [4]. This molecular subtype accounts for 10% to 20% of all breast cancer cases [3,5]. It is often considered to be the most aggressive with the majority of these tumors being high grade at time of diagnosis and basal-like in their appearance [6]. This aggressiveness is especially alarming considering the patient populations most commonly diagnosed with this form of cancer.

TNBC is most common among women under 50 years of age, among women that are African American or Hispanic, and among women that harbor breast cancer gene 1 (BRAC1) mutations [3,7,8] (Figure 1). The aggressiveness of TNBC could be due to its heterogeneous nature and the limited treatment options, given that targeted therapies for breast cancer primarily focus on ERα and HER2. TNBCs are currently treated with a combination of surgery, chemotherapy and radiation [3]. Given the propensity of TNBC to be of a higher grade and more aggressive, there is an increased likelihood of metastasis within this subtype [5].

ER+BC is the most common type of breast cancer observed in the clinic, accounting for approximately 60% to 75% of all breast cancer diagnoses [9]. These tumors express ERα and their growth and proliferation is driven by estrogen [10]. ER+ tumors are typically slow-growing and found in older patients, as most patients with ER+BC are post-menopausal [7]. This subtype also has the best prognosis, especially at early stages, due to targeted therapies that have been developed [3] (Figure 1).

These therapies target different aspects of estrogen processing and can be classified into three major categories: aromatase inhibitors, selective estrogen receptor modulator (SERM), and selective estrogen receptor 2 Figure 1: General subtypes of Breast Cancer. This schematic illustrates patient outcomes according to breast cancer subtypes. Adapted from Dai et al, 2015 [8]. Abbreviations: Estrogen Receptor (ER), Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2), Triple Negative Breast Cancer (TNBC), Estrogen Receptor Positive Breast Cancer (ER+BC).

The different categories of endocrine therapy all target estrogen but do so via several different mechanisms (Figure 2). Aromatase Inhibitors, such as letrozole, prevent the conversion of androgens to estrogens [11]. Tamoxifen is a SERM and its primary mechanism of action is through competitive binding against estrogens for the estrogen receptor resulting in the inhibition of the estrogen driven pro-proliferative transcription program [12,13]. Fulvestrant binds to the estrogen receptor and attenuates estrogen signaling through blocking estrogen binding and increased ER protein degradation [12].

Tamoxifen was the first of these targeted therapies and became the standard first-line therapy in the 1990s [9]. Since its introduction, the mortality rates associated with breast cancer have decreased significantly [3]. Much of this trend has been attributed to Tamoxifen, and its later designated replacements, that target estrogen signaling in ER+ tumors as well as improvements in detection methods [3]. With these advances in therapies and detection techniques, the current overall survival rate for female breast cancer has improved and is approaching 90%; however, metastatic disease, where the overall 5-year survival rate is 22% [2] is still an overwhelming problem.

Metastases or advanced recurrent disease will develop in approximately 30% of patients despite treatment with standard and advanced therapies [14] and is responsible for 90% of breast cancer-related deaths [15]. Metastasis occurs when cells disseminate from the primary tumor site 4 Figure 2: Endocrine Therapy Mechanisms of Action. This schematic shows the mechanisms of action for common endocrine therapies. Aromatase inhibitors inhibit the conversion of testosterone to estrogen.

Selective estrogen receptor modulators (SERMs), represented by Tamoxifen, compete with estrogen for binding to the estrogen receptor and block estrogen signaling. Selective estrogen receptor degraders, represented by Fulvestrant, inhibit estrogen signaling and unlike SERMs also degrade the estrogen receptor. Adapted from Schmid 2017 [9]. 5 and populate a distant site.