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University of Massachusetts Medical School

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GSBS Dissertations and Theses

2017-12-12

Graduate School of Biomedical Sciences

RUNX1 Control of Mammary Epithelial and Breast Cancer Cell Phenotypes

Deli Hong

University of Massachusetts Medical School

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Hong D. (2017). RUNX1 Control of Mammary Epithelial and Breast Cancer Cell Phenotypes. GSBS Dissertations and Theses. https://doi.org/10.13028/M21Q2F. Retrieved from

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RUNX1 CONTROL OF MAMMARY EPITHELIAL AND BREAST CANCER
CELL PHENOTYPES

A Dissertation Presented
By
Deli Hong

Submitted to the Faculty of the
University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY
December 12, 2017
Program of Cell Biology
RUNX1 CONTROL OF MAMMARY EPITHELIAL AND BREAST CANCER CELL
PHENOTYPES

A Dissertation Presented By
Deli Hong

This work was undertaken in the Graduate School of Biomedical Sciences
Program of Cell Biology
The signature of the Thesis Advisor signifies validation of Dissertation content

Gary S. Stein, Ph. D., Thesis Advisor
The signatures of the Dissertation Defense Committee signify completion and approval as to style and content of the Dissertation

Leslie Shaw, Ph. D., Member of Committee Kendall Knight, Ph. D., Member of Committee Jeffery Nickerson, Ph. D., Member of Committee Robert Weinberg, Ph. D., Member of Committee
The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee

Hong Zhang, Ph.D., Chair of Committee
The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the School.

Anthony Carruthers, Ph.D., Dean of the Graduate School of
Biomedical Sciences

December 12, 2017

Acknowledgements

Foremost, I would like to express my deepest gratitude to my thesis advisor Prof. Gary Stein, Dr. Jane Lian and Dr. Janet Stein for their continuous guidance and generous support throughout my doctoral study, without which this experience would not have been the positive experience it was. They always support me to pursue my own scientific interests in the lab. This great experience working in their lab helped me to mature into a well-rounded scientist.
I would like to give thanks to Jason Dobson for his guidance during the initial stage of my doctoral study doctoral study. Specific thanks go to Dr. Andrew Fritz, who is my primary collaborator in the lab. Additional thanks to Dr. Coralee Tye and Natali Page for their help in RNA-seq analysis, Joseph Boyd for his assistance with bioinformatics analysis, and Kristiaan Finstad and Morgan Czaja for their help in animal studies. Thanks to Terri Messier for her kind help in the lab. Additional thanks to all current and previous members of the Stein-Lian laboratory, Nick, Gillian, Prachi, Kaleem, Jonathan, Kirsten, Mingu, Areg, Josh, Helena, Gileade, Phil, Hai, Xuhui, Rodrigo, Jennifer, Mark, Cesear, Ryan, Alex for their help with my projects and scientific discussions.

I would like to thank my thesis research advisory committee members Dr. Leslie Shaw,
Dr. Kendall Knight, Dr. Jeffrey Nickerson, Dr. Stephen Jones and my external committee Dr. Robert Weinberg and my committee chair Dr. Hong Zhang for their time, valuable input and support for my graduate studies and career development.
I would like to extend my gratitude to my beloved parents Dr. Xiuping Hong and Dr.
Yiping Sun for their unconditional love and support throughout my life. I also want to thank all of my friends for their support and company.

iii

Abstract

Breast cancer remains the most common malignant disease in women worldwide. Despite the advantages of early detection and improved treatments, studies into the

mechanisms that initiate and drive breast cancer progression are still required. Recent studies have identified RUNX1, which is an essential transcription factor for hematopoiesis, is one of the most frequently mutated genes in breast cancer patients. However, the role of RUNX1 in the mammary gland is understudied.

In this dissertation, we examined the role of RUNX1 in both normal mammary epithelial and breast cancer cells. Our in vitro studies demonstrated that RUNX1 inhibits epithelial to mesenchymal transition (EMT), migration, and invasion, reflecting its tumor suppressor activity, which was confirmed in vivo. Moreover, RUNX1 also contributes significantly to inhibition of the phenotypes of breast cancer stem cells (CSC), which is responsible for metastasis and tumor relapse. We showed that Runx1 overexpression reduces the tumorsphere formation and cancer stem cell population. Overall, our studies provide mechanistic evidence for RUNX1 repression of EMT in mammary cells, anti-tumor activity in vivo and regulation of CSC-like properties in breast cancer.

Our results highlight crucial roles for RUNX1 in preventing epithelial to mesenchymal transition and tumor progression in breast cancer. This RUNX1 mediated mechanism points to novel intervention strategies for early stage breast cancer.

iv

Table of Contents

Acknowledgements……………………………………….…………….……..……………iii Abstract………………………………………………………………….……...……….…...iv Table of Contents...…………………………………………………………………............v List of Figures……………………………………………………………...…………......….x List of Tables…………………………………………………..…………………….……..xiii List of Symbols and Abbreviations………..……..…………………..……………….….xiv Preface……………………………………………….……………………………………..xvi CHAPTER I. Introduction…………………………………...…………………………...1-52

  • 1.1
  • The Introduction of Breast Cancer …………………………………………………1

  • 1.1.1
  • Breast cancer overview………………………………………...……………1

Breast cancer molecular subtypes………………………………………....1 The origin of breast cancer and breast cancer subtypes………..………7 Cell line models used in breast cancer study………….........…………..11
1.1.2 1.1.3 1.1.4

  • 1.2
  • The Runx Family………………………………………………..……………....….12

  • 1.2.1
  • Runx family overview……………………………..………...…………......12

Structure of Runx………………………………..………………………....14 Evolutionary role of Runx………………………..………………………...17
1.2.2 1.2.3

  • 1.3
  • The Runx Family and Development in Mammals…….........…………………..20

  • 1.3.1
  • Overview……………………………………….……………………………20

Runx1………………………………………………..……………………….21 Runx2……………………………………………….……………………….24 Runx3………………………………………………….…………………….26
1.3.2 1.3.3 1.3.4

v

1.4 1.5 1.6
The Runx Family in Disease and Cancer…………………….………………….27

  • 1.4.1
  • Overview………………………………………….…………………………27

Runx1………………………………………….…………………………….28 Runx2 and Runx3…………………………………………………....……..31
1.4.2 1.4.3
Runx1 and Mammary Gland Development and Breast Cancer……………....32

  • 1.5.1
  • Mammary gland development and hierarchy……………………………32

Runx1 and mammary gland development…………………...…………..33 Runx1 and breast cancer………….…………………………...………….34
1.5.2 1.5.3
Epithelial Mesenchymal Transition in Breast Cancer…………..………………38

  • 1.6.1
  • Overview of epithelial mesenchymal transition …………..…………….38

Epithelial mesenchymal transition in development…………..…………39 Epithelial mesenchymal transition in cancer………………….…………41 Runx and EMT………………………………………………….…………..43
1.6.2 1.6.3 1.6.4

  • 1.7
  • Cancer Stem Cell and Breast Cancer……………..………………..……………45

  • 1.7.1
  • Intra-tumor heterogeneity………………………………….………………45

Cancer stem cells………………………………………….……………….47 EMT and plasticity and cancer stem cells……………………....……….49
1.7.2 1.7.3
1.8 Rationale for the Dissertation………………………………………..…………….…50 CHAPTER II. Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition……………………………...………53-101 2.1 Abstract……………………………………………………….………….……….…….54 2.2 Introduction…………………………………………….….……………….…………...55 2.3 Materials and Methods……………………………….……….……….….………..…58

vi

2.4 Results……………………………………………………………..…………....…...…66
2.4.1 Runx1 expression is decreased in breast cancer …………..……...….….…66 2.4.2 TGFβ induced EMT decreases Runx1 expression in MCF10A cells …..….69 2.4.3 Runx1 reverses the TGFβ-induced EMT phenotype……………..….….……72 2.4.4 Decreased expression of Runx1 during TGFβ independent EMT in MCF10A cells…………………………………………..………………..................…..……..74
2.4.5 Gene expression profiling of growth factor-depleted MCF10A cells reveals the spectrum of EMT markers………………………………………..…………..……77
2.4.6 Directly Depleting Runx1 in MCF10A cells results in loss of epithelial morphology and activation of EMT.………………….……………………………….……....…82
2.4.7 Depleting Runx1 in MCF7 breast cancer cells promotes EMT.…...….….…88 2.4.8 Overexpressing Runx1 in mesenchymal like breast cancer cells drives mesenchymal to epithelial transition (MET).…….……………………...…….…89
2.4.9 Runx1 expression in breast tumors correlates with metastasis, tumor subtype and survival…………………………………...…….…………………...……….…92
2.5 Discussion for Chapter II…………………………………………….….……...……..96 Chapter III RUNX1 Genome-wide Regulation of Normal Mammary Epithelial Cells: Novel Functions for Mitosis and Genome Stability……………………………………....102-146 3.1 Introduction……………………………………………...……………….……………106 3.2 Materials and Methods……………………………………………..…….....……….106 3.3 Results…………………………………………………………….………………......111
3.3.1. RUNX1 knockdown in normal-like mammary epithelial cells results in aberrant gene regulation …………………………………………………………….……..111

vii

3.3.2. Runx1 ChIP-seq analysis identifies enriched binding at promoters……...119 3.3.3. Runx1 binds to up- or down-regulated genes ………………………...…..122 3.3.4. Loss of RUNX1 affects cell cycle-related genes ……………..……….......127 3.3.5. Loss of RUNX1 decreases the proportion of mitotic cells……..……….…130 3.3.6. Loss of RUNX1 decreases genomic stability…………………….………..134
3.4 Discussion for Chapter III……………………………………………….…………...136 Chapter IV RUNX1 suppresses breast cancer stemness and tumor growth.…147-194 4.1 Abstract…………………………………………………………………….…...……..148 4.2 Introduction……………………………………………………………….…...………149 4.3 Materials and Methods…………………………………………………….....…..….152 4.4 Results……………………………………………………………………..……….....160
4.4.1. Reduced RUNX1 expression is associated with decreased survival probability in breast cancer patients ………………………………………………...………160
4.4.2. RUNX1 is decreased in tumors formed in mouse mammary fat pad…....165 4.4.3. RUNX1 reduces the aggressive phenotype of breast cancer cells……....168 4.4.4. RUNX1 represses tumor growth in vivo ……………..…………….............172 4.4.5. RUNX1 level is decreased in breast cancer stem cells (BCSC).……..….176 4.4.6. RUNX1 inhibits stemness properties in breast cancer cells……………..181 4.4.7. RUNX1 represses the expression of Zeb1 in breast cancer cells.…..…..185
4.5Discussion for Chapter IV………………………. …………………………...……..190 Chapter V Discussion and future direction….….………………...…….…………195-212 5.1 Results summary………………………………………………...…….……………..195 5.2 Significant and clinical impact …………………………………………..…………..197

viii

5.3. Open questions and future directions…..……..…………………………………..199

5.4. Concluding Remarks……………………………………….………………………………..212

Bibliography……………………………………………….……………………………….213

ix

List of Figures

Figure 1.1 Schematic model of mammary epithelial hierarchy and potential relationship with breast tumor subtypes. ………………………….….….……..………10 Figure 1.2 Structure of the CBF-b: Runt domain: DNA complex. ……..…….………..16 Figure 2.1. Decreased Runx1 expression is related to breast cancer progression in cell models…………………………...………………………………………………………..…68 Figure 2.2. Runx1 decreases during TGFβ-induced EMT. MCF10A cells treated with 10 ng/ml TGFβ for 6 days. ……………………………………………..………………….....71 Figure 2.3. Runx1 reverses TGFβ induced EMT ……………………………...….....…74 Figure 2.4. Decreased Runx1 during TGFβ-independent EMT ………………………76 Figure 2.5. RNA-Seq reveals MCF10A cells undergo EMT upon growth factor removal. ………………...…………………………………………………………………………......80 Figure 2.6. Increased Runx2 during growth factor depleted induced EMT. ………………………………...……………………………….……...……………………..82 Figure 2.7. Depleting Runx1 in MCF10A cells promotes a mesenchymal-like phenotype.………………………………………………………………………..….……...84 Figure 2.8. . Schematic diagram of ChIP qPCR primers and amplicons over the tested gene for ChIP-qPCR.……………… ……………………………………………………...87 Figure 2.9. Runx1 consensus sequences in CDH1 are coincident with H3K4Ac peaks in MCF10A cells ………………………………..……………………………………………..88 Figure 2.10. Runx1 controls EMT-MET in non-metastatic breast cancer cells. …………………………………….………………………………………………………….90

x

Figure 2.11. Runx1 expression in breast tumors correlates with metastasis, tumor subtype and survival …………………………………………………………………………...……93 Figure 2.12 Runx1 tissue microarray show that Runx1 is associated with early stage tumor …………………………………………………………………...…………………...95 Figure 3.1 RNA-seq in RUNX1 depleted MCF10A cells. …………………….………113 Figure 3.2 The expression of mesenchymal genes is increased in RUNX1 depleted MCF10A cells……………………………………………………………..………..……...115 Figure 3.3 Defining differentially expressed genes in RUNX1 knockdown in MCF10A cells showing in Venn diagram. ………………………………………………………...116 Figure 3.4 IPA canonical pathway analyses from each tier of core analysis. ………………………………………………………………………………………………118 Figure 3.5 RUNX1 ChIP-seq in parental MCF10A cells. ………………………....….121 Figure 3.6 RUNX1 regulates up- and down- regulated genes in a different pattern.123 Figure 3.7 HOMER de novo motif analysis of the RUNX1 peaks in un-differentially expresses genes…………………………………………………………………………..126 Figure 3.8 RUNX1 alters the expression of cell cycle genes……….. ……………....129 Figure 3.9 Loss of RUNX1 reduces the mitotic population. ………………….………131 Figure 3.10 Runx1 is a direct regulator of Bub1b, MAD2L1 and APC…………..…..133 Figure 3.11 Loss of Runx1 slows DNA repair………………………………………….135 Figure 3.12 Runx1 is a direct regulator of Bub1b, MAD2L1 and APC………………142 Figure 3.13 Possible mechanisms of Runx1-controlled mitotic entry………………..146 Figure 4.1. Reduced RUNX1 expression is associated with decreased survival probability in breast cancer patients………………………………………………………………….162

xi

Figure 4.2. RUNX1 mRNA is decreased during breast cancer progression..……..164 Figure 4.3. RUNX1 is decreased in tumors formed in mouse mammary fat pad….166 Figure 4.4. RUNX1 reduces the aggressive phenotype of breast cancer cells………… in vitro………………………………………………………………………………...……169 Figure 4.5. RUNX1 overexpression does not change cell proliferation……………..171 Figure 4.6. RUNX1 represses tumor growth in vivo…………………………………..174 Figure 4.7. RUNX1 represses tumor growth in mammary fat pad………………….175 Figure 4.8. Gate for MCF10AT1 sorting and MCF10CA1a cells have high BCSC population………………………………………………………………………………….177 Figure 4.9. RUNX1 level is decreased in BCSC……………………………………...178 Figure 4.10. CD24high Cells have high RUNX1 expression in MCF10AT1 cells.…180 Figure 4.11. Loss of RUNX1 promotes stemness in MCF10A and MCF7 cells……182 Figure 4.12. RUNX1 reduces BCSC sub-population………………………………….184 Figure 4.13. Overexpression RUNX1 in MCF10CA1a cells does not change BCSC population………………………………………………………………………………….185 Figure 4.14. RUNX1 negatively regulates Zeb1 expression…………………………187 Figure 4.15. Zeb1 is expressed at low level in MCF10CA1a cells…………………..189 Figure 4.16. Schematic diagram of ChIP qPCR primers and amplicons over Zeb1 for ChIP-qPCR………………………………………………………………………………...190 Figure 5.1 Potential Runx1 regulators locate within 1kb upstream of Runx1 promoter…………………………………………………………………………………....202 Figure 5.2 Heat map of changes in ribosome protein mRNAs……………………….205

xii

List of Tables

Table 1.1 Features of molecular subtypes of breast cancer..………………… ............3 Table 5.1. Table 5.1 List of LncRNAs which expression is changed upon Runx1 knockdown in MCF10A cells and their involvement in human breast cancer.…………………………………………………………….……...…………..……211

xiii

List of Symbols and Abbreviations

Acute myeloid leukemia (AML) Acute lymphoblastic leukemia (ALL) Aldehyde dehydrogenase (ALDH) Anaphase-promoting complex (APC) complex Breast cancer stem cell (BCSC)

Caenorhabditis elegans (C.elegans)

Cancer stem cell (CSC) Circulating tumor cell (CTC) Cleidocaranial dysplasia (CCD) Core-binding factor b (CBFb) Cyclin-dependent kinase 1 (CDK1)

Drosophila melanogaster (Dm)

Ductal carcinoma in situ (DCIS) Endothelial to hematopoietic transition (EHT) Epidermal growth factor (EGF) Epithelial-mesenchymal transition (EMT) Estrogen receptor (ER) Fibroblast-specific protein 1 (Fsp1) Hematopoietic stem cells (HSCs)

xiv

Hepatocyte growth factor (HGF) Human epidermal growth factor receptor 2 (HER2) Integrin-like kinase (ILK) Invasive ductal carcinoma (IDC) Long noncoding RNAs (lncRNAs) Mammary stem cells (MSC) Mesenchymal to epithelial transition (MET) Mitotic checkpoint complex (MCC) Myelodysplastic syndrome (MDS) Nervy homology regions (NHR) Polyoma enhancer-binding protein-2α (PEBP2α) Progesterone receptor (PR) Propidium iodide (PI) Standard error of the mean (SEM)

Strongylocentrotus purpuratus (Sp)

Sub-nuclear matrix-targeting signal (NMTS) The Cancer Genome Atlas (TCGA) Transcripts per million (TPM) Transforming growth factor beta (TGF-b) Triple-negative breast cancer (TNBC)

xv

Preface Portions of this thesis have appeared in the following published works: Chapter II:

Hong D, Messier TL, Tye CE, Dobson JR, Fritz AJ, Sikora, KR, Browne G, Stein JL, Lian JB, Stein GS

Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition. Oncotarget. 2017; 8:17610-17627

Other published work during graduate study that are not presented in this thesis:

1. Zaidi SK, Frietze SE, Gordon JA, Heath JL, Messier TL, Hong D, Boyd JR, Kang M, Imbalzano AN, Lian JB, Stein JL, Stein GS

Bivalent Epigenetic ControlD of Oncofetal Gene Expression in Cancer. Molecular and Cellular Biology 2017 In press PMID: 28923849

2. Fritz AJ, Ghule PN, Boyd JR, Tye CE, Page NA, Hong D, Weinheimer AS, Barutcu AR, Gerrard DL, Frietze SE, Zaidi SK, Imbalzano AN, Lian JB, Stein JL, Stein GS

Intranuclear and higher‐order chromatin organization of the major histone gene cluster in breast cancer Journal of cellular physiology 2017 In press PMID: 28504305

3. Barutcu AR, Hong D, Lajoie BR, McCord RP, van Wijnen AJ, Lian JB, Stein JL, Dekker J, Imbalzano AN, Stein GS

RUNX1 associates with TAD boundaries and organizes higher order chromatin

structure in breast cancer cells. Biochimica et Biophysica Acta (BBA) - Gene Regulatory

Mechanisms 2016; 1859(11): 1389 4. Dobson JR, Hong D, Barutcu AR, Hai W, Anthony N Imbalzano, Lian JB, Stein JL, van Wijnen AJ, Nickerson JA, Stein GS

Isolation and characterization of nuclear matrix-associated DNA in breast cancer cell lines. J Cell Physiol. 2017;232(6):1295.

5. Browne G, Dragon JA, Hong D, Messier TL, Gordon JA, Farina NH, Boyd JR, VanOudenhove JJ, Perez AW, Zaidi SK, Stein JL, Stein GS, Lian JB

MicroRNA-378-mediated suppression of Runx1 alleviates the aggressive phenotype of triple-negative MDA-MB-231 human breast cancer cells. Tumour Biol. 2016; 37(7): 8825

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  • Characterization and Assay of Progesterone Receptor in Human Mammary Carcinoma1

    Characterization and Assay of Progesterone Receptor in Human Mammary Carcinoma1

    [CANCER RESEARCH 37, 464-471 , February 1977] Characterization and Assay of Progesterone Receptor in Human Mammary Carcinoma1 M. F. Pichon and E. Milgrom Groupe de Recherches sur Ia Biochimie Endocrinienne et Ia Reproduction (INSERM U.135), FacultédeMédecine,Paris-Sud, 94270 Bicétre,France ever, only some patients are improved by such treatments. It SUMMARY would thus be of great practical importance to predict in advance which patient will respond and to nationalize the [3H]Pregn-4-ene-3,20-dione ([3Hjpmogestenane)-recepton choice of the surgical or pharmacological technique to be complexes from human mammary carcinoma were found to used. During the last 15 years, the detection and quantifica be stabilized in the presence of glycerol. The dissociation tion of estrogen receptors in mammary tumors have allowed rate constant was lowered and the equilibrium dissociation marked progress in this direction (11, 12, 16, 18, 21, 22, 34). constant was decreased (KD = 3 nM in the absence of However, the growth of mammary carcinoma may be can glycerol and 1.1 nM in the presence of 30% glycerol), traIled not only by estrogens but also by other hormones whereas no clean-cut effect on the association rate was including observed and no change occurred in the concentration andragens, glucacorticaids, pragestagens , and of binding sites. Gortisol was found to compete with prolactin (13). The study of specific receptors for these [3Hjpmagesterone only at concentrations higher than 1 @M. hormones should shed some bight on the problem of tumor This made it possible to distinguish [3H]pmogestemanebind hormonal dependence . The characterization and measu me ing to the receptor from binding to carticosteroid-binding ment of progesterone receptors have been hampered by the globulin.
  • Genetic Alterations of Histone Lysine Methyltransferases and Their Significance in Breast Cancer

    Genetic Alterations of Histone Lysine Methyltransferases and Their Significance in Breast Cancer

    www.impactjournals.com/oncotarget/ Oncotarget, Vol. 6, No.4 Genetic alterations of histone lysine methyltransferases and their significance in breast cancer Lanxin Liu1,*, Sarah Kimball1,*, Hui Liu1, Andreana Holowatyj1 and Zeng-Quan Yang1 1 Department of Oncology, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA * These authors contributed equally to this work Correspondence to: Zeng-Quan Yang, email: [email protected] Keywords: breast cancer, histone lysine methyltransferase, gene amplification, deletion, mutation Received: August 27, 2014 Accepted: December 10, 2014 Published: December 11, 2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Histone lysine methyltransferases (HMTs), a large class of enzymes that catalyze site-specific methylation of lysine residues on histones and other proteins, play critical roles in controlling transcription, chromatin architecture, and cellular differentiation. However, the genomic landscape and clinical significance of HMTs in breast cancer remain poorly characterized. Here, we conducted a meta-analysis of approximately 50 HMTs in breast cancer and identified associations among recurrent copy number alterations, mutations, gene expression, and clinical outcome. We identified 12 HMTs with the highest frequency of genetic alterations, including 8 with high-level amplification, 2 with putative homozygous deletion, and 2 with somatic mutation. Different subtypes of breast cancer have different patterns of copy number and expression for each HMT gene. In addition, chromosome 1q contains four HMTs that are concurrently or independently amplified or overexpressed in breast cancer. Copy number or mRNA expression of several HMTs was significantly associated with basal- like breast cancer and shorter patient survival.
  • Diagnostic Value of Progesterone Receptor and P53 Expression in Uterine Smooth Muscle Tumors Iman H Hewedi*, Nehal a Radwan and Lobna S Shash

    Diagnostic Value of Progesterone Receptor and P53 Expression in Uterine Smooth Muscle Tumors Iman H Hewedi*, Nehal a Radwan and Lobna S Shash

    Hewedi et al. Diagnostic Pathology 2012, 7:1 http://www.diagnosticpathology.org/content/7/1/1 RESEARCH Open Access Diagnostic value of progesterone receptor and p53 expression in uterine smooth muscle tumors Iman H Hewedi*, Nehal A Radwan and Lobna S Shash Abstract Background: The diagnosis of uterine smooth muscle tumors depends on a combination of microscopic features. However, a small number of these tumors still pose difficult diagnostic challenges. Aim: To investigate progesterone receptor (PR) and p53 expression in leiomyomas (LMs), atypical leiomyomas (ALMs), smooth muscle tumors of uncertain malignant potential (STUMP), and leiomyosarcomas (LMSs) and to evaluate the potential utility of the selected immunohistochemical markers in differentiating these tumors. Materials and methods: Immunohistochemical expression of PR and p53 was investigated in 41 uterine smooth muscle tumors comprising: 15 LMS, 4 STUMP, 6 ALM and 16 LM. Quantitative evaluation of PR and p53 expression was graded on a scale from 0 to 3+. Results: Leiomyosarcomas showed reduced PR expression. All LMs as well as ALMs and STUMP were stained intensely for PR. Conversely, LMS was strongly stained with p53, while the three non-sarcomatous groups (STUMP, ALM, LM) were either entirely negative or weakly stained for p53. Regarding both PR and p53 expression, the difference between the LMS group and the three non-sarcomatous groups was highly significant (p < 0.001). Combined high PR - low p53 expression was seen in all the 26 examined cases of the non-sarcomatous group including the STUMP cases and none of the LMS cases. Therefore, it represents a “benign” profile with 100% specificity in diagnosis of a non-sarcomatous tumor.
  • Progesterone Receptor-A Isoform Interaction with RUNX Transcription Factors Controls

    Progesterone Receptor-A Isoform Interaction with RUNX Transcription Factors Controls

    bioRxiv preprint doi: https://doi.org/10.1101/2021.06.17.448908; this version posted June 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Progesterone receptor-A isoform interaction with RUNX transcription factors controls 2 chromatin remodelling at promoters during ovulation 3 Authors: Dinh DT 1 (*), Breen J 2, Nicol B 3, Smith KM 1, Nicholls M 1, Emery A 1, 4 Wong YY 1, Barry SC 1, Yao HHC 3, Robker RL 1, Russell DL 1 (*) 5 Affiliations: 6 1. Robinson Research Institute, Adelaide Medical School, University of Adelaide, Australia 7 2. South Australian Genomics Centre (SAGC), South Australian Health and Medical 8 Research Institute (SAHMRI), Adelaide, South Australia, Australia 9 3. Reproductive Developmental Biology Group, National Institute of Environmental Health 10 Sciences, Research Triangle Park, NC 27709, USA 11 12 Summary: 13 Progesterone receptor (PGR) plays diverse roles in reproductive tissues and thus coordinates 14 mammalian fertility. In the ovary, acutely induced PGR is the key determinant of ovulation 15 through transcriptional control of a unique set of genes that culminates in follicle rupture. 16 However, the molecular mechanisms for PGR’s specialised function in ovulation is poorly 17 understood. To address this, we assembled a detailed genomic profile of PGR action through 18 combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR 19 null mice.
  • Maintenance of Mammary Epithelial Phenotype by Transcription Factor Runx1 Through Mitotic Gene Bookmarking Joshua Rose University of Vermont

    Maintenance of Mammary Epithelial Phenotype by Transcription Factor Runx1 Through Mitotic Gene Bookmarking Joshua Rose University of Vermont

    University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2019 Maintenance Of Mammary Epithelial Phenotype By Transcription Factor Runx1 Through Mitotic Gene Bookmarking Joshua Rose University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Biochemistry Commons, and the Genetics and Genomics Commons Recommended Citation Rose, Joshua, "Maintenance Of Mammary Epithelial Phenotype By Transcription Factor Runx1 Through Mitotic Gene Bookmarking" (2019). Graduate College Dissertations and Theses. 998. https://scholarworks.uvm.edu/graddis/998 This Thesis is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. MAINTENANCE OF MAMMARY EPITHELIAL PHENOTYPE BY TRANSCRIPTION FACTOR RUNX1 THROUGH MITOTIC GENE BOOKMARKING A Thesis Presented by Joshua Rose to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Master of Science Specializing in Cellular, Molecular, and Biomedical Sciences January, 2019 Defense Date: November 12, 2018 Thesis Examination Committee: Sayyed Kaleem Zaidi, Ph.D., Advisor Gary Stein, Ph.D., Advisor Seth Frietze, Ph.D., Chairperson Janet Stein, Ph.D. Jonathan Gordon, Ph.D. Cynthia J. Forehand, Ph.D. Dean of the Graduate College ABSTRACT Breast cancer arises from a series of acquired mutations that disrupt normal mammary epithelial homeostasis and create multi-potent cancer stem cells that can differentiate into clinically distinct breast cancer subtypes. Despite improved therapies and advances in early detection, breast cancer remains the leading diagnosed cancer in women.
  • Progesterone Receptor Transcription and Non-Transcription Signaling Mechanisms Susan A

    Progesterone Receptor Transcription and Non-Transcription Signaling Mechanisms Susan A

    Steroids 68 (2003) 761–770 Progesterone receptor transcription and non-transcription signaling mechanisms Susan A. Leonhardt a, Viroj Boonyaratanakornkit a, Dean P. Edwards b,∗ a Department of Pathology B216, School of Medicine University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Campus Box B216, Denver, CO 80262, USA b Department of Pathology and Program in Molecular Biology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Campus Box B216, Denver, CO 80262, USA Abstract The diverse effects of progesterone on female reproductive tissues are mediated by the progesterone receptor (PR), a member of the nuclear receptor family of ligand-dependent transcription factors. Thus, PR is an important therapeutic target in female reproduction and in certain endocrine dependent cancers. This paper reviews our understanding of the mechanism of action of the most widely used PR antagonist RU486. Although RU486 is a competitive steroidal antagonist that can displace the natural hormone for PR, it’s potency derives from additional “active antagonism” that involves inhibiting the activity of PR hormone agonist complexes in trans through heterodimerization and competition for binding to progesterone response elements on target DNA, and by recruitment of corepressors that have the potential to actively repress gene transcription. An additional functional role for PR has recently been defined whereby a subpopulation of PR in the cytoplasm or cell membrane is capable of mediating rapid progesterone induced activation of certain signal transduction pathways in the absence of gene transcription. This paper also reviews recent results on the mechanism of the extra-nuclear action of PR and the potential biological roles and implications of this novel PR signaling pathway.
  • (NGS) for Primary Endocrine Resistance in Breast Cancer Patients

    (NGS) for Primary Endocrine Resistance in Breast Cancer Patients

    Int J Clin Exp Pathol 2018;11(11):5450-5458 www.ijcep.com /ISSN:1936-2625/IJCEP0084102 Original Article Impact of next-generation sequencing (NGS) for primary endocrine resistance in breast cancer patients Ruoyang Li1*, Tiantian Tang1*, Tianli Hui1, Zhenchuan Song1, Fugen Li2, Jingyu Li2, Jiajia Xu2 1Breast Center, Fourth Hospital of Hebei Medical University, Shijiazhuang, China; 2Institute of Precision Medicine, 3D Medicines Inc., Shanghai, China. *Equal contributors. Received August 15, 2018; Accepted September 22, 2018; Epub November 1, 2018; Published November 15, 2018 Abstract: Multiple mechanisms have been detected to account for the acquired resistance to endocrine therapies in breast cancer. In this study we retrospectively studied the mechanism of primary endocrine resistance in estrogen receptor positive (ER+) breast cancer patients by next-generation sequencing (NGS). Tumor specimens and matched blood samples were obtained from 24 ER+ breast cancer patients. Fifteen of them displayed endocrine resistance, including recurrence and/or metastases within 24 months from the beginning of endocrine therapy, and 9 pa- tients remained sensitive to endocrine therapy for more than 5 years. Genomic DNA of tumor tissue was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks. Genomic DNA of normal tissue was extracted from peripheral blood mononuclear cells (PBMC). Sequencing libraries for each sample were prepared, followed by target capturing for 372 genes that are frequently rearranged in cancers. Massive parallel
  • Expression of Human Estrogen Receptor

    Expression of Human Estrogen Receptor

    Proc. Natl. Acad. Sci. USA Vol. 96, pp. 5722–5727, May 1999 Medical Sciences Expression of human estrogen receptor-a and -b, progesterone receptor, and androgen receptor mRNA in normal and malignant ovarian epithelial cells (mutationysequence deletionyestrogen actionyandrogen actionyprogesterone action) KIN-MANG LAU*, SAMUEL C. MOK†, AND SHUK-MEI HO*‡ *Department of Biology, Tufts University, Medford, MA 02155; and †Laboratory of Gynecological Oncology, Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115 Communicated by Elwood V. Jensen, Karolinska Institute, Huddinge, Sweden, March 15, 1999 (received for review September 15, 1998) b a ABSTRACT Our understanding of the roles played by sex estradiol-17 (E2), progesterone, 20 -hydroxyprogesterone, hormones in ovarian carcinogenesis has been limited by a lack testosterone, and androstenedione have all been shown to of data concerning the mode of sex hormone action in human correlate with OC tumor masses (9–11). Taken together, these ovarian surface epithelial (HOSE) cells, the tissue of origin of findings suggest that steroid hormones are likely involved in >90% of ovarian cancers. We have compared the relative the genesis and progression of the disease, yet their mecha- abundance of estrogen receptor (ER)a,ERb, progesterone nisms of action remain unclear. receptor (PR), and androgen receptor (AR) mRNA in four The classical estrogen receptor (ER), recently renamed ERa primary cultures of HOSE cells obtained from postmeno- (12), and the progesterone receptor (PR) were found in ,50% pausal women to those found in late serous adenocarcinoma of OC specimens, whereas androgen receptor (AR) was de- primary cell cultures and established ovarian cancer cell lines.