ERalpha isoforms modulate the tumorigenicity of 24R,25(OH)2D3 in estrogen-responsive cancer

Abstract

....................................................................................................................... 17 Chapter 1. ..................................................................................................................... 19 Introduction .........................................................................................................................19 Specific Aim 1: Determine the estrogen receptor profile and estrogen responsiveness in laryngeal cancer and clinical outcomes. ............................................................................24 Specific Aim 2: Determine if 24R,25-Dihydroxyvitamin D3 regulates breast cancer cells in vitro and in vivo. ................................................................................................................25 Specific Aim 3: Determine if 24R,25(OH)2D3 differentially regulates tumorigenicity in estrogen receptor \uf061 dependent manner. ..........................................................................................26 Chapter 2. ..................................................................................................................... 28 Background and Literature Review ...................................................................................28 Nature of steroid-hormone membrane receptors ...............................................................28 Classical nuclear receptor trafficking .................................................................................29 Estrogen Receptor α and its isoforms ................................................................................30 Estrogen and estrogen receptors in laryngeal cancer ........................................................32 Estrogen receptor signaling ...............................................................................................33 Modulation of 24R,25(OH)2D3 Tumorigenicity by ER\uf061 Isoforms in E2-Responsive Cancer • Spring 2019 Anjali Verma • Ph.D. Dissertation • Biomedical Engineering • Virginia Commonwealth University 6 Rapid Estrogen Signaling ..................................................................................................35 24R,25(OH)2D3 signaling ...................................................................................................38 Vitamin D3 supplementation in cancer. .............................................................................42 Chapter 3. ..................................................................................................................... 44 Estradiol Receptor Profile and Estrogen Responsiveness in Laryngeal Cancer and Clinical Outcomes ...............................................................................................................44 Introduction .......................................................................................................................44 Materials and Methods ......................................................................................................48 Results ..............................................................................................................................53 Discussion .........................................................................................................................62 Loss of estrogen receptors is associated with increased tumor aggression in laryngeal squamous cell carcinoma...................................................................................................69 Introduction .......................................................................................................................69 Materials and Methods ......................................................................................................72 Results ..............................................................................................................................84 Discussion .........................................................................................................................97 Chapter 5. ................................................................................................................... 108 24R,25-Dihydroxyvitamin D3 regulates breast cancer cells in vitro and in vivo ........... 108 Introduction ..................................................................................................................... 108 Materials & Methods ........................................................................................................ 112 Results ............................................................................................................................ 122 Discussion ....................................................................................................................... 140 Chapter 6. ................................................................................................................... 151 24R,25-dihydroxyvitamin D3 regulates breast cancer progression in an estrogen receptor α 66-dependent manner. .................................................................................... 151 Modulation of 24R,25(OH)2D3 Tumorigenicity by ER\uf061 Isoforms in E2-Responsive Cancer • Spring 2019 Anjali Verma • Ph.D. Dissertation • Biomedical Engineering • Virginia Commonwealth University 7 Introduction ..................................................................................................................... 151 Materials & Methods ........................................................................................................ 153 Results ............................................................................................................................ 166 Discussion ....................................................................................................................... 194 Conclusion ...................................................................................................................... 200 Chapter 7. ................................................................................................................... 201 Conclusions and Future Perspectives............................................................................. 201 References ................................................................................................................. 206 Vita .............................................................................................................................. 246 Modulation of 24R,25(OH)2D3 Tumorigenicity by ER\uf061 Isoforms in E2-Responsive Cancer • Spring 2019 Anjali Verma • Ph.D. Dissertation • Biomedical Engineering • Virginia Commonwealth University 8 List of Tables Table 3.1 Human primers used in real-time PCR analysis. ........................................... 49 Table 4.1 Laryngeal squamous cell carcinoma samples and patient demographics ..... 76 Table 4.2 Summary of clinical laryngeal squamous cell carcinoma samples. Preparation of Lysates ...................................................................................................................... 77 Table 4.3 Estrogen receptor protein expression in clinical laryngeal squamous cell carcinoma samples. ...................................................................................................... 82 Table 4.4 Xenograft models of LSCC tumor growth curve: linear fit, weeks 2-8. .......... 85 Table 4.5 Xenograft models of LSCC tumor growth curve: linear fit, weeks 4-8. .......... 86 Table 4.6 Xenograft models of LSCC tumor growth curve: exponential fit, weeks 2-8. . 89 Table 5.1 Human primers used in real-time PCR analysis. ......................................... 119 Table 6.1 Antibodies used in western blots. ................................................................ 156 Table 6.2 Gene expression primers used in quantitative real-time PCR. .................... 161 Modulation of 24R,25(OH)2D3 Tumorigenicity by ER\uf061 Isoforms in E2-Responsive Cancer • Spring 2019 Anjali Verma • Ph.D. Dissertation • Biomedical Engineering • Virginia Commonwealth University 9 List of Figures Figure 1.1 Effect of vitamin D supplementation on breast cancer recurrence in ERand ER+ breast cancer. ...................................................................................................... 19 Figure 2.1 Western blot data of ERα isoforms in breast cancer cells. ........................... 31 Figure 2.2 E2 upregulates the expression of ERα36 and ERα66 in UM-SCC-12, a laryngeal cancer cell line. .............................................................................................. 34 Figure 2.3 Mechanism of E2 signaling through ERα36. ................................................ 37 Figure 2.4 24R,25 signaling through PLD. .................................................................... 41 Figure 3.1 Estrogen receptor expression in whole cell lysates of UM-SCC-12 and UMSCC-11A cells. .............................................................................................................. 54 Figure 3.2 UM-SCC-12 and UM-SCC-11A cells were serum-starved and treated with E2 for 9 minutes. ................................................................................................................ 56 Figure 3.3 UM-SCC-12 and UM-SCC-11A cells were treated with E2 for 9 minutes. ... 57 Figure 3.4 UM-SCC-12 cells were treated with methyl-β-cyclodextrin for 30 minutes, then treated with 10-8M E2 for 9 minutes. Media were changed, and 24 hours later total intracellular p53 content was quantified with a sandwich ELISA assay. ........................ 58 Figure 3.5 E2 stimulates PKC activity in UM-SCC-12 cell layer lysates. ....................... 60 Figure 3.6 E2 stimulates PLD activity in UM-SCC-12 cell layer lysates. ....................... 61 Figure 3.7 17β-estradiol activates PKC in UM-SCC-11A cells; and PKC and PLD in UMSCC-12 cells by binding to ERα36 in the caveolae. ........................