University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-7-2016 Role of ECDYSONELESS in ERBB2/HER2 Mediated Breast Oncogenesis Shalis A. Ammons University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Biology Commons, and the Cancer Biology Commons Recommended Citation Ammons, Shalis A., "Role of ECDYSONELESS in ERBB2/HER2 Mediated Breast Oncogenesis" (2016). Theses & Dissertations. 107. https://digitalcommons.unmc.edu/etd/107 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. ROLE OF ECDYSONELESS IN ERBB2/HER2 MEDIATED BREAST ONCOGENESIS By Shalis Ammons A DISSERTATION Presented to the Faculty of the University of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Genetics, Cell Biology & Anatomy Graduate Program Under the supervision of Professor Vimla Band University of Nebraska Medical Center Omaha, Nebraska April, 2016 Supervisory Committee: Karen Gould, Ph.D Kay-Uwe Wagner, Ph.D Hamid Band, Ph.D Kaustubh Datta, Ph.D ROLE OF ECDYSONELESS IN ERBB2/HER2 MEDIATED BREAST ONCOGENESIS By Shalis Ammons University of Nebraska Medical Center, 2016 Advisor: Vimla Band, PhD Breast cancer is the second leading cause of cancer related deaths in women in the United States. The human Epidermal Growth Factor 2 (ErbB2) gene amplification and/or receptor overexpression subtype of breast cancer accounts for 25% of all breast cancers. A crucial regulator of the ErbB2 signaling pathway is the heat shock protein 90 (Hsp90) and its interacting protein complex. One such complex is the R2TP/Prefoldin-like complex that is composed of four proteins, RUVBL1, RUVBL2, PIH1D1, and RPAP3 and seven prefoldin-like proteins. This complex has been shown to be involved in telomere elongation, ribosome biogenesis, protein stability; etc. We and others have recently shown that Ecdysoneless (ECD) protein functions as a mediator for interaction of HSP90 and the R2TP complex and determines which intracellular molecules the chaperone complex will regulate. Ecdysoneless, was first discovered as a Drosophila fly mutation and we later identified the mammalian ortholog of ECD in human epithelial cells as a binding partner of human papilloma virus 16 E6 oncoprotein. Using knockout gene strategy we demonstrated that ECD deletion is embryonic lethal and its knockdown or knockout (using fl/fl mouse embryonic fibroblasts and adenovirus cre mediated deletion) in vitro led to block in cell cycle progression. Subsequently, we demonstrated ECD is overexpressed in breast cancers, specifically in ErbB2+ breast cancers and its overexpression correlates with poor prognosis and poor survival in these patients. As part of my thesis work, I investigated how ECD regulates cell cycle progression and the role of ECD in ErbB2-driven oncogenesis. We showed that ECD is phosphorylated on several serine residues by CK2 that are important for ECD’s cell cycle function. In second goal, we have shown a novel interaction between ErbB2 and ECD, and knockdown of ECD deregulates the stability of the ERBB2 and HSP90 complex and leads to downregulation of ErbB2 and consequently decreased expression of downstream effectors. We speculate ECD functions as a co-oncogene in ErbB2-driven breast cancer and future studies using transgenic models will explore this possibility. i Table of Contents Page No. Table of Contents…………………………………………………………….……………………….i List of Figures………………………………………………………………………..….………………iv List of Tables………………………………………………………………………….…………….….viii Abbreviations………………………………………………………………….……………………..xvii Acknowledgements………………………………………………………………..…………………vi i: Chapter 1: Introduction……………………………………………………..………….............1 1.1: The Ecdysoneless protein: Discovery and History.………………………………2 1.2: Domains of Ecdysoneless Protein……………………………………………………….4 1:3: Expression of ECD in Different Cancers……………………………………………….5 1.4: ECD Known Protein Interactions and Pathways………………………………….13 1.5: ECD potential roles through different protein-protein interactions.....29 1.6: Rationale, Hypothesis, and Specific Aims……………………………………….....39 ii: Chapter 2: ECD Regulates HER2 Signaling in Breast Cancers……………………42 2.1: Abstract……………………………………………………………..………………………….....44 2.2: Introduction…………………………………………………………….……………………....45 ii 2.3: Results ……………………………………………………………………………………………..47 2.3.1: ECD associates with ErbB2 protein………………..………………………47 2.3.2: ECD regulates levels of ErbB2 protein……….………………………….48 2.3.3: Knockdown of ECD decreases ErbB2 interactions with HSP90…………………………………………………………………………………………….49 2.3.4: ECD downregulates ErbB2 mRNA by altering its stability………50 2.3.5: ECD knockdown dramatically inhibits invasion and migration of ErbB2+ breast cancer cells………………………………………………………………51 2.4: Discussion………………………………………………………………………...................51 2.5: Experimental procedures………………………………………………………………….55 2.6: Figure Legends……………………………………………………………….....................59 2.7: Figures:……………………………………………………………………………...................64 2.8: Supplementary Figures Legends and Figures……………………………………..69 Iii: Chapter 3: Ongoing Studies and Future Directions…………………………………73 3.1: ECD localization and expression in ERBB2 exogenous overexpressed cell lines.……………………………………………………………….………………………………………74 iii 3.2: Potential role of ECD and the R2TP complex in ErbB2 signaling pathway………………………………………………………………………………………………..83 3.3: Role of ECD in downstream ErbB2 Signaling Pathway……………………..110 3.4: Potential Role of ECD in Different Subtypes of Breast of Cancer………112 3.5: Role of ECD in Endoplasmic Reticulum Stress Pathway…………………….116 3.6: ECD potential role as a therapy target in cervical cancer………………….134 iv: Chapter 4: Summary and Conclusions…………………………………………………137 v: Chapter 5: References………………………………………………………………………….144 vi: Appendix…………………………………………………………………..………………………….170 Appendix A: Transcriptional Regulation of ERBB2…………………………………..170 Appendix B: Casein kinase 2-dependent phosphorylation of Ecdysoneless and its interaction with R2TP complex component RUVBL1 regulates its function in cell cycle progression……………………………………….…………….…….200 iv List of Figures Page No. Figure 1.1: Ecd is overexpressed in various breast cancer cell lines.……. 12 Figure 1.2: Ecd is overexpressed in various prostate cancer cell lines.... 14 Figure 1.3: ECD and its Different Interacting partners………………………….15 Figure 1.4: Endogenous association of ECD with HSP90, and the four R2TP components using Proximity Ligation Assay …………………………….………...31 Figure 1.5: Potential Functions of ECD with the HSP90 and the R2TP/Prefoldin-like Complex…………………………………………………………………34 Figure 2.1: ECD associates with ErbB2……………………………………………...….64 Figure 2.2 ECD regulates levels of ErbB2 protein……..………………………...….65 Figure 2.3: Knockdown of ECD decreases interaction of ErbB2 with HSP90………………………………………………………….……………………………………….... 66 Figure 2.4: ECD knockdown decreases ErbB2 mRNA levels by enhancing its turn over……………………………………………………………..……………………………….….67 Figure 2.5: Knockdown of ECD decreases invasion and migration ability of ErbB2 positive breast cancer cell lines ……………………………………………….….68 v Figure Supplementary Figure 1 ..………………………………………….………………….71 Figure Supplementary Figure 2……………………………………………………………….72 Figure 3.1: ECD and ERBB2 Localization in ERBB2+ Breast Cancer Cell Lines………………………………………………………………………………………………………..76 Figure 3.2: ECD is overexpressed in overexpressed ERBB2 transfected cell lines………………………………………………………………...…………………………………..80 Figure 3.3: ECD mRNA is unaffected in overexpressed ERBB2 transfected cell lines.…………………………………………………………………………………………..….82 Figure 3.4: ECD and HSP90 Co-localization in SKBR3 cells.…………………….84 Figure 3.5: ECD, HSP90, and members of the R2TP complex is overexpressed in various breast cancer cell lines..…………………………………85 Figure 3.6: ECD siRNA mediated knockdown does not downregulate members HSP90/R2TP complex in various breast cancer cell lines…….…..88 Figure 3.7: ERBB2+ cell lines treated with Trastuzumab causes downregulation of ECD and members of the R2TP complex.………………….92 Figure 3.8: ERBB2+ Trastuzumab resistant cell lines treated with Trastuzumab does not change ECD and members of the R2TP complex levels…………………………………………………………………………………..………………….95 vi Figure 3.9: ERBB2+ cell lines treated with Lapatinib causes downregulation of ECD and members of the R2TP complex……………………………………………99 Figure 3.10: MCF7 and MDA MB 231 cell lines treated with Lapatinib causes no downregulation of ECD and members of the R2TP complex………………102 Figure 3.11: ERBB2+ cell lines treated with 17-AAG leads to downregulation of ECD…………………………………………………………………………..105 Figure 3.12: BT474 ERBB2+ breast cancer cell line treated with 17-AAG lead to downregulation of ECD and members of the R2TP complex……………….106 Figure 3.13: ERBB2+ cell lines treated with 17-AAG leads to downregulation of ECD and members of the R2TP complex………………………………..…..………108 Figure 3.14: ERBB2+ cell lines treated with Rapamycin causes downregulation of ECD and members of the R2TP complex………..…..…….113 Figure 3.15: ERBB2+ cell lines treated with MK-2206 HCl causes downregulation of ECD…………………………………………………………………………115 Figure 3.16: Proximity Ligation Assay depicting Interaction between ECD and ER Stress Components……………………………………………………………………..128