Non-canonical Triple-negative Breast Cancer Cell Death Induction by Reassortant Oncolytic Reovirus Generated by Forward Genetics 公开
Rodriguez Stewart, Roxana M. (Summer 2020)
Abstract
Triple-negative breast cancer (TNBC) constitutes 10-15% of all breast cancer and is associated with worse prognosis when compared to other subtypes of breast cancer. Because of limited current therapy options, there is a need for targeted therapeutics to improve outcomes for TNBC patients. Mammalian orthoreovirus (reovirus) is a non-enveloped, segmented, dsRNA virus in the Reoviridae family. Reovirus selectively kills transformed cells and a serotype 3 reovirus is in clinical trials to assess its efficacy as an oncolytic agent against several cancers. To engineer reovirus with enhanced infective and cytopathic properties against TNBC cells, we coinfected TNBC MDA- MB-231 cells with prototype strains from three reovirus serotypes, Type 1 Lang (T1L), Type 2 Jones (T2J), and Type 3 Dearing (T3D). Following serial passage, we isolated two reassortant reoviruses, r1Reovirus and r2Reovirus, which contain gene segments predominately from T1L, with one (r2Reovirus) or three (r1Reovirus) gene segments from T3D and synonymous and nonsynonymous point mutations. Both reassortant reoviruses display enhanced infective and cytotoxic properties in TNBC cells. Additionally, combinatorial treatment with DNA damaging topoisomerase inhibitors enhances reovirus infectivity and cytotoxicity of TNBC cells. In a second study, we found that r2Reovirus infection of TNBC cells of a mesenchymal-stem like (MSL) lineage downregulates MAPK/ERK signaling and induces non-canonical cell death that is caspase-dependent, but caspase 3-independent. Furthermore, r2Reovirus blocks caspase 3 activity in a replication-dependent manner. Infection of other MSL lineage TNBC cells with r2Reovirus results in caspase 3-dependent cell death. We mapped the enhanced oncolytic properties of r2Reovirus in both TNBC cells to the T3D M2 gene segment in an otherwise T1L virus. Together, our findings suggest that the genetic composition of the host cell and interactions between host and viral gene products impact the mechanism of reovirus-induced cell death in TNBC cells. These studies identify a reassortant reovirus engineered by forward genetics with enhanced non-canonical, cell-dependent oncolytic properties in TNBC cells. Understanding how reovirus induces cell death will help define host and viral factors that promote enhanced oncolysis against TNBC, which will help generate a more effective and targeted viral oncolytic therapy that enhances the prognosis of TNBC patients.
Table of Contents
Abstract
Acknowledgments
Table of Contents
List of Figures and Tables Author Contributions
Table of Contents
Chapter I. Introduction................................................................................................................................................................1
Introduction ................................................................................................................................................................................1 References....................................................................................................................................................................................19
Chapter II. Enhanced Killing of Triple-Negative Breast Cancer Cells by Reassortant Reovirus and Topoisomerase Inhibitors...31
Abstract .......................................................................................................................................................................................32
Importance ..................................................................................................................................................................................33
Introduction ................................................................................................................................................................................34 Results.........................................................................................................................................................................................36 Discussion....................................................................................................................................................................................46
Materials and Methods................................................................................................................................................................52 Acknowledgements......................................................................................................................................................................59
Figures and Tables.......................................................................................................................................................................60 References...................................................................................................................................................................................74
Supplemental Information..........................................................................................................................................................79
Chapter III. Unconventional oncolysis by reassortant reovirus..................................................................................................82
Abstract ......................................................................................................................................................................................83
Importance .................................................................................................................................................................................84
Introduction ...............................................................................................................................................................................84 Results.........................................................................................................................................................................................87 Discussion...................................................................................................................................................................................96
Materials and Methods...............................................................................................................................................................101 Acknowledgements....................................................................................................................................................................107
Figures .......................................................................................................................................................................................108 References..................................................................................................................................................................................117
Chapter IV. Discussion...............................................................................................................................................................125
Discussion .................................................................................................................................................................................125 References..................................................................................................................................................................................140
Chapter V. Appendix..................................................................................................................................................................146
Unpublished Data.......................................................................................................................................................................146 References..................................................................................................................................................................................162
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