Dissertation
RETROVIRAL VECTOR MUTAGENESIS SCREENS TO IDENTIFY CANCER DRIVER GENES AND PROGNOSTIC BIOMARKERS
Doctor of Philosophy (PhD), Washington State University
01/2016
Handle:
https://hdl.handle.net/2376/117178
Abstract
Cancer remains a major cause of mortality and morbidity and novel therapies are needed to improve patient outcomes. Targeting specific oncogenes can lead to improved therapies and whole genomic sequencing has been used to identify cancer driver genes, but distinguishing driver genes from passenger genes remains challenging. Replication-incompetent retroviral vector mutagenesis screens are promising alternative. In this approach, a stably integrated vector provirus dysregulates nearby proto-oncogenes or tumor suppressors. Cell clones with an integrated vector that provides a selective advantage by dysregulating a nearby driver gene become dominant over time, and the vector provirus tags the candidate cancer driver gene. We used a replication-incompetent gammaretroviral (γRV) vector to identify breast cancer (BC) metastasis driver genes. γRV transduced cells were xenotranplanted into the mammary fat pad of immunodeficient mice and metastatic tumors were obtained and analyzed for retroviral integration sites (RIS) to identify nearby genes that provided a selective advantage to form metastasis. Candidate BC metastasis genes SHARPIN, WWTR1, RIN1 and MAF1 were identified. SHARPIN was validated as a BC metastasis driver gene and prognostic biomarker.
Replication-incompetent γRV and lentiviral (LV) vectors were then directly compared to evaluate their ability to identify driver genes that mediate androgen-independent prostate cancer (PC) progression. γRV and LV vectors have distinct integration profiles and genotoxicity that makes them potentially complementary vectors to identify cancer driver genes. Transduced LNCaP PC cells were injected into the prostate gland of immunodeficient mice. The mice were castrated to model androgen deprivation therapy in PC patients. Metastatic tumors that developed under androgen-independent conditions were analyzed using a high-throughput modified genomic sequencing PCR (MGS-PCR) approach. TAOK3, MBNL2, SERBP1, SLC7A1, SLC25A24, MAN1A2, PLEKHA2, SPTAN1, and ABCC1 candidate PC genes were identified. TAOK3 and ABCC1 were validated by showing that their expression increased the clonogenic potential of PC cells. TAOK3 and ABCC1 expression predicted disease recurrence in PC patients after androgen deprivation therapy. The work presented here shows that replication-incompetent retroviral vector insertional mutagenesis screens are a powerful approach to identify cancer driver genes in diverse cancer types.
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Details
- Title
- RETROVIRAL VECTOR MUTAGENESIS SCREENS TO IDENTIFY CANCER DRIVER GENES AND PROGNOSTIC BIOMARKERS
- Creators
- Victor Malakwen Bii
- Contributors
- Grant D Trobridge (Advisor)Gary G Meadows (Committee Member)Mary Sanchez Lanier (Committee Member)James K Pru (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- College of Pharmacy and Pharmaceutical Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 171
- Identifiers
- 99900581830401842
- Language
- English
- Resource Type
- Dissertation