Dissertation
Role of RABL6A in pancreatic neuroendocrine tumor development and progression
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2021
DOI: 10.17077/etd.006079
Abstract
Pancreatic neuroendocrine tumors (pNETs) are slow-growing neoplasms whose incidence has increased more than four-fold over the past four decades. Localized pNETs can be treated by surgical resection, but majority of pNETs are not diagnosed until their advanced stages associated with metastasis to liver and other tissues. This is alarming because effective targeted therapies to treat advanced pNETs and improve the patient overall survival are still lacking. Greater understanding of mechanisms driving pNET growth and progression is urgently required to identify more reliable disease markers and therapeutic targets. RABL6A, an oncogenic RAB-like GTPase, is highly expressed in patient pNETs and required for pNET cell survival and proliferation in vitro. Mechanistic studies reveal that RABL6A promotes pNETs partly by promoting the oncogenic Akt/mTOR signaling and inhibiting the RB1 tumor suppressor pathway. These in vitro findings led to our hypothesis that RABL6A promotes pNET angiogenesis and progression in vivo. Our first aim investigates the in vivo role of RABL6A in pNET angiogenesis and progression using a mouse model of the disease, whereas the second aim uses an unbiased, forward genetic mutagenesis screen called Sleeping Beauty to identify novel RABL6A targets.Aim1 explores the in vivo role of RABL6A in pNET pathogenesis using an established pNET mouse model called RIP-Tag2 (RT2). RT2 mice are genetically engineered to express the oncogenic SV40 large T-antigen in pancreatic islet β cells. This causes β cell hyperproliferation leading to rapid development of insulinomas (functional pNETs). Normally, these animals die at a young age of 13-17 weeks from their tumors. We crossed the RT2 mice with RABL6A knockout (KO) mice to determine if loss of RABL6A disrupts pNET development and angiogenesis. Our findings are exciting and provide compelling evidence that RABL6A drives pNET pathogenesis in vivo. Specifically, RABL6A loss in RT2 mice was found to delay pNET formation, reduce tumor angiogenesis and mitosis, and improve their overall survival. These biological effects were consistent with downregulation of oncogenic c-Myc and upregulation of the p19ARF tumor suppressor in RABL6A-deficient islets and tumors.
Aim2 employs a powerful genetic screen called Sleeping Beauty (SB) transposition to identify undefined RABL6A-regulated drivers or suppressors of pNET pathogenesis. The SB system involves random insertion of a mutagenic DNA segment called transposon into the genomic DNA by virtue of an enzyme called transposase. Depending on the distribution and orientation of the mutagenic transposon insertion, the SB system can induce gain- or loss-of-function mutations. Our SB screen selects for specific mutational events that enable pNET cells to grow in the absence of RABL6A. We sequenced the growing, SB-mutagenized RABL6A-depleted cells and subsequently performed the gene-centric common insertion site analysis to identify 26 candidate RABL6A effector genes that were predicted to control pNET proliferation. Three genes, LCOR, TENM3 and AHRR, contained primarily sense-orientation insertions that suggest their overexpression promotes the growth of RABL6A-depleted pNET cells. In contrast, most genes (including RORA, NPAS3, and ARHGAP35) had randomly oriented insertions suggesting they are tumor suppressors whose disruption enables pNET proliferation despite RABL6A loss. Functional studies evaluating the relationship of these genes with RABL6A and their biological role in pNETs are expected to expand our understanding of pNET pathogenesis and identify new targets for therapy.
Details
- Title: Subtitle
- Role of RABL6A in pancreatic neuroendocrine tumor development and progression
- Creators
- Chandra Kumar Maharjan
- Contributors
- Dawn E Quelle (Advisor)Frederick W Quelle (Committee Member)Rory A Fisher (Committee Member)Adam J Dupuy (Committee Member)Benjamin W Darbro (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Pharmacology
- Date degree season
- Spring 2021
- Publisher
- University of Iowa
- DOI
- 10.17077/etd.006079
- Number of pages
- xv. 133 pages
- Copyright
- Copyright 2021 Chandra Kumar Maharjan
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (pages 113-133).
- Public Abstract (ETD)
Pancreatic neuroendocrine tumors (pNETs) are devastating cancers whose incidence is rising globally. This is concerning because therapies to effectively treat this disease are still not available. My thesis project investigates how pNETs form and progress as this will contribute to developing more efficient pNET therapies. We study a tumor-promoting (oncogenic) protein called RABL6A, which has previously been shown to enable pNET cells to survive and grow by activating different tumor promoting signaling pathways.
My first project seeks to obtain direct evidence from a pNET animal model called RIPTag2 (RT2) mice that RABL6A promotes pNET development and progression because that will strengthen the biological relevance of studying RABL6A signaling in this disease. RT2 mice rapidly develop pNETs and as a result die at a very young age. We found that genetically eliminating RABL6A from RIP-Tag2 mice slows tumor formation and blood vessel growth (required for tumor progression), consistent with improvement in survival. This suggests RABL6A promotes pNET development and progression in vivo.
Our knowledge of how RABL6A drives pNET pathways is limited. To better understand RABL6A-regulated signaling pathways in pNETs, we used a powerful genetic screen called Sleeping Beauty. Our SB screen selects for specific genetic mutations like activation of an oncogene or inhibition of tumor suppressor that enable pNET cells to grow in the absence of RABL6A. Normally, pNET cells die or stop growing following RABL6A loss. DNA sequencing in growing, SB-treated RABL6A-depleted pNET cells revealed new candidate genes, which could contribute to the oncogenic role of RABL6A in pNETs.
- Academic Unit
- Neuroscience and Pharmacology
- Record Identifier
- 9984097367502771
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