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Investigating the interaction between K-Ras and HuR in pancreatic cancer
Dissertation   Open access

Investigating the interaction between K-Ras and HuR in pancreatic cancer

Matthew C. Stout
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2021
DOI:
https://doi.org/10.17918/00000573
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Abstract

Pancreas--Cancer Cytology
Poised to become the 2nd leading cause of cancer death in the US, pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of 10% and a median survival rate after diagnosis of only 6 months. New therapeutics are desperately needed for PDAC treatment. Oncogenic K-Ras, a small membrane-bound GTPase, is the main driver of PDAC, being mutated in up to 95% of all PDAC tumor. One mechanism by which K-Ras-dependent signaling may influence tumor cell biology is by controlling the transcription and/or translation of genes that drive an altered phenotype. Gene expression is partly controlled by the activity of a class of proteins called mRNA binding proteins. HuR is a ubiquitously expressed mRNA binding protein that binds to and regulates the stability of hundreds of pro-tumorigenic transcripts. HuR is overexpressed in many cancer types including pancreatic cancer and cytoplasmic accumulation of HuR correlates with a worse prognosis and tumor aggressiveness. Additionally, HuR is proteolytically cleaved, and these small forms tend to promote an apoptotic transcriptional program. We investigated whether these two important mediators of PDAC interact in a feedback mechanism to promote tumorigenesis to increase our understanding of PDAC biology and aid the development of novel therapeutics. We hypothesize that a positive feedback loop exists between K-Ras and HuR to drive PDAC tumorigenesis. We hypothesize that K-Ras signaling modulates HuR expression, localization, and function and in return HuR regulates K-Ras and K-Ras associated proteins expression. Our results show that K-Ras signaling prevents HuR cleavage under hypoxia, allowing for HuR cytoplasmic accumulation that stabilizes a pro-tumorigenic transcriptome. Loss of oncogenic K-Ras or inhibition of the downstream signaling pathways of MEK-ERK or PI3K-AKT lead to increases in HuR cleavage. For the first time ever, these results link oncogenic K-Ras signaling to HuR cleavage regulation. Our results imply that K-Ras inhibits HuR-CPs generation as a pro-tumorigenic survival mechanism in PDAC. This is substantiated by an increase in PDAC cell death with HuR-CP1 overexpression in cells lacking HuR. In turn, HuR enhances the expression of K-Ras associated proteins necessary for proper oncogenic K-Ras signaling. We show that HuR loss increases K-Ras expression under normal conditions but K-Ras activation is hampered when PDAC cell lacking HuR are stressed. Proteins necessary for proper K-Ras signaling, including GAPs, GEFs, adaptor, and scaffold proteins, are downregulated under stressed conditions with HuR loss. These results show how HuR regulates K-Ras signaling as part of the pro-tumorigenic transcriptome to drive tumorigenesis in PDAC. From our results, we see that oncogenic K-Ras inhibits HuR cleavage, increasing the amount of cytoplasmic full length HuR, which allows HuR to bind to and up regulate the translation of hundreds of pro-tumorigenic transcripts that include K-Ras associated proteins. We conclude that oncogenic K-Ras and HuR work in concert in a positive feedback mechanism to maintain a pro-tumorigenic transcriptome to drive tumor development.

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