Abstract 1368: RABL6A, a novel critical regulator of Akt-mTOR signaling in pancreatic neuroendocrine tumor cells
Authors: Salma Shaik Amjad Ume , Hagen Jussara , Reilly Jacki , Sheehy Ryan , Tiwari Nitija , Nteeba Jackson , Sherman Scott K. , O'Dorisio Thomas M. , Howe James R. , Bellizzi Andrew M. , Darbro Benjamin W. , Quelle Dawn E. 
Published: July 2017

Introduction: A better molecular understanding of pancreatic neuroendocrine tumors (PNETs) is needed to improve patient diagnosis and treatment. Everolimus (mTOR inhibitor) is a standard-of-care therapy for PNET patients based on aberrant activation of the PI3K/Akt/mTOR kinase pathway in tumors. However, sustained mTOR inhibition paradoxically promotes Akt kinase hyperactivation due to loss of negative feedback regulation and tumors become drug resistant. Our data reveal that RABL6A, a novel oncoprotein amplified in PNETs, is a key regulator of this clinically relevant pathway.

Methods: RABL6A and Akt protein levels were manipulated using viral shRNAs in BON1 PNET cells. Transcript levels were assayed by microarray and qRT-PCR, proteins assessed by western blotting, and cell proliferation and survival measured by cell counts, trypan blue exclusion and EdU incorporation. Effect of RABL6A expression on sensitivity to clinically relevant drugs, MK2206 (Akt inhibitor) and everolimus, were tested.

Results: Silencing of RABL6A in PNET cells causes G1 and G2/M cell cycle arrest, and pathway analysis of microarray data suggested inactivation of Akt signaling in the arrested cells. Immunoblotting confirmed dramatic loss of Akt phosphorylation at Ser-473 along with impaired phosphorylation and activation of its targets, PRAS40 and FOXO-1/3. Phosphorylation of S6K, a downstream target of Akt-mTOR signaling, was also reduced by RABL6A deficiency. The mechanism by which RABL6A controls Akt-S473 phosphorylation is currently not known although we demonstrated that mTORC2 (the kinase that phosphorylates Akt at Ser473) remains active in RABL6A deficient cells since the phosphorylation of other mTORC2 substrates (SGK1 and PKCα) is unaffected. Given the central role of Akt1 in tumorigenesis, we hypothesized that reinstating its activity may rescue the arrest phenotype caused by RABL6A loss. Restoration of Akt1 in RABL6A-depleted cells partially rescued the G1 phase arrest and induced S phase entry but was insufficient to allow mitosis, suggesting RABL6A regulates other factors required for cell division. Finally, drug response assays showed that RABL6A loss desensitizes PNET cells to Akt and mTOR inhibitors.

Conclusion: Our previous work showed RABL6A promotes G1 progression in PNET cells by inactivating Rb1, an established suppressor of PNET pathogenesis. We now show that RABL6A also controls Akt phosphorylation and is essential for Akt-mTOR activation. Thus, RABL6A controls multiple cancer pathways necessary for PNET cell cycle progression and survival. We are testing if RABL6A status in PNETs predicts responsiveness to combination therapies targeting Akt and mTOR. Overall, this work identifies RABL6A as a new essential activator of Akt1-mTOR signaling, suggesting it is a new potential biomarker and target for anticancer therapy in PNET patients.

Citation Format: Shaik Amjad Ume Salma, Jussara Hagen, Jacki Reilly, Ryan Sheehy, Nitija Tiwari, Jackson Nteeba, Scott K. Sherman, Thomas M. O'Dorisio, James R. Howe, Andrew M. Bellizzi, Benjamin W. Darbro, Dawn E. Quelle. RABL6A, a novel critical regulator of Akt-mTOR signaling in pancreatic neuroendocrine tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1368. doi:10.1158/1538-7445.AM2017-1368