RABL6A inhibits tumor-suppressive PP2A/AKT signaling to drive pancreatic neuroendocrine tumor growth
Shaikamjad Umesalma, … , Frederick W. Quelle, Dawn E. Quelle
Shaikamjad Umesalma, … , Frederick W. Quelle, Dawn E. Quelle
Published February 5, 2019
Citation Information: J Clin Invest. 2019;129(4):1641-1653. https://doi.org/10.1172/JCI123049.
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RABL6A inhibits tumor-suppressive PP2A/AKT signaling to drive pancreatic neuroendocrine tumor growth

Abstract

Hyperactivated AKT/mTOR signaling is a hallmark of pancreatic neuroendocrine tumors (PNETs). Drugs targeting this pathway are used clinically, but tumor resistance invariably develops. A better understanding of factors regulating AKT/mTOR signaling and PNET pathogenesis is needed to improve current therapies. We discovered that RABL6A, a new oncogenic driver of PNET proliferation, is required for AKT activity. Silencing RABL6A caused PNET cell-cycle arrest that coincided with selective loss of AKT-S473 (not T308) phosphorylation and AKT/mTOR inactivation. Restoration of AKT phosphorylation rescued the G1 phase block triggered by RABL6A silencing. Mechanistically, loss of AKT-S473 phosphorylation in RABL6A-depleted cells was the result of increased protein phosphatase 2A (PP2A) activity. Inhibition of PP2A restored phosphorylation of AKT-S473 in RABL6A-depleted cells, whereas PP2A reactivation using a specific small-molecule activator of PP2A (SMAP) abolished that phosphorylation. Moreover, SMAP treatment effectively killed PNET cells in a RABL6A-dependent manner and suppressed PNET growth in vivo. The present work identifies RABL6A as a new inhibitor of the PP2A tumor suppressor and an essential activator of AKT in PNET cells. Our findings offer what we believe is a novel strategy of PP2A reactivation for treatment of PNETs as well as other human cancers driven by RABL6A overexpression and PP2A inactivation.

Authors

Shaikamjad Umesalma, Courtney A. Kaemmer, Jordan L. Kohlmeyer, Blake Letney, Angela M. Schab, Jacqueline A. Reilly, Ryan M. Sheehy, Jussara Hagen, Nitija Tiwari, Fenghuang Zhan, Mariah R. Leidinger, Thomas M. O’Dorisio, Joseph Dillon, Ronald A. Merrill, David K. Meyerholz, Abbey L. Perl, Bart J. Brown, Terry A. Braun, Aaron T. Scott, Timothy Ginader, Agshin F. Taghiyev, Gideon K. Zamba, James R. Howe, Stefan Strack, Andrew M. Bellizzi, Goutham Narla, Benjamin W. Darbro, Frederick W. Quelle, Dawn E. Quelle

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Figure 1

RABL6A depletion impairs AKT-S473 phosphorylation and AKT-mTOR signaling in PNETs.

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RABL6A depletion impairs AKT-S473 phosphorylation and AKT-mTOR signaling...
(A) Schematic showing that RABL6A promotes PNET cell proliferation and survival through multiple mechanisms, including inhibition of RB1 signaling as well as regulation of other undefined (X, Y) pathways. Arrow, activating event; perpendicular bar, inhibitory event. (B) BON-1 cells expressing vector control (CON: with RABL6A) or shRNAs targeting RABL6A (KD1 and KD2: without RABL6A) were examined by microarray analyses. Heat map shows that RABL6A depletion significantly alters the expression of genes involved in AKT signaling; data from 3 experiments, designated A–C. Genes were categorized by IPA software and displayed 2-fold or greater changes in expression (P < 0.05). Red, relatively increased expression; blue, relatively decreased expression. (C) Representative Western blots showing that RABL6A knockdown (KD1, KD2) in BON-1 cells specifically reduces the activating phosphorylation of AKT at S473, not T308. Vinculin served as loading control. Relative phosphorylation of each residue relative to total AKT was quantified by ImageJ. (D) Representative Western blots showing that inactivation of AKT in RABL6A knockdown BON-1 cells coincides with reduced phosphorylation of AKT substrates, PRAS40-T246 and FoxO1-T24/FoxO3a-T32, with GAPDH as loading control. (E) Representative Western blots showing that inactivation of AKT in RABL6A knockdown BON-1 cells coincides with reduced activation of mTORC1, as measured by decreased S6K phosphorylation at T389. GAPDH was the loading control. S6K-T389 phosphorylation relative to total S6K was quantified by ImageJ. Experiments in C–E represent at least 3 independent experimental repeats.