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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Research Article Cell biology Oncology

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 4

PP2A reactivation with SMAP reduces AKT-S473 phosphorylation, downregulates RABL6A, and induces PNET cell death in a RABL6A-dependent manner.

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PP2A reactivation with SMAP reduces AKT-S473 phosphorylation, downregula...
(A) Western blots (repeated 3 or more times) of lysates from BON-1 and Qgp1 cells treated 20 hours with SMAP showing effective reduction of pAKT-S473 and pERK-T202/Y204. Loading, vinculin (BON-1), and GAPDH (Qgp1). (B) Representative images from clonogenic assays of BON-1 and Qgp1 cells treated with 0 (vehicle), 5, 10, or 20 μM SMAP for 3 weeks. (C) Quantification of clonogenic assay results, normalized to 100% for vehicle-treated control cells, from 2 separate experiments performed in triplicate. *P < 0.001, 2-way ANOVA and adjusted for multiple comparisons using the Bonferroni method. Identical results were obtained in 4 additional repeats per cell line for cells plated at different densities. (D) Representative experiment (repeated twice) showing dose response curves for BON-1 and Qgp1 cells following exposure for 3 days to indicated concentrations of SMAP. Data represent the mean ± SD for triplicate samples and were normalized to values from untreated cells. (E) Relative cell number (assayed by Cell-Quant) of BON-1 cells expressing CON, KD1, or KD2 shRNAs after exposure for 3 days to increasing concentrations of SMAP. Data (from 3 experiments done in triplicate) were normalized to untreated controls. *P < 0.001 for KD1 and KD2 compared with CON, 2-way ANOVA and adjusted for multiple comparisons using the Bonferroni method. Overall differences between curves were assessed using generalized linear regression. (F) BON-1 CON, KD1, and KD2 cells were treated with 10 μM SMAP for 3 days. Dark and light exposures of Western blots show PP2A activation decreased RABL6A levels and altered its migration on gels. *P < 0.05 for untreated versus treated samples, Student’s t test. Error bars in E and F indicate SEM for data from 3 independent experiments.