RABL6A inhibits tumor-suppressive PP2A/AKT signaling to drive pancreatic neuroendocrine tumor growth
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
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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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 2

RABL6A-AKT signaling is required for PNET cell cycle progression and response to AKT inhibitors.

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RABL6A-AKT signaling is required for PNET cell cycle progression and res...
(A) BON-1 cells expressing vector (Vec) or constitutively activated myristoylated AKT (Myr-AKT) were infected with control (CON) or RABL6A shRNAs (KD1, KD2). Representative Western blots of phosphorylated AKT-S473 detect endogenous AKT (bar, lower band) and Myr-AKT (arrow, upper band only in lanes 4-6). Heightened activity of Myr-AKT was assessed by levels of PRAS40-T246 phosphorylation. β-actin served as the loading control. Cell ratios (i.e., relative cell numbers normalized to CON cells) are indicated for each sample. (B) The percentage of BrdU-positive cells was quantified in BON-1 control and RABL6A knockdown cells expressing vector (V) versus Myr-AKT (M). Data were quantified from 3 independent experiments; *P < 0.02 for V versus M comparison, 2-way ANOVA. (C) Schematic of RABL6A promoting G1-to-S phase progression and PNET cell proliferation by activating AKT signaling. (D) Dose response curves, shown as relative cell number, in BON-1 control and RABL6A knockdown cells treated for 5 days with increasing concentrations of the AKT inhibitor, MK-2206. Data represent the mean ± SEM for triplicate samples from 3 separate experiments, in which results were normalized to values for untreated cells within each group. *P < 0.001 for KD1 or KD2 compared with CON, 2-way ANOVA and adjusted for multiple comparisons using the Bonferroni method. Overall differences between the curves were assessed by generalized linear regressions. (E) Percentage of cell death in BON-1 control and RABL6A knockdown cells following treatment with MK-2206 (10 μM) for 3 days. Data represent the mean ± SEM from 3 independent experiments. *P < 0.001 for (+) versus (–) comparison, 2-way ANOVA and adjusted for multiple comparisons using the Bonferroni method.