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. 2016 Apr 5;7(14):18694-704.
doi: 10.18632/oncotarget.7909.

Suppression of B-Raf(V600E) cancers by MAPK hyper-activation

Rawan Atiq  1 , Rachel Hertz  1 , Sophia Eldad  1 , Elia Smeir  1 , Jacob Bar-Tana  1
Affiliations

Suppression of B-Raf(V600E) cancers by MAPK hyper-activation

Rawan Atiq et al. Oncotarget. .

Abstract

B-Raf(V600E) activates MEK/MAPK signalling and acts as oncogenic driver of a variety of cancers, including melanoma, colorectal and papillary thyroid carcinoma. Specific B-Raf(V600E) kinase inhibitors (e.g., Vemurafenib) prove initial efficacy in melanoma followed shortly by acquired resistance, while failing in most other B-Raf(V600E) cancers due to primary resistance. Resistance is due to acquired mutations in the Ras/Raf/MEK/MAPK pathway and/or other oncogenic drivers that bypass B-Raf(V600E). Surprisingly, hyper-activation of MAPK by inhibiting its protein phosphatase 2A by a synthetic long-chain fatty acid analogue (MEDICA), results in oncogene-induced growth arrest and apoptosis of B-Raf(V600E) cancer cells. Growth arrest is accompanied by MAPK-mediated serine/threonine phosphorylation and suppression of a variety of oncogenic drivers that resist treatment by B-Raf(V600E) kinase inhibitors, including ErbB members, c-Met, IGFR, IRS, STAT3 and Akt. The combined activities of mutated B-Raf and MEDICA are required for generating hyper-activated MAPK, growth arrest and apoptosis, implying strict specificity for mutated B-Raf cancer cells.

Keywords: B-Raf(V600E); MAPK; colorectal cancer; melanoma; papillary thyroid carcinoma.

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Conflict of interest statement

CONFLICTS OF INTEREST

JBT is director in SyndromeX, a company that develops drugs for the Metabolic Syndrome.

Figures

Figure 1
Figure 1. Growth Inhibition and apoptosis of B-Raf(V600E) cell types by MEDICA
(A–C) Cell growth (A), anchorage-independent colonies (B), and cell cycle distribution (48 h) (C). Mean ± SD. *Significant as compared to control. Inset-representative micrographs. (D) Senescence-associated β-galactosidase. Mean ± SE. *Significant as compared to control. Inset-representative micrographs. (E, F) Cyclin D1, p21, P-Rb(Ser807/811), cleaved PARP and caspase 3. Representative blots.
Figure 2
Figure 2. Suppression of PLX4032-resistant B-Raf(V600E) cell types by MEDICA
(A) EGF- and HGF-induced resistance to PLX4032. Mean ± SD. *Significant as compared to control. (B) Suppression of PLX4032-resistant B-Raf(V600E) cell types by MEDICA. Mean ± SD. #Significance of added cytokine as compared to its absence. *Significant as compared to respective control. (C) Suppression of EGF- and HGF-induced phosphorylation of EGFR, ErbB2/HER, MET, Erk and Akt by MEDICA. Representative blots. (D) Suppression of gp130, IL6- and HGF-induced phosphorylation of STAT3, and constitutive STAT3 by MEDICA. Representative blots. (E) Suppression of transfected STAT3 reporter by MEDICA (HT29 cells). Mean ± SE. #Significant as compared to control. *Significant as compared to control IL-6.
Figure 3
Figure 3. Growth suppression of B-Raf(V600E) cells by MEDICA is B-Raf(V600E)-dependent
(A) Suppression of HT29 cell growth (48 h) by MEDICA is abrogated by infected shB-Raf(V600E). Mean ± SD. *Significant as compared to respective control. #Significant as compared to respective scramble. (B) MEDICA-induced p21 and cleaved PARP in HT29 cells is abrogated by infected shB-Raf(V600E). (C) Suppression of EGF-activated EGFR, Akt and Erk in HT29 cells by MEDICA is abrogated by infected shB-Raf(V600E). (D) Suppression of EGF-activated EGFR and Akt in HT29 cells by MEDICA is abrogated by inhibiting MEK by U0126. (E, F) Suppression of IL-6-activated STAT3 in HT29 cells by MEDICA is abrogated by infected shB-Raf(V600E), or upon inhibiting MEK by U0126 or PD0325901. (G) Suppression of constitutive STAT3 and Erk in A375 cells by MEDICA is abrogated by inhibiting B-Raf(V600E) by PLX4032, or by inhibiting MEK by PD0325901. (B-G) Representative blots.
Figure 4
Figure 4. Erk activation by MEDICA
(A, B) MEDICA activates Erk and its downstream targets RSK(Thr359/Ser363), LKB1(Ser428), CREB(Ser133), EGFR(Thr669), STAT3(Ser727), FAK(Ser910) and IRS1(Ser636/639), while inhibiting basal Akt(Ser473). (C) shRNA B-Raf(V600E) or U0126 abrogates EGFR(Thr669) phosphorylation and inhibition of basal Akt(Ser473) by MEDICA (HT29 cells). (D) Activation of Erk and its canonical downstream targets by MEDICA is MEK-independent (HT29 cells). (E) Growth suppression, Akt inhibition and apoptosis by MEDICA are AMPK-independent (HT29 cells). (A-E) Representative blots.
Figure 5
Figure 5. MEDICA-induced ROS production in HT29 cells
(A) MEDICA-induced ROS production as verified by DCFDA fluorescence and decrease in GSH/GSSG ratio. Mean ± SE. *Significant as compared to respective control. #Significant as compared to the respective MEDICA concentrations. (B) MEDICA-induced mitochondrial ROS production as verified by mitoSOX and succinate-dependent Amplex Red fluorescence. Mean ± SD. *Significant as compared to control. (C) Suppression of PP2A activity by MEDICA-induced ROS. Mean ± SE. *Significant as compared to respective control. #Significant as compared to respective control. (D–F) Activation of Erk downstream targets (D), increase in p21 (E), and suppression of EGF- and IL-6-induced STAT3 and Akt (F) by MEDICA are abrogated by NAC. Representative blots. (G) Suppression of cell growth (48 h) by MEDICA is abrogated by NAC. Mean ± SD. *Significant as compared to growth in NAC absence. (H) Suppression of clonogenic growth by MEDICA is abrogated by PEG-SOD (100 u/ml). Mean ± SD. *Significant as compared to respective control.
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