A novel MTORC2-AKT-ROS axis triggers mitofission and mitophagy-associated execution of colorectal cancer cells upon drug-induced activation of mutant KRAS.

RAS is one of the most commonly mutated oncogenes associated with multiple cancer hallmarks. Notably, RAS activation induces intracellular reactive oxygen species (ROS) generation, which we previously demonstrated as a trigger for autophagy-associated execution of mutant KRAS-expressing cancer cells. Here we report that drug (merodantoin; C1)-induced activation of mutant KRAS promotes phospho-AKT S473-dependent ROS-mediated S616 phosphorylation and mitochondrial localization of DNM1L/DRP1 (dynamin 1 like) and cleavage of the fusion-associated protein OPA1 (OPA1 mitochondrial dynamin like GTPase). Interestingly, accumulation of the outer mitochondrial membrane protein VDAC1 (voltage dependent anion channel 1) is observed in mutant KRAS-expressing cells upon exposure to C1. Conversely, silencing VDAC1 abolishes C1-induced mitophagy, and gene knockdown of either KRAS, AKT or DNM1L rescues ROS-dependent VDAC1 accumulation and stability, thus suggesting an axis of mutant active KRAS-phospho-AKT S473-ROS-DNM1L-VDAC1 in mitochondrial morphology change and cancer cell execution. Importantly, we identified MTOR (mechanistic target of rapamycin kinsase) complex 2 (MTORC2) as the upstream mediator of AKT phosphorylation at S473 in our model. Pharmacological or genetic inhibition of MTORC2 abrogated C1-induced phosphorylation of AKT S473, ROS generation and mitophagy induction, as well as rescued tumor colony forming ability and migratory capacity. Finally, increase in thermal stability of KRAS, AKT and DNM1L were observed upon exposure to C1 only in mutant KRAS-expressing cells. Taken together, our work has unraveled a novel mechanism of selective targeting of mutant KRAS-expressing cancers via MTORC2-mediated AKT activation and ROS-dependent mitofission, which could have potential therapeutic implications given the relative lack of direct RAS-targeting strategies in cancer.Abbreviations: ACTB/ß-actin: actin beta; AKT: AKT serine/threonine kinase; C1/merodantoin: 1,3-dibutyl-2-thiooxo-imidazoldine-4,5-dione; CAT: catalase; CETSA: cellular thermal shift assay; CHX: cycloheximide; DKO: double knockout; DNM1L/DRP1: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H2O2: hydrogen peroxide; HSPA1A/HSP70-1: heat shock protein family A (Hsp70) member 1A; HSP90AA1/HSP90: heat shock protein 90 alpha family class A member 1; KRAS: KRAS proto-oncogene, GTPase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; LC3B-I: unlipidated form of LC3B; LC3B-II: phosphatidylethanolamine-conjugated form of LC3B; MAPKAP1/SIN1: MAPK associated protein 1; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MFI: mean fluorescence intensity; MiNA: Mitochondrial Network Analysis; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; O2.-: superoxide; OMA1: OMA1 zinc metallopeptidase; OPA1: OPA1 mitochondrial dynamin like GTPase; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR/raptor: regulatory associated protein of MTOR complex 1; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; SQSTM1/p62: sequestosome 1; VDAC1: voltage dependent anion channel 1; VDAC2: voltage dependent anion channel 2.

Serine-70 phosphorylated Bcl-2 prevents oxidative stress-induced DNA damage by modulating the mitochondrial redox metabolism.

Bcl-2 phosphorylation at serine-70 (S70pBcl2) confers resistance against drug-induced apoptosis. Nevertheless, its specific mechanism in driving drug-resistance remains unclear. We present evidence that S70pBcl2 promotes cancer cell survival by acting as a redox sensor and modulator to prevent oxidative stress-induced DNA damage and execution. Increased S70pBcl2 levels are inversely correlated with DNA damage in chronic lymphocytic leukemia (CLL) and lymphoma patient-derived primary cells as well as in reactive oxygen species (ROS)- or chemotherapeutic drug-treated cell lines. Bioinformatic analyses suggest that S70pBcl2 is associated with lower median overall survival in lymphoma patients. Empirically, sustained expression of the redox-sensitive S70pBcl2 prevents oxidative stress-induced DNA damage and cell death by suppressing mitochondrial ROS production. Using cell lines and lymphoma primary cells, we further demonstrate that S70pBcl2 reduces the interaction of Bcl-2 with the mitochondrial complex-IV subunit-5A, thereby reducing mitochondrial complex-IV activity, respiration and ROS production. Notably, targeting S70pBcl2 with the phosphatase activator, FTY720, is accompanied by an enhanced drug-induced DNA damage and cell death in CLL primary cells. Collectively, we provide a novel facet of the anti-apoptotic Bcl-2 by demonstrating that its phosphorylation at serine-70 functions as a redox sensor to prevent drug-induced oxidative stress-mediated DNA damage and execution with potential therapeutic implications.

Resveratrol attenuates TLR-4 mediated inflammation and elicits therapeutic potential in models of sepsis.

Sepsis is a potentially fatal condition triggered by systemic inflammatory response to infection. Due to the heightened immune reactivity and multi-organ pathology, treatment options are limited and several clinical trials have not produced the desired outcome, hence the interest in the discovery of novel therapeutic strategies. The polyphenol resveratrol (RSV) has shown promise against several pathological states, including acute and chronic inflammation. In this study, we evaluated its therapeutic potential in a murine model of sepsis and in patients undergoing transrectal ultrasound biopsy. RSV was able to inhibit lipopolysaccharide (LPS) stimulated inflammatory responses through blocking Phospholipase D (PLD) and its downstream signaling molecules SphK1, ERK1/2 and NF-κB. In addition, RSV treatment resulted in the downregulation of MyD88, an adaptor molecule in the TLR4 signaling pathway, and this effect at least in part, involved RSV-induced autophagy. Notably, RSV protected mice against polymicrobial septic shock induced upon cecal ligation and puncture, and inhibited pro-inflammatory cytokine production by human monocytes from transrectal ultrasound (TRUS) biopsy patients. Together, these findings demonstrate the immune regulatory activity of RSV and highlight its therapeutic potential in the management of sepsis.

Peroxynitrite promotes serine-62 phosphorylation-dependent stabilization of the oncoprotein c-Myc.

Stabilization of c-Myc oncoprotein is dependent on post-translational modifications, especially its phosphorylation at serine-62 (S62), which enhances its tumorigenic potential. Herein we report that increase in intracellular superoxide induces phospho-stabilization and activation of c-Myc in cancer cells. Importantly, sustained phospho-S62 c-Myc was necessary for promoting superoxide dependent chemoresistance as non-phosphorylatable S62A c-Myc was insensitive to the redox impact when subjected to chemotherapeutic insults. This redox-dependent sustained S62 phosphorylation occurs through nitrative inhibition of phosphatase, PP2A, brought about by peroxynitrite, a reaction product of superoxide and nitric oxide. We identified a conserved tyrosine residue (Y238) in the c-Myc targeting subunit B56α of PP2A, which is selectively amenable to nitrative inhibition, further preventing holoenzyme assembly. In summary, we have established a novel mechanism wherein the pro-oxidant microenvironment stimulates a pro-survival milieu and reinforces tumor maintenance as a functional consequence of c-Myc activation through its sustained S62 phosphorylation via inhibition of phosphatase PP2A. SIGNIFICANCE STATEMENT: Increased peroxynitrite signaling in tumors causes sustained S62 c-Myc phosphorylation by PP2A inhibition. This is critical to promoting c-Myc stabilization and activation which promotes chemoresistance and provides significant proliferative and growth advantages to osteosarcomas.

Correction: Simultaneous Induction of Non-Canonical Autophagy and Apoptosis in Cancer Cells by ROS-Dependent ERK and JNK Activation.

[This corrects the article DOI: 10.1371/journal.pone.0009996.].

Synthetic Lethality of a Novel Small Molecule Against Mutant KRAS-Expressing Cancer Cells Involves AKT-Dependent ROS Production.

AIMS: We recently reported the death-inducing activity of a small-molecule compound, C1, which triggered reactive oxygen species (ROS)-dependent autophagy-associated apoptosis in a variety of human cancer cell lines. In this study, we examine the ability of the compound to specifically target cancer cells harboring mutant KRAS with minimal activity against wild-type (WT) RAS-expressing cells. RESULTS: HCT116 cells expressing mutated KRAS are susceptible, while the WT-expressing HT29 cells are resistant. Interestingly, C1 triggers activation of mutant RAS, which results in the downstream phosphorylation and activation of AKT/PKB. Gene knockdown of KRAS or AKT or their pharmacological inhibition resulted in the abrogation of C1-induced ROS production and rescued tumor colony-forming ability. We also made use of HCT116 mutant KRAS knockout (KO) cells, which express only a single WT KRAS allele. Exposure of KO cells to C1 failed to increase mitochondrial ROS and cell death, unlike the parental cells harboring mutant KRAS. Similarly, mutant KRAS-transformed prostate epithelial cells (RWPE-1-RAS) were more sensitive to the ROS-producing and death-inducing effects of C1 than the vector only expressing RWPE-1 cells. An in vivo model of xenograft tumors generated with HCT116 KRAS(WT/MUT) or KRAS(WT/-) cells showed the efficacy of C1 treatment and its ability to affect the relative mitotic index in tumors harboring KRAS mutant. INNOVATION AND CONCLUSION: These data indicate a synthetic lethal effect against cells carrying mutant KRAS, which could have therapeutic implications given the paucity of KRAS-specific chemotherapeutic strategies. Antioxid. Redox Signal. 24, 781-794.

Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers.

Simultaneous induction of non-canonical autophagy and apoptosis in cancer cells by ROS-dependent ERK and JNK activation.

BACKGROUND: Chemotherapy-induced reduction in tumor load is a function of apoptotic cell death, orchestrated by intracellular caspases. However, the effectiveness of these therapies is compromised by mutations affecting specific genes, controlling and/or regulating apoptotic signaling. Therefore, it is desirable to identify novel pathways of cell death, which could function in tandem with or in the absence of efficient apoptotic machinery. In this regard, recent evidence supports the existence of a novel cell death pathway termed autophagy, which is activated upon growth factor deprivation or exposure to genotoxic compounds. The functional relevance of this pathway in terms of its ability to serve as a stress response or a truly death effector mechanism is still in question; however, reports indicate that autophagy is a specialized form of cell death under certain conditions. METHODOLOGY/PRINCIPAL FINDINGS: We report here the simultaneous induction of non-canonical autophagy and apoptosis in human cancer cells upon exposure to a small molecule compound that triggers intracellular hydrogen peroxide (H(2)O(2)) production. Whereas, silencing of beclin1 neither inhibited the hallmarks of autophagy nor the induction of cell death, Atg 7 or Ulk1 knockdown significantly abrogated drug-induced H(2)O(2)-mediated autophagy. Furthermore, we provide evidence that activated extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) are upstream effectors controlling both autophagy and apoptosis in response to elevated intracellular H(2)O(2). Interestingly, inhibition of JNK activity reversed the increase in Atg7 expression in this system, thus indicating that JNK may regulate autophagy by activating Atg7. Of note, the small molecule compound triggered autophagy and apoptosis in primary cells derived from patients with lymphoma, but not in non-transformed cells. CONCLUSIONS/SIGNIFICANCE: Considering that loss of tumor suppressor beclin 1 is associated with neoplasia, the ability of this small molecule compound to engage both autophagic and apoptotic machineries via ROS production and subsequent activation of ERK and JNK could have potential translational implications.

Hydrogen peroxide-mediated cytosolic acidification is a signal for mitochondrial translocation of Bax during drug-induced apoptosis of tumor cells.

Absence of the proapoptotic protein Bax renders tumor cells resistant to drug-induced apoptosis. We have shown that hydrogen peroxide (H(2)O(2))-mediated cytosolic acidification is an effector mechanism during drug-induced apoptosis of tumor cells. Here, we report that Bax is critical in determining the sensitivity of tumor cells to H(2)O(2)-induced apoptosis. More importantly, exposure of colorectal carcinoma (HCT116) and leukemia cells (HL60 and CEM) to H(2)O(2) or its intracellular production during drug-induced apoptosis is a signal for mitochondrial translocation of Bax. Furthermore, we provide evidence that drug-induced H(2)O(2)-mediated Bax translocation in tumor cells is caspase independent but involves cytosolic acidification. Inhibiting cytosolic acidification prevents Bax translocation, and contrarily enforced acidification of the intracellular milieu results in mitochondrial recruitment of Bax, even in the absence of a trigger. These findings provide a novel mechanism for mitochondrial translocation of Bax and directly implicate H(2)O(2)-mediated cytosolic acidification in the recruitment of the mitochondrial pathway during drug-induced apoptosis of tumor cells.