Hyperphosphorylation of BCL-2 family proteins underlies functional resistance to venetoclax in lymphoid malignancies.

The B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as BCL2 mutations have been described, this probably only explains a subset of resistant cases. Using 2 complementary functional precision medicine techniques - BH3 profiling and high-throughput kinase activity mapping - we found that hyperphosphorylation of BCL-2 family proteins, including antiapoptotic myeloid leukemia 1 (MCL-1) and BCL-2 and proapoptotic BCL-2 agonist of cell death (BAD) and BCL-2 associated X, apoptosis regulator (BAX), underlies functional mechanisms of both intrinsic and acquired resistance to venetoclax in CLL and DLBCL. Additionally, we provide evidence that antiapoptotic BCL-2 family protein phosphorylation altered the apoptotic protein interactome, thereby changing the profile of functional dependence on these prosurvival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs rewired these dependencies, thus restoring sensitivity to venetoclax in a panel of venetoclax-resistant lymphoid cell lines, a resistant mouse model, and in paired patient samples before venetoclax treatment and at the time of progression.

Erratum: LAMA4 upregulation is associated with high liver metastasis potential and poor survival outcome of Pancreatic Cancer: Erratum.

[This corrects the article DOI: 10.7150/thno.47001.].

PIK3CB is involved in metastasis through the regulation of cell adhesion to collagen I in pancreatic cancer.

Introduction: Pancreatic adenocarcinoma (PAAD) is an aggressive malignancy, with a major mortality resulting from the rapid progression of metastasis. Unfortunately, no effective treatment strategy has been developed for PAAD metastasis to date. Thus, unraveling the mechanisms involved in PAAD metastatic phenotype may facilitate the treatment for PAAD patients. Objectives: PIK3CB is an oncogene implicated in cancer development and progression but less is known about whether PIK3CB participates in PAAD metastasis. Therefore, the objective of this study is to explore the mechanism(s) of PIK3CB in PAAD metastasis. Methods: In our study, we examined the PIK3CB expression pattern using bioinformatic analysis and clinical material derived from patients with PAAD. Subsequently, a series of biochemical experiments were conducted to investigate the role of PIK3CB as potential mechanism(s) underlying PAAD metastasis in vivo using nude mice and in vitro using cell lines. Results: We observed that PIK3CB was involved in PAAD progression. Notably, we identified that PIK3CB was involved in PAAD metastasis. Downregulation of PIK3CB significantly reduced PAAD metastatic potential in vivo. Furthermore, a series of bioinformatic analyses showed that PIK3CB was involved in cell adhesion in PAAD. Notably, PIK3CB depletion inhibited invasion potential specifically via suppressing cell adhesion to collagen I in PAAD cells. Conclusion: Collectively, our findings indicate that PIK3CB is involved in PAAD metastasis through cell-matrix adhesion. We proposed that PIK3CB is a potential therapeutic target for PAAD therapy.

Sustained IKKß phosphorylation and NF-κB activation by superoxide-induced peroxynitrite-mediated nitrotyrosine modification of B56γ3 and PP2A inactivation.

Apart from its physiological role in inflammation and immunity, the nuclear factor-kappa B (NF-κB) protein complex has been implicated in tumorigenesis and its progression. Here, we provide evidence that a pro-oxidant milieu is an upstream effector of oncogenic NF-κB signaling. Through pharmacological or genetic inhibition of SOD1, we show that elevated intracellular superoxide (O2-) mediates sustained IKK phosphorylation, and induces downstream degradation of IκBα, leading to the nuclear localization and transcriptional activation of NF-κB. Mechanistically, we show that such sustained NF-κB signaling is a function of protein phosphatase 2A (PP2A) inactivation brought about by the nitrative modification of its substrate-binding sub-unit B56γ. Importantly, the pro-oxidant driven NF-κB activation enhances the migratory and invasive potential of cancer cells. In summary, our work highlights the critical involvement of O2--dependent peroxynitrite production in inhibiting PP2A-mediated dephosphorylation of IKK, thereby facilitating cancers to acquire an invasive phenotype. Given that NF-κB is a key player of chronic inflammation and carcinogenesis, our work unravels a novel synergistic node involving O2--driven redox milieu and deregulated PP2A as a potential therapeutic target.

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.

LAMA4 upregulation is associated with high liver metastasis potential and poor survival outcome of Pancreatic Cancer.

Rationale: Pancreatic cancer is one of the most difficult cancers to manage and its poor prognosis stems from the lack of a reliable early disease biomarker coupled with its highly metastatic potential. Liver metastasis accounts for the high mortality rate in pancreatic cancer. Therefore, a better understanding of the mechanism(s) underlying the acquisition of the metastatic potential in pancreatic cancer is highly desirable. Methods: Microarray analysis in wild-type and highly liver metastatic human pancreatic cancer cell lines was performed to identify gene expression signatures that underlie the metastatic process. We validated our findings in patient samples, nude mice, cell lines and database analysis. Results: We identified a metastasis-related gene, laminin subunit alpha 4 (LAMA4), that was upregulated in highly liver metastatic human pancreatic cancer cell lines. Downregulation of LAMA4 reduced the liver metastatic ability of pancreatic cancer cells in vivo. Furthermore, LAMA4 expression was positively correlated with tumor severity and in silico analyses revealed that LAMA4 was associated with altered tumor microenvironment. In particular, our in vitro and in vivo results showed that LAMA4 expression was highly correlated with cancer-associated fibroblasts (CAFs) level which may contribute to pancreatic cancer metastasis. We further found that LAMA4 had a positive effect on the recruitment and activity of CAFs. Conclusions: These data provide evidence for LAMA4 as a possible biomarker of disease progression and poor prognosis in pancreatic cancer. Our findings indicate that LAMA4 may contribute to pancreatic cancer metastasis via recruitment or activation of CAFs.

Noncanonical Cell Fate Regulation by Bcl-2 Proteins.

Bcl-2 proteins are widely known as key controllers of mitochondrial outer membrane permeabilization, arguably the most important step of intrinsic apoptosis. Accumulating evidence indicate that most, if not all, members of the Bcl-2 protein family also mediate a number of apoptosis-unrelated functions. Intriguingly, many of these functions ultimately impinge on cell fate decisions via apoptosis-dependent or -independent mechanisms, delineating a complex network through which Bcl-2 family members regulate cell survival and death. Here, we critically discuss the mechanisms through which Bcl-2 proteins influence cell fate as they regulate autophagy, cellular senescence, inflammation, bioenergetic metabolism, Ca2+ fluxes, and redox homeostasis.

A feedforward relationship between active Rac1 and phosphorylated Bcl-2 is critical for sustaining Bcl-2 phosphorylation and promoting cancer progression.

Active GTPase-Rac1 is associated with cellular processes involved in carcinogenesis and expression of Bcl-2 endows cells with the ability to evade apoptosis. Here we provide evidence that active Rac1 and Bcl-2 work in a positive feedforward loop to promote sustained phosphorylation of Bcl-2 at serine-70 (S70pBcl-2), which stabilizes its anti-apoptotic activity. Pharmacological and genetic inactivation of Rac1 prevent interaction with Bcl-2 and reduce S70pBcl-2. Similarly, BH3-mimetic inhibitors of Bcl-2 could disrupt Rac1-Bcl-2 interaction and reduce S70pBcl-2. This effect of active Rac1 could also be rescued by scavengers of intracellular superoxide (O2.-), thus implicating NOX-activating activity of Rac1 in promoting S70pBcl-2. Moreover, active Rac1-mediated redox-dependent S70pBcl-2 involves the inhibition of phosphatase PP2A holoenzyme assembly. Sustained S70pBcl-2 in turn secures Rac1/Bcl-2 interaction. Importantly, inhibiting Rac1 activity, scavenging O2.- or employing BH3-mimetic inhibitor significantly reduced S70pBcl-2-mediated survival in cancer cells. Notably, Rac1 expression, and its interaction with Bcl-2, positively correlate with S70pBcl-2 levels in patient-derived lymphoma tissues and with advanced stage lymphoma and melanoma. Together, we provide evidence of a positive feedforward loop involving active Rac1, S70pBcl-2 and PP2A, which could have potential diagnostic, prognostic and therapeutic implications.

Reactive Oxygen Species and Oncoprotein Signaling-A Dangerous Liaison.

SIGNIFICANCE: There is evidence to implicate reactive oxygen species (ROS) in tumorigenesis and its progression. This has been associated with the interplay between ROS and oncoproteins, resulting in enhanced cellular proliferation and survival. Recent Advances: To date, studies have investigated specific contributions of the crosstalk between ROS and signaling networks in cancer initiation and progression. These investigations have challenged the established dogma of ROS as agents of cell death by demonstrating a secondary function that fuels cell proliferation and survival. Studies have thus identified (onco)proteins (Bcl-2, STAT3/5, RAS, Rac1, and Myc) in manipulating ROS level as well as exploiting an altered redox environment to create a milieu conducive for cancer formation and progression. CRITICAL ISSUES: Despite these advances, drug resistance and its association with an altered redox metabolism continue to pose a challenge at the mechanistic and clinical levels. Therefore, identifying specific signatures, altered protein expressions, and modifications as well as protein-protein interplay/function could not only enhance our understanding of the redox networks during cancer initiation and progression but will also provide novel targets for designing specific therapeutic strategies. FUTURE DIRECTIONS: Not only a heightened realization is required to unravel various gene/protein networks associated with cancer formation and progression, particularly from the redox standpoint, but there is also a need for developing more sensitive tools for assessing cancer redox metabolism in clinical settings. This review attempts to summarize our current knowledge of the crosstalk between oncoproteins and ROS in promoting cancer cell survival and proliferation and treatment strategies employed against these oncoproteins. Antioxid. Redox Signal.

The anti-oxidant and pro-oxidant dichotomy of Bcl-2.

Across a wide spectrum of cellular redox status, there emerges a dichotomy of responses in terms of cell survival/proliferation and cell death. Of note, there is emerging evidence that the anti-apoptotic protein, Bcl-2, in addition to its conventional activity of titrating the pro-apoptotic effects of proteins such as Bax and Bak at the mitochondria, also impacts cell fate decisions via modulating cellular redox metabolism. In this regard, both pro- and anti-oxidant effects of Bcl-2 overexpression have been described under different conditions and cellular contexts. In this short review, we attempt to analyze existing observations and present a probable explanation for the seemingly conflicting redox regulating activity of Bcl-2 from the standpoint of its pro-survival function. The consequential effect(s) of the dual redox functions of Bcl-2 are also discussed, particularly from the viewpoint of developing novel therapeutic strategies against cancers rendered refractory due to the aberrant expression of Bcl-2.

Overexpression of Bcl-2 induces STAT-3 activation via an increase in mitochondrial superoxide.

We recently reported a novel interaction between Bcl-2 and Rac1 and linked that to the ability of Bcl-2 to induce a pro-oxidant state in cancer cells. To gain further insight into the functional relevance of this interaction, we utilized computer simulation based on the protein pathway dynamic network created by Cellworks Group Inc. STAT3 was identified among targets that positively correlated with Rac1 and/or Bcl-2 expression levels. Validating this, the activation level of STAT3, as marked by p-Tyr705, particularly in the mitochondria, was significantly higher in Bcl-2-overexpressing cancer cells. Bcl-2-induced STAT3 activation was a function of GTP-loaded Rac1 and NADPH oxidase (Nox)-dependent increase in intracellular superoxide (O2•-). Furthermore, ABT199, a BH-3 specific inhibitor of Bcl-2, as well as silencing of Bcl-2 blocked STAT3 phosphorylation. Interestingly, while inhibiting intracellular O2•- blocked STAT3 phosphorylation, transient overexpression of wild type STAT3 resulted in a significant increase in mitochondrial O2•- production, which was rescued by the functional mutants of STAT3 (Y705F). Notably, a strong correlation between the expression and/or phosphorylation of STAT3 and Bcl-2 was observed in primary tissues derived from patients with different sub-sets of B cell lymphoma. These data demonstrate the presence of a functional crosstalk between Bcl-2, Rac1 and activated STAT3 in promoting a permissive redox milieu for cell survival. Results also highlight the potential utility of a signature involving Bcl-2 overexpression, Rac1 activation and STAT3 phosphorylation for stratifying clinical lymphomas based on disease severity and chemoresistance.

Mitochondrial ROS and involvement of Bcl-2 as a mitochondrial ROS regulator.

Mitochondria are the major intracellular source of reactive oxygen species (ROS). While excessive mitochondrial ROS (mitoROS) production induces cell injury and death, there is accumulating evidence that non-toxic low levels of mitoROS could serve as important signaling molecules. Therefore, maintenance of mitoROS at physiological levels is crucial for cell homeostasis as well as for survival and proliferation. This review describes the various mechanisms that keep mitoROS in check, with particular focus on the role of the onco-protein Bcl-2 in redox regulation. In addition to its canonical anti-apoptotic activity, Bcl-2 has been implicated in mitoROS regulation by its effect on mitochondrial complex IV activity, facilitating the mitochondrial incorporation of GSH and interaction with the small GTPase-Rac1 at the mitochondria. We also discuss some of the plausible mechanism(s) which allows Bcl-2 to sense and respond to the fluctuations in mitoROS.