p66ShcA promotes malignant breast cancer phenotypes by alleviating energetic and oxidative stress.

Significant efforts have focused on identifying targetable genetic drivers that support the growth of solid tumors and/or increase metastatic ability. During tumor development and progression to metastatic disease, physiological and pharmacological selective pressures influence parallel adaptive strategies within cancer cell sub-populations. Such adaptations allow cancer cells to withstand these stressful microenvironments. This Darwinian model of stress adaptation often prevents durable clinical responses and influences the emergence of aggressive cancers with increased metastatic fitness. However, the mechanisms contributing to such adaptive stress responses are poorly understood. We now demonstrate that the p66ShcA redox protein, itself a ROS inducer, is essential for survival in response to physiological stressors, including anchorage independence and nutrient deprivation, in the context of poor outcome breast cancers. Mechanistically, we show that p66ShcA promotes both glucose and glutamine metabolic reprogramming in breast cancer cells, to increase their capacity to engage catabolic metabolism and support glutathione synthesis. In doing so, chronic p66ShcA exposure contributes to adaptive stress responses, providing breast cancer cells with sufficient ATP and redox balance needed to withstand such transient stressed states. Our studies demonstrate that p66ShcA functionally contributes to the maintenance of aggressive phenotypes and the emergence of metastatic disease by forcing breast tumors to adapt to chronic and moderately elevated levels of oxidative stress.

Sarm1 activation produces cADPR to increase intra-axonal Ca++ and promote axon degeneration in PIPN.

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a "dying-back" axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium-modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating paclitaxel-induced peripheral neuropathy (PIPN).

Reprogramming of Nucleotide Metabolism Mediates Synergy between Epigenetic Therapy and MAP Kinase Inhibition.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but often lethal cancer that is diagnosed at a median age of 24 years. Optimal management of patients is not well defined, and current treatment remains challenging, necessitating the discovery of novel therapeutic approaches. The identification of SMARCA4-inactivating mutations invariably characterizing this type of cancer provided insights facilitating diagnostic and therapeutic measures against this disease. We show here that the BET inhibitor OTX015 acts in synergy with the MEK inhibitor cobimetinib to repress the proliferation of SCCOHT in vivo Notably, this synergy is also observed in some SMARCA4-expressing ovarian adenocarcinoma models intrinsically resistant to BETi. Mass spectrometry, coupled with knockdown of newly found targets such as thymidylate synthase, revealed that the repression of a panel of proteins involved in nucleotide synthesis underlies this synergy both in vitro and in vivo, resulting in reduced pools of nucleotide metabolites and subsequent cell-cycle arrest. Overall, our data indicate that dual treatment with BETi and MEKi represents a rational combination therapy against SCCOHT and potentially additional ovarian cancer subtypes.

Methotrexate elicits pro-respiratory and anti-growth effects by promoting AMPK signaling.

One-carbon metabolism fuels the high demand of cancer cells for nucleotides and other building blocks needed for increased proliferation. Although inhibitors of this pathway are widely used to treat many cancers, their global impact on anabolic and catabolic processes remains unclear. Using a combination of real-time bioenergetics assays and metabolomics approaches, we investigated the global effects of methotrexate on cellular metabolism. We show that methotrexate treatment increases the intracellular concentration of the metabolite AICAR, resulting in AMPK activation. Methotrexate-induced AMPK activation leads to decreased one-carbon metabolism gene expression and cellular proliferation as well as increased global bioenergetic capacity. The anti-proliferative and pro-respiratory effects of methotrexate are AMPK-dependent, as cells with reduced AMPK activity are less affected by methotrexate treatment. Conversely, the combination of methotrexate with the AMPK activator, phenformin, potentiates its anti-proliferative activity in cancer cells. These data highlight a reciprocal effect of methotrexate on anabolic and catabolic processes and implicate AMPK activation as a metabolic determinant of methotrexate response.

Comparison of N-ethyl maleimide and N-(1-phenylethyl) maleimide for derivatization of biological thiols using liquid chromatography-mass spectrometry.

The ratio between reduced and oxidized thiols, mainly glutathione and oxidized glutathione, is one of the biomarkers for the evaluation of oxidative stress. The accurate measurement of thiol concentrations is challenging because reduced thiols are easily oxidized during sample manipulation. Derivatization is commonly used to protect thiols from oxidation. The objective of this work was to systematically compare two cell-permeable derivatizing agents: N-ethyl maleimide (NEM) and (R)-(+)-N-(1-phenylethyl)maleimide (NPEM) in terms of derivatization efficiency, ionization enhancement, side product formation, reaction selectivity for thiols, pH dependence of the reaction, and derivative stability. All thiol measurements and the characterization of side products were performed using a biphenyl reversed phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Four thiols, cysteine (CYS), homocysteine, N-acetylcysteine (NAC), and glutathione (GSH), were used for the evaluation. Using 1:10 ratio of thiol:derivatizing agent, complete derivatization was obtained within 30 min for both agents tested with the exception of CYS-NEM, where 97% efficiency was obtained. The more hydrophobic NPEM provided better ionization of the thiols, with enhancement ranging from 2.1x for GSH to 5.7x for CYS in comparison to NEM. NPEM derivatization led to more extensive side reactions, such as double derivatization and ring opening, which hindered the accurate measurement of the thiol concentrations. Both NEM and NPEM also showed poor stability of CYS derivative due to its time-dependent conversion to cyclic cysteine-maleimide derivative. Both reagents also showed significant reactivity with amine-containing metabolites depending on the pH used during derivatization, but overall NEM was found to be more selective towards thiol group than NPEM. Taking into account all evaluation criteria, NEM was selected as a more suitable reagent for the thiol protection and derivatization, but strict control of pH 7.0 is recommended to minimize the side reactions. This work illustrates the importance of the characterization of side products and derivative stability during the evaluation of thiol derivatizing agents and contributes fundamental understanding to improve the accuracy of thiol determinations. The key sources of errors during maleimide derivatization include the derivatization of amine-containing metabolites, poor derivative stability of certain thiols (CYS and NAC), and the side reactions especially if ring opening of the reagent is not minimized. Graphical abstract.