Use of CRISPR-based screens to identify mechanisms of chemotherapy resistance.

Despite the development of new classes of targeted anti-cancer drugs, the curative treatment of metastatic solid tumors remains out of reach owing to the development of resistance to current chemotherapeutics. Although many mechanisms of drug resistance have been described, there is still a general lack of understanding of the many means by which cancer cells elude otherwise effective chemotherapy. The traditional strategy of isolating resistant clones in vitro, defining their mechanism of resistance, and testing to see whether these mechanisms play a role in clinical drug resistance is time-consuming and in many cases falls short of providing clinically relevant information. In this review, we summarize the use of CRISPR technology, including the promise and pitfalls, to generate libraries of cancer cells carrying sgRNAs that define novel mechanisms of resistance. The existing strategies using CRISPR knockout, activation, and inhibition screens, and combinations of these approaches are described. In addition, specialized approaches to identify more than one gene that may be contributing to resistance, as occurs in synthetic lethality, are described. Although these CRISPR-based approaches to cataloguing drug resistance genes in cancer cells are just beginning to be utilized, appropriately used they promise to accelerate understanding of drug resistance in cancer.

Low dose HSP90 inhibition with AUY922 blunts rapid evolution of metastatic and drug resistant phenotypes induced by TGF-ß and paclitaxel in A549 cells.

OBJECTIVES: Despite advances in cancer treatment, drug resistance and metastasis continue to contribute to treatment failure. Since drug resistance and metastasis in cancer are features that often occur toward the late stages in the disease after withstanding numerous selective pressures, they may rely on a shared adaptive mechanism in order to persist. The heat shock response is one of the most well conserved adaptive responses to cellular stress found in nature. A major player in the heat shock response is HSP90, with some studies suggesting that it can facilitate the molecular evolution of drug resistance and metastasis in cancer. Non-small cell lung cancers (NSCLCs) are strongly associated with drug resistance and metastasis either at the time of diagnosis or early in the treatment process. MATERIALS AND METHODS: We explored the role of HSP90 in the evolution of metastatic and drug resistant features in NSCLC by treating A549 cells with AUY922, a clinically relevant HSP90 inhibitor, and inducing metastatic and drug resistant phenotypes via treatment with TGF-ß and paclitaxel, respectively. We measured phenotypic plasticity in E-Cadherin, a marker for epithelial to mesenchymal transition and two ABC transporters associated with drug resistant lung cancers. RESULTS: We found that metastatic and efflux dependent drug resistant features negatively correlated with AUY922 treatment. We followed our results with functional assays relevant to metastasis and ABC transporters to confirm our results. Specifically we found the expression of E-cadherin was significantly increased in A549 cultures pretreated with AUY922 prior to exposure to paclitaxel, while expression of the drug transporters ABCB1 and ABCC1 was significantly reduced under similar conditions. CONCLUSION: Together our data indicates that HSP90 inhibition with AUY922 can limit the acquisition of metastatic and drug resistant phenotypes in A549 cells at low, clinically appropriate doses.

Effects of Intermittent Fasting and Physical Activity on Salivary Expression of Reduced Glutathione and Interleukin-1ß.

Previous research has consistently demonstrated that regular exercise promotes antioxidant production and decreases the expression of inflammation markers. However, there is very little research examining the effects of intermittent fasting (IF) on oxidative stress and inflammation. The present study investigated the hypothesis that a combination of IF and physical activity will reduce the need for glutathione (GSH) production by decreasing oxidative stress. In addition, it was hypothesized that a combination of IF and physical activity will significantly reduce inflammation, as indicated by a decrease in interleukin-1ß (IL-1ß) concentration. For three months, subjects practicing IF (n=7) ate only during an eight-hour window each day and fasted for the next 16 hours. A standard diet control group (n=18) maintained a normal, balanced diet spread out over the course of 14-18 hours each day. Based on data obtained from fitness-tracking devices, subjects were placed into one of three activity level groups: minimum, moderate, and maximum physical activity. Subjects provided fasting saliva samples monthly. The samples were subjected to a glutathione microplate assay and an interleukin ELISA test to determine salivary concentrations of GSH and IL-1ß, respectively. For GSH concentration, there were no significant differences between the diets at any physical activity level. However, moderate to maximum physical activity, in conjunction with fasting, led to significant decreases in IL-1ß concentration. In summary, results suggest that a combination of moderate physical activity and intermittent fasting promotes the maintenance of antioxidant function while inhibiting the inflammatory process.