Glioblastoma (GBM) is often treated with the cytotoxic drug temozolomide (TMZ) but the disease inevitably recurs in a drug-resistant form after initial treatment. Here we report that in GBM cells even a modest decrease in the mismatch repair (MMR) components MSH2 and MSH6 have profound effects on TMZ sensitivity. RNAi-mediated attenuation of MSH2 and MSH6 showed that such modest decreases provided an unexpectedly strong mechanism of TMZ resistance. In a mouse xenograft model of human GBM, small changes in MSH2 were sufficient to suppress TMZ-induced tumor regression. Using the Cancer Genome Atlas to analyze mRNA expression patterns in tumors from TMZ-treated GBM patients, we found that MSH2 transcripts in primary GBM could predict patient responses to initial TMZ therapy. In recurrent disease, the absence of microsatellite instability (the standard marker for MMR deficiency) suggests a lack of involvement of MMR in the resistant phenotype of recurrent disease. However, more recent studies reveal that decreased MMR protein levels occur often in recurrent GBM. In accordance with our findings, these reported decreases may constitute a mechanism by which GBM evades TMZ sensitivity while maintaining microsatellite stability. Overall, our results highlight the powerful effects of MSH2 attenuation as a potent mediator of TMZ resistance, and argue that MMR activity offers a predictive marker for initial therapeutic response to TMZ treatment.
The appetite suppressant actions of estradiol are due to its ability to attenuate orexigenic signals and potentiate anorexigenic signals. The work from my laboratory has shown that male guinea pigs are more sensitive to the hyperphagic and hypothermic effects of cannabinoids than their female counterparts. Cannabinoid sensitivity is further dampened by the activational effects of estradiol. This occurs via the hypothalamic feeding circuitry, where estradiol rapidly attenuates the cannabinoid CB1 receptor-mediated presynaptic inhibition of glutamatergic input onto anorexigenic proopiomelanocortin (POMC) neurons in the arcuate nucleus. This disruption is blocked by the estrogen receptor antagonist ICI 182,780, and associated with increased expression of phosphatidylinositol-3-kinase (PI3K). Moreover, the ability of estradiol to reduce both the cannabinoid-induced hyperphagia and glutamate release onto POMC neurons is abrogated by the PI3K inhibitor PI 828.
The peptide orphanin FQ/nociceptin (OFQ/N) activates opioid receptor-like (ORL)1 receptors to hyperpolarize and inhibit POMC neurons via the activation of postsynaptic G protein-gated, inwardly-rectifying (GIRK) channels. We have demonstrated that the fasting-induced hyperphagia observed in ORL1-null mice is blunted compared to wild type controls. In addition, the ORL1 receptor-mediated activation of GIRK channels in POMC neurons from ovariectomized female rats is markedly impaired by estradiol. The estrogenic attenuation of presynaptic CB1 and postsynaptic ORL1 receptor function may be part of a more generalized mechanism through which anorexigenic hormones suppress orexigenic signaling. Indeed, we have found that leptin robustly suppresses the OFQ/N-induced activation of GIRK channels in POMC neurons. Furthermore, its ability to augment excitatory input onto POMC neurons is blocked by PI 828. Thus, estradiol and other hormones like leptin reduce energy intake at least partly by activating PI3K to disrupt the pleiotropic functions of Gi/o-coupled receptors that inhibit anorexigenic POMC neurons.
<p>Supplementary Methods and Discussion. Description of additional methods and procedures used in the study. Also includes Supplementary References.</p>
<p>Supplementary Figures S1-S14. p53 levels in p53 knockdown GBM cells (S1); TMZR3 cells obtained from a p53 deficient background display increased ploidy (S2); H2AX activation in temozolomide treated parental and TMZR3 GBM cells (S3); In-cell Host cell reactivation (HCR) assays used to assess the MGMT and MMR repair capacity of GBM cells (S4); MSH6 and MSH2 levels in MSH6 and MSH2 knockdown GBM cells (S5); Cell cycle profiles and quantitation of cell cycle changes in TMZ-treated MSH6 knockdown cells two cell cycle times post-TMZ treatment (S6); Cell cycle profiles and quantitation of cell cycle changes in TMZ-treated MSH2 knockdown cells two cell cycle times post-TMZ treatment (S7); Minor decreases in MSH2 levels correlate with acquired TMZ resistance in LN229 and A172 GBM cells (S8); Effects of MSH6 or MSH2 knockdown on the protein level of its dimerization partner (S9); Msh2 knockdown in GL261 glioblastoma cells (S10); Moderate decreases in Msh2 confer a growth advantage to GL261 GBM cells after TMZ, but not BCNU, exposure in vitro (S11); Distribution of patient survival in TMZ treated TCGA GBM patients and effects of MSH2 transcript levels on the survival of TMZ treated patients in the 95th percentile for patient survival after TMZ therapy (S12); Overall survival of GBM patients stratified by MSH3, MLH1 and PMS2 tumor transcript levels (S13); PMS2 transcript levels correlate with increased chromosome 7 copy number (S14).</p>
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