RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. The standard treatment achieves a median overall survival for GBM patients of only 15 months. Hence, novel therapies based on an increased understanding of the mechanistic underpinnings of GBM are desperately needed. In this study, we show that elevated expression of 28S rRNA (cytosine-C(5))-methyltransferase NSUN5, which methylates cytosine 3782 of 28S rRNA in GBM cells, is strongly associated with the poor survival of GBM patients. Moreover, we demonstrate that overexpression of NSUN5 increases protein synthesis in GBM cells. NSUN5 knockdown decreased protein synthesis, cell proliferation, sphere formation, migration, and resistance to temozolomide in GBM cell lines. NSUN5 knockdown also decreased the number and size of GBM neurospheres in vitro. As a corollary, mice harboring U251 tumors wherein NSUN5 was knocked down survived longer than mice harboring control tumors. Taken together, our results suggest that NSUN5 plays a protumorigenic role in GBM by enabling the enhanced protein synthesis requisite for tumor progression. Accordingly, NSUN5 may be a hitherto unappreciated target for the treatment of GBM.

Pharmacological Inhibition of p38 MAPK by SB203580 Increases Resistance to Carboplatin in A2780cp Cells and Promotes Growth in Primary Ovarian Cancer Cells.

Chemoresistance renders current chemotherapy regimens ineffective against advanced epithelial ovarian cancer (EOC). Carboplatin (the first-line chemotherapeutic agent to treat EOC) induces cell death by regulating multiple signaling pathways. The objective of this study is to identify the signaling pathways that contribute to carboplatin resistance in EOC. To this end, we performed a proteome profiler human phospho-kinase array experiment and compared the phosphorylation profiles between the cisplatin-sensitive A2780s versus its derivative cisplatin-resistant A2780cp cells. The phospho-kinase array revealed that A2780s and A2780cp cells displayed different profiles in basal and carboplatin-induced phosphorylation. Phosphorylation of p38 MAPK was increased by carboplatin more markedly in A2780s cells compared to A2780cp cells. Inhibition of p38 MAPK activity by its specific inhibitor SB203580 increased resistance to carboplatin in A2780cp cells, but not in A2780s cells or in ascites-derived high-grade serous EOC cells. Interestingly, SB203580 increased the number of viable cells in the primary EOC cells, which was concomitant with an increase in survivin expression. In conclusion, inhibition of p38 MAPK by SB203580 increases resistance to carboplatin in A2780cp cells and the number of viable cells in the primary EOC cells, suggesting that pharmacological inhibition of p38 MAPK might not be an effective therapeutic strategy for EOC.

Notch and TGFß form a positive regulatory loop and regulate EMT in epithelial ovarian cancer cells.

Epithelial-mesenchymal transition (EMT) plays a critical role in the progression of epithelial ovarian cancer (EOC). However, the mechanisms that regulate EMT in EOC are not fully understood. Here, we report that activation of Notch1 induces EMT in EOC cells as evidenced by downregulation of E-cadherin and cytokeratins, upregulation of Slug and Snail, as well as morphological changes. Interestingly, activation of Notch1 increases TGFß/Smad signaling by upregulating the expression of TGFß and TGFß type 1 receptor. Time course experiments demonstrate that inhibition of Notch by DAPT (a γ-secretase inhibitor) decreases TGFß-induced phosphorylation of receptor Smads at late, but not at early, timepoints. These results suggest that Notch activation plays a role in sustaining TGFß/Smad signaling in EOC cells. Furthermore, inhibition of Notch by DAPT decreases TGFß induction of Slug and repression of E-cadherin and knockdown of Notch1 decreases TGFß-induced repression of E-cadherin, indicating that Notch is required, at least in part, for TGFß-induced EMT in EOC cells. On the other hand, TGFß treatment increases the expression of Notch ligand Jagged1 and Notch target gene HES1 in EOC cells. Functionally, the combination of Notch1 activation and TGFß treatment is more potent in promoting motility and migration of EOC cells than either stimulation alone. Taken together, our results indicate that Notch and TGFß form a reciprocal positive regulatory loop and cooperatively regulate EMT and promote EOC cell motility and migration.