e K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks KRas signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely, supplementation with exogenous cholesterol restores K-Ras4A but not K-Ras4B nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly, delivery of recombinant ASM or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization. Ras proteins are small guanine nucleotide binding proteins that oscillate between active GTP-bound and inactive GDP-bound states. Activated Ras proteins transmit signals for cell proliferation and cell survival. Importantly, ϳ15% of all human tumors express mutant Ras proteins that are locked in the GTP-bound state (1). Of the three ubiquitously expressed Ras isoforms, H-, N-, and K-Ras, oncogenic mutant K-Ras is the most prevalent, being expressed in ϳ95% of pancreatic, ϳ45% of colorectal, and ϳ35% of lung cancers (1). Despite its importance, there are currently no clinically approved drugs that directly target oncogenic K-Ras. To date, Ras drug discovery efforts have focused largely on inhibitors of Ras downstream effectors, including B-Raf, C-Raf, phosphatidylinositol 3-kinase (PI3K), MEK, and extracellular signal-regulated kinase (ERK) (2). For example, B-Raf-specific inhibitors produce excellent albeit often short-lived responses in patients with B-Raf mutant melanoma (3), in part because of a perturbation of complex negative-feedback control loops (2). B-Raf inhibitors also paradoxically activate the mitogen-activated protein kinase (MAPK) cascade in melanoma cells expressing oncogenic mutant N-or K-Ras (4-6). Other highly promising approaches include compounds that covalently modify K-Ras proteins with a G12C mutation to abrogate effector interactions (7,8) and allosteric modulators that directly bind Ras to inhibit guanine nucleotide exchange factor (GEF)-mediated nucleotide exchange (9-11). Chronic i...

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KRAS must localize to the plasma membrane (PM) for biological activity. We show here that multiple acid sphingomyelinase (ASM) inhibitors, including tricyclic antidepressants, mislocalized phosphatidylserine (PtdSer) and KRASG12V from the PM; resulting in abrogation of KRASG12V signaling and potent, selective growth inhibition of mutant KRAS transformed cancer cells. Concordantly, in nude mice, the ASM inhibitor fendiline decreased the rate of growth of oncogenic KRAS-expressing MiaPaCa-2 tumors, but had no effect on the growth of the wild-type KRAS-expressing BxPC-3 tumors. ASM inhibitors also inhibited activated LET-60 (a KRAS ortholog) signaling in as evidenced by suppression of the induced multi-vulva phenotype. Using RNAis against genes encoding other enzymes in the sphingomyelin (SM) biosynthetic pathway, we identified 14 enzymes whose knockdown strongly or moderately suppressed the LET-60 multi-vulva phenotype. In mammalian cells, pharmacological agents that target these enzymes all depleted PtdSer from the PM and caused KRASG12V mislocalization. These effects correlated with changes in SM levels or subcellular distribution. Selected compounds, including sphingosine kinase inhibitors, potently inhibited the proliferation of oncogenic KRAS-expressing pancreatic cancer cells. In conclusion, these results show that normal SM metabolism is critical for KRAS function, which may present therapeutic options for the treatment of KRAS-driven cancers.

BackgroundThe study of cyclooxygenase-2 (COX-2) inhibitors is now mired in controversy. We performed a meta-analysis to assess the efficacy and safety profile of COX-2 inhibitors in patients with advanced non-small-cell lung cancer (NSCLC).Patients and methodsA literature search of PubMed, EMBASE, the Cochrane Central databases, and ClinicalTrials.gov, up until March 26, 2017, identified relevant randomized controlled trials. Data analysis was performed using Stata 12.0.ResultsSix eligible trials (1,794 patients) were selected from the 407 studies that were identified initially. A significant difference, favoring COX-2 inhibitors plus chemotherapy over chemotherapy alone, was observed in the overall response rate (relative risk [RR] =1.25, 95% confidence interval [CI]: 1.06–1.48). Further, we conducted two subgroup analyses according to the type of COX-2 inhibitors (celecoxib, rofecoxib, or apricoxib) and treatment line (first or second chemotherapy). The first-line treatment includes: NP (changchun red bean + cisplatin or carboplatin), GP (double fluorine cytidine + cisplatin or carboplatin), or TP (paclitaxel + cisplatin or carboplatin, docetaxel + cisplatin or carboplatin). The second-line treatment includes two internationally recognized compounds, one is docetaxel and the other is the pemetrexed, both of which are individually selected. In subgroup analysis, significantly increased overall response rate (ORR) results were found for rofecoxib plus chemotherapy (RR =1.56, 95% CI: 1.08–2.25) and COX-2 inhibitor given with first-line chemotherapy (RR =1.27, 95% CI: 1.07–1.50). However, there was no difference between COX-2 inhibitors plus chemotherapy and chemotherapy alone in overall survival (hazard ratio [HR] =1.04, 95% CI: 0.91–1.18), progression-free survival (HR =0.97, 95% CI: 0.86–1.10), and 1-year survival rate (RR =1.03, 95% CI: 0.89–1.20). Toxicity did not differ significantly between COX-2 inhibitors plus chemotherapy and chemotherapy alone with the exception of leukopenia (RR =1.21, 95% CI: 1.03–1.42), thrombocytopenia (RR =1.32, 95% CI: 1.04–1.67), and cardiovascular events (RR =2.39, 95% CI: 1.06–5.42). The results of the Egger’s test indicated no significant difference in primary outcomes.ConclusionCOX-2 inhibitors improved ORR of advanced NSCLC with chemotherapy, but had no effect on survival indices. Moreover, COX-2 inhibitors may lead to higher rates of hematologic toxicities and cardiovascular events.

The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We, therefore, investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidyl-4-phosphate synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion significantly reduced proliferation and anchorage-independent growth of multiple KRAS-dependent cancer cell lines, and attenuated KRAS signaling in vivo. Similarly, functionally inhibiting ORP5 and ORP8 by inhibiting PI4KIIIα-mediated synthesis of phosphatidyl-4-phosphate at the PM selectively inhibited the growth of KRAS-dependent cancer cell lines over normal cells. Inhibiting KRAS function through regulating PM lipid PtdSer content may represent a viable strategy for KRAS-driven cancers.

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Objective:To analyze the clinical significance of COX-2, GLUT-1 and VEGF expressions in endometrial cancer tissues.Methods:One hundred and eight tissue samples from the patients with endometrial cancer enrolled in our hospital from August 2011 to July 2014 were selected, including 60 normal tissue samples (normal group), 60 neoplastic tissue samples (neoplastic group) and 60 cancer tissue samples (cancer group). All the samples were subjected to immunohistochemical assay to detect the expressions of COX-2, GLUT-1 and VEGF. The clinical data were also investigated for correlation analysis.Results:The positive rates of COX-2 in normal group, neoplastic group and cancer groups were 3.3%, 21.7% and 55.0% respectively. The positive rates of GLUT-1 in normal group, neoplastic group and cancer groups were 3.3%, 25.0% and 70.0% respectively. The positive rates of VEGF in normal group, neoplastic group and cancer groups were 1.7%, 23.3% and 63.3% respectively. With increasing stage of such cancer, decreasing degree of differentiation and lymphatic metastasis, the positive expression rates of COX-2, GLUT-1 and VEGF proteins were raised significantly (P<0.05). Spearman’s correlation analysis showed that the expressions of COX-2 and GLUT-1 (r=0.207, P<0.05), COX-2 and VEGF (r=0.243, P<0.05), as well as GLUT-1 and VEGF (r=0.758, P<0.05) were positively correlated.Conclusion:COX-2, GLUT-1 and VEGF were highly prominent in endometrial cancer, especially in the patients with low degree of differentiation, late stage and metastasis. They functioned synergistically in the onset and progression of this cancer.