sildenafil-citrate and Carcinoma--Non-Small-Cell-Lung

Pemetrexed is an approved therapeutic in NSCLC and ovarian cancer. Our studies focused on the ability of [pemetrexed + sildenafil] exposure to alter the immunogenicity of lung and ovarian cancer cells. Treatment of lung and ovarian cancer cells with [pemetrexed + sildenafil] in vitro rapidly reduced the expression of PD-L1, PD-L2 and ornithine decarboxylase (ODC), and increased the expression of class I MHCA. In a cell-specific fashion, some cells also released the immunogenic nuclear protein HMGB1 into the extracellular environment. [Pemetrexed + sildenafil] reduced the expression of multiple histone deacetylases that was blocked by knock down of autophagy regulatory proteins. [Pemetrexed + sildenafil] lethality was enhanced by the histone deacetylase inhibitors AR42 and sodium valproate; AR42 and valproate as single agents also rapidly reduced the expression of PD-L1, PD-L2 and ODC, and increased expression of MHCA and CerS6. Nitric oxide and CerS6 signaling was required for drug-induced death receptor activation and tumor cell killing. In vivo, [pemetrexed + sildenafil] lethality against lung cancer cells was enhanced by sodium valproate. Using syngeneic mouse lung cancer cells [pemetrexed + sildenafil] enhanced the anti-tumor effects of antibodies directed to inhibit PD-1 or CTLA4. [Pemetrexed + sildenafil] interacted with the anti-PD-1 antibody to strongly enhance tumor infiltration by M1 macrophages; activated NK cells and activated T cells. Our data demonstrate that treatment of tumor cells with [pemetrexed + sildenafil] results in tumor cell killing and via autophagy-dependent downregulation of HDACs, it opsonizes the remaining tumor cells to anti-tumor immunotherapy antibodies.

Topics: Animals; Antineoplastic Agents; Autophagy; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Disease Models, Animal; Drug Synergism; Female; Histone Deacetylase Inhibitors; Humans; Immunotherapy; Lung Neoplasms; Mice; Pemetrexed; Signal Transduction; Sildenafil Citrate; Xenograft Model Antitumor Assays

K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function.

Topics: AMP-Activated Protein Kinases; Animals; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Cyclic GMP-Dependent Protein Kinase Type II; Dogs; Endocytosis; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Madin Darby Canine Kidney Cells; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphorylation; ras Proteins; Serine; Signal Transduction; Sildenafil Citrate