sirolimus and lysophosphatidic-acid

Increased cytoplasmic lipid droplets (LDs) and elevated AKT/mTOR signaling are characteristics of clear cell renal cell carcinoma (ccRCC). Lysophosphatidic acid (LPA), a potent lipid mitogen generated via autotaxin (elevated in ccRCC), can modulate tumor progression but its role in altering chemotherapeutic sensitivity to mTOR inhibitors is unclear and thus is the focus of the studies presented herein. Using malignant (A-498, 769-P and 786-O) and normal immortalized kidney (HK-2) cell lines, we investigated their cellular responsiveness to Temsirolimus (TEMS, mTOR inhibitor) in the absence or presence of LPA by monitoring alterations in AKT/mTOR pathway mediators (via western blotting), LDs (using LipidTOX and real-time PCR to assess transcript changes in modulators of LD biogenesis/turnover), mitochondrial networks (via immunofluorescence staining for TOM20 and TOM70), as well as cellular viability. We identified that TEMS reduced cellular viability in all renal cell lines, with increased sensitivity in the presence of an autophagy inhibitor. TEMS also altered activation of AKT/mTOR pathway mediators, abundance of LDs, and fragmentation of mitochondrial networks. We observed that these effects were antagonized by LPA. In HK-2 cells, LPA markedly increased LD size and abundance, coinciding with phospho-MAPK and phospho-S6 activation, increased diacylglycerol O-acetyltransferase 2 (DGAT2) mRNA (which produces triacylglycerides), and survival. Inhibiting MAPK partially antagonized LPA-induced LD changes. Collectively, we have identified that LPA can reverse the effects of TEMS by increasing LDs in a MAPK-dependent manner; these results suggest that LPA may contribute to the pathogenesis and chemotherapeutic resistance of ccRCC.

Topics: Antineoplastic Agents; Autophagy; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Humans; Hydroxychloroquine; Kidney Neoplasms; Lipid Droplets; Lysophospholipids; Mitochondria; Phosphoric Diester Hydrolases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Acquisition of mesenchymal properties by cancer cells is critical for their malignant behaviour, but regulators of the mesenchymal molecular machinery and how it is activated remain elusive. Here we show that clear cell renal cell carcinomas (ccRCCs) frequently utilize the Arf6-based mesenchymal pathway to promote invasion and metastasis, similar to breast cancers. In breast cancer cells, ligand-activated receptor tyrosine kinases employ GEP100 to activate Arf6, which then recruits AMAP1; and AMAP1 then binds to the mesenchymal-specific protein EPB41L5, which promotes epithelial-mesenchymal transition and focal adhesion dynamics. In renal cancer cells, lysophosphatidic acid (LPA) activates Arf6 via its G-protein-coupled receptors, in which GTP-Gα12 binds to EFA6. The Arf6-based pathway may also contribute to drug resistance. Our results identify a specific mesenchymal molecular machinery of primary ccRCCs, which is triggered by a product of autotaxin and it is associated with poor outcome of patients.

Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Adult; Aged; Aged, 80 and over; Amides; Animals; Antineoplastic Agents; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Female; GTP-Binding Protein alpha Subunits, G12-G13; Guanine Nucleotide Exchange Factors; HEK293 Cells; Humans; Immunohistochemistry; Indoles; Isoxazoles; Kidney Neoplasms; Lysophospholipids; Male; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Nerve Tissue Proteins; Propionates; Pyridines; Pyrroles; Receptors, Lysophosphatidic Acid; Signal Transduction; Sirolimus; Sunitinib; Triazoles

Impaired endothelial recovery after the implantation of drug-eluting stents is a major concern because of the increased risk for late stent thrombosis. The disruption of the chemokine axis CXCL12/CXCR4 inhibits neointima formation by blocking the recruitment of smooth muscle progenitor cells. To directly compare a CXCR4-targeting treatment strategy with drugs that are currently used for stent coating, we studied the effects of the CXCR4 antagonist POL5551 and the drug sirolimus on neointima formation. Apolipoprotein E-deficient mice were treated with POL5551 or sirolimus continuously for 28 days after a carotid wire injury. POL5551 inhibited neointima formation by 63% (for a dosage of 2 mg/kg/day) and by 70% (for a dosage of 20 mg/kg/day). In comparison, sirolimus reduced the neointimal area by 69%. In contrast to treatment with POL5551 during the first three days after injury, injection of POL5551 (20 mg/kg) once per day for 28 days diminished neointimal hyperplasia by 53%. An analysis of the cellular composition of the neointima showed a reduction in the relative smooth muscle cell (SMC) and macrophage content in mice that had been treated with a high dose of POL5551. In contrast, the diminished SMC content after sirolimus treatment was associated with a neointimal enrichment of macrophages. Furthermore, endothelial recovery was impaired by sirolimus, but not by POL5551. Therefore, the inhibition of CXCR4 by POL5551 is equally effective in preventing neointima formation as sirolimus, but POL5551 might be more beneficial because treatment with it results in a more stable lesion phenotype and because it does not impair re-endothelialisation.

Topics: Angioplasty; Animals; Apolipoproteins E; Blood Vessel Prosthesis Implantation; Carotid Arteries; Cell Movement; Coronary Restenosis; Disease Models, Animal; Drug-Eluting Stents; Endothelium, Vascular; Humans; Lysophospholipids; Macrophages; Mice; Mice, Knockout; Myocytes, Smooth Muscle; Neointima; Postoperative Complications; Proteins; Receptors, CXCR4; Sirolimus