Malignant mesothelioma (MM) are highly aggressive asbestos-related neoplasms, which show strong chemotherapy resistance, and there is no effective cure for MM so far. Calretinin (CR) is widely used as a diagnostic marker for epithelioid and mixed (biphasic) mesothelioma; however, little is known about CR's putative functions in tumorigenesis. CR protects against asbestos-induced acute cytotoxicity mediated by the AKT/PI3K pathway, and furthermore, SV40 early region genes are able to upregulate CR in mesothelial cells. However, the precise role of CR in mesothelioma is still unknown. Downregulation of CR via lentiviral-mediated short-hairpin RNA significantly decreased the viability and proliferation of mesothelioma cells in vitro.The effect was strong in epithelioid-dominated cell lines (ZL55 and MSTO-211H). A weaker and delayed effect was observed in mesothelioma cells with prevalent sarcomatoid morphology (SPC111, SPC212 and ZL34). The specificity of the effect was confirmed by stable enhanced green fluorescent protein-CR expression in mesothelioma cell lines and subsequent downregulation. Depletion of CR led these cancer cell lines to enter apoptosis within 72 hr postinfection via strong activation of the intrinsic caspase 9-dependent pathway. Downregulation of CR in immortalized mesothelial cells LP9/TERT-1 strongly blocked proliferation and caused a G 1 block without decreasing viability or activating apoptosis pathways. These results demonstrate that downregulation of CR had a strong effect on the viability of MM cells and that CR is essential for cells derived from MM. The authors anticipate these findings to reveal CR as a highly interesting new putative therapeutic target for mesothelioma treatment of especially the epithelioid, as well as of the mixed and sarcomatoid type.Malignant mesothelioma (MM) is a neoplasm developing from mesothelial cells of the pleural, pericardial and peritoneal cavities and is prevalently associated with asbestos exposure. In the United States alone, more than 20 million people were exposed to asbestos and could potentially develop MM.

Chronic exposure to intraperitoneal asbestos triggered a marked response in the mesothelium well before tumor development. Macrophages, mesothelial precursor cells, cytokines, and growth factors accumulated in the peritoneal lavage. Transcriptome profiling revealed YAP/TAZ activation in inflamed mesothelium with further activation in tumors, paralleled by increased levels of cells with nuclear YAP/TAZ. Arg1 was one of the highest upregulated genes in inflamed tissue and tumor. Inflamed tissue showed increased levels of single-nucleotide variations, with an RNA-editing signature, which were even higher in the tumor samples. Subcutaneous injection of asbestos-treated, but tumor-free mice with syngeneic mesothelioma tumor cells resulted in a significantly higher incidence of tumor growth when compared to naïve mice supporting the role of the environment in tumor progression.

Vanilloids including capsaicin and resiniferatoxin are potent transient receptor potential vanilloid type 1 (TRPV1) agonists. TRPV1 overstimulation selectively ablates capsaicin-sensitive sensory neurons in animal models in vivo. The cytotoxic mechanisms are based on strong Na(+) and Ca(2+) influx via TRPV1 channels, which leads to mitochondrial Ca(2+) accumulation and necrotic cell swelling. Increased TRPV1 expression levels are also observed in breast and prostate cancer and derived cell lines. Here, we examined whether potent agonist-induced overstimulation mediated by TRPV1 might represent a means for the eradication of prostate carcinoma (PC-3, Du 145, LNCaP) and breast cancer (MCF7, MDA-MB-231, BT-474) cells in vitro. While rat sensory neurons were highly vanilloid-sensitive, normal rat prostate epithelial cells were resistant in vivo. We found TRPV1 to be expressed in all cancer cell lines at mRNA and protein levels, yet protein expression levels were significantly lower compared to sensory neurons. Treatment of all human carcinoma cell lines with capsaicin didn't lead to overstimulation cytotoxicity in vitro. We assume that the low vanilloid-sensitivity of prostate and breast cancer cells is associated with low expression levels of TRPV1, since ectopic TRPV1 expression rendered them susceptible to the cytotoxic effect of vanilloids evidenced by plateau-type Ca(2+) signals, mitochondrial Ca(2+) accumulation and Na(+)- and Ca(2+)-dependent membrane disorganization. Moreover, long-term monitoring revealed that merely the ectopic expression of TRPV1 stopped cell proliferation and often induced apoptotic processes via strong activation of caspase-3 activity. Our results indicate that specific targeting of TRPV1 function remains a putative strategy for cancer treatment.

SummaryChronic exposure to intraperitoneal asbestos triggered a marked response in the mesothelium well before tumor development. Macrophages, mesothelial precursor cells, cytokines and growth factors accumulated in the peritoneal lavage. Transcriptome profiling revealed YAP/TAZ activation in inflamed mesothelium with further activation in tumors, paralleled by increased levels of cells with nuclear YAP/TAZ. Arg1 was one of the highest upregulated genes in inflamed tissue and tumor. Inflamed tissue showed increased levels of single nucleotide variations, with an RNA-editing signature, which were even higher in the tumor samples. Subcutaneous injection of asbestos-treated, but tumor-free mice with syngeneic mesothelioma tumor cells resulted in a significantly higher incidence of tumor growth when compared to naïve mice supporting the role of the environment in tumor progression.

Calretinin (CR) is used as a positive marker for human malignant mesothelioma (MM) and is essential for mesothelioma cell growth/survival. Yet, the putative role(s) of CR during MM formation in vivo, binding partners or CR’s influence on specific signaling pathways remain unknown. We assessed the effect of CR overexpression in the human MM cell lines MSTO-211H and SPC111. CR overexpression augmented the migration and invasion of MM cells in vitro. These effects involved the activation of the focal adhesion kinase (FAK) signaling pathway, since levels of total FAK and phospho-FAK (Tyr397) were found up-regulated in these cells. CR was also implicated in controlling epithelial-to-mesenchymal transition (EMT), evidenced by changes of the cell morphology and up-regulation of typical EMT markers. Co-IP experiments revealed FAK as a new binding partner of CR. CR co-localized with FAK at focal adhesion sites; moreover, CR-overexpressing cells displayed enhanced nuclear FAK accumulation and an increased resistance towards the FAK inhibitor VS-6063. Finally, CR downregulation via a lentiviral shRNA against CR (CALB2) resulted in a significantly reduced tumor formation in vivo in an orthotopic xenograft mouse model based on peritoneal MM cell injection. Our results indicate that CR might be considered as a possible target for MM treatment.