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.

Mesothelial cells are susceptible to asbestos fiber-induced cytotoxicity and on longer time scales to transformation; the resulting mesothelioma is a highly aggressive neoplasm that is considered as incurable at the present time Zucali et al. (Cancer Treatment Reviews 37:543–558, 2011). Only few murine cell culture models of immortalized mesothelial cells and mesothelioma cell lines exist to date. We generated SV40-immortalized cell lines derived from wild-type (WT) and neurofibromatosis 2 (merlin) heterozygote (Nf2+/−) mice, both on a commonly used genetic background, C57Bl/6J. All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum. Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate. The tumor suppressor gene NF2 is one of the most frequently mutated genes in human mesothelioma, but its detailed function is still unknown. Thus, these genotypically distinct cell lines likely relevant for malignant mesothelioma formation are expected to serve as useful in vitro models, in particular to compare with in vivo studies in mice of the same genotype. Furthermore, we generated a novel murine mesothelioma cell line RN5 originating from an Nf2+/− mouse subjected to repeated crocidolite exposure. RN5 cells are highly tumorigenic.

SummaryMalignant mesothelioma (MM) is an aggressive neoplasm characterized by a poor patient survival rate, because of rapid tumor recurrence following first-line therapy. Cancer stem cells (CSCs) are assumed to be responsible for initiating tumorigenesis and driving relapse after therapeutic interventions. CSC-enriched MM cell subpopulations were identified by an OCT4/SOX2 reporter approach and were characterized by (1) increased resistance to cisplatin, (2) increased sensitivity toward the FAK inhibitor VS-6063 in vitro, and (3) a higher tumor-initiating capacity in vivo in orthotopic xenograft and allograft mouse models. Overexpression of NF2 (neurofibromatosis 2, merlin), a tumor suppressor often mutated or lost in MM, did not affect proliferation and viability of CSC-enriched MM populations but robustly decreased the viability of reporter-negative cells. In contrast, downregulation of calretinin strongly decreased proliferation and viability of both populations. In summary, we have enriched and characterized a small MM cell subpopulation that bears the expected CSC characteristics.

Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a pain-sensing, ligand-gated, non-selective cation channel expressed in peripheral sensory neurons. Prolonged activation of TRPV1 by capsaicin leads to cell swelling and formation of membrane blebs in rat dorsal root ganglion (DRG) neurons. Similar results were obtained in NIH3T3 fibroblast cells stably expressing TRPV1. Here, we assessed the contribution of Ca(2+) and Na(+) ions to TRPV1-mediated changes. Cell swelling was caused by a substantial influx of extracellular Na(+) via TRPV1 channels, causing concomitant transport of water. In the absence of extracellular Na(+), the membrane blebbing was completely inhibited, but Ca(2+) influx did not change under these conditions. Na(+) influx was modulated by the intracellular Ca(2+) concentration ([Ca(2+)]i). Elevation of [Ca(2+)]i by ionomycin sensitized/activated TRPV1 channels causing cell swelling in TRPV1-positive cells. In the absence of extracellular Ca(2+), capsaicin caused only little increase in [Ca(2+)]i indicating that the increase in [Ca(2+)]i observed after capsaicin application is derived essentially from extracellular Ca(2+) and not from internal Ca(2+) stores. In the absence of extracellular Ca(2+) also the process of cell swelling was considerably slower. Calretinin is a Ca(2+) buffer protein, which is expressed in a subset of TRPV1-positive neurons. Calretinin decreased the amplitude, but slowed down the decay of Ca(2+) signals evoked by ionomycin. Cells co-expressing TRPV1 and calretinin were less sensitive to TRPV1-mediated, capsaicin-induced volume increases. In TRPV1-expressing NIH3T3 cells, calretinin decreased the capsaicin-induced Ca(2+) and Na(+) influx. Swelling and formation of membrane blebs resulted in impaired plasma membrane integrity finally leading to cell death. Our results hint towards a mechanistic explanation for the apoptosis-independent capsaicin-evoked neuronal loss and additionally reveal a protective effect of calretinin; we propose that the Ca(2+)-buffering capacity of calretinin reduces the susceptibility of calretinin-expressing DRG neurons against cell swelling/death caused by overstimulation of TRPV1 channels. This article is part of a Special Issue entitled:12th European Symposium on Calcium.