Epigenomic changes are an important feature of malignant tumors. How tumor aggressiveness is affected by DNA methylation of specific loci is largely unexplored. In genome-wide DNA methylation analyses, we identified the K Ca 3.1 channel gene (KCNN4) promoter to be hypomethylated in an aggressive non-small-cell lung carcinoma (NSCLC) cell line and in patient samples. Accordingly, K Ca 3.1 expression was increased in more aggressive NSCLC cells. Both findings were strong predictors for poor prognosis in lung adenocarcinoma. Increased K Ca 3.1 expression was associated with aggressive features of NSCLC cells. Proliferation and migration of pro-metastatic NSCLC cells depended on K Ca 3.1 activity. Mechanistically, elevated K Ca 3.1 expression hyperpolarized the membrane potential, thereby augmenting the driving force for Ca 21 influx. K Ca 3.1 blockade strongly reduced the growth of xenografted NSCLC cells in mice as measured by positron emission tomography-computed tomography. Thus, loss of DNA methylation of the KCNN4 promoter and increased K Ca 3.1 channel expression and function are mechanistically linked to poor survival of NSCLC patients.Lung cancer is a leading cause of cancer-related death. 1 The 5-year survival rate of patients with non-small-cell lung cancer (NSCLC), which accounts for 80% of lung cancers, may be as low as $10%. 2-4 Unfortunately, many patients develop local or distant metastasis relapse even after complete resection. Thus, high aggressiveness is an intrinsic feature of many NSCLC tumors. Adjuvant and palliative chemotherapy are of limited benefit in NSCLC, and novel therapeutic targets are needed.Individual NSCLC cancers harbor multiple mutations in protein-coding genes, most of which cannot be targeted therapeutically. 5 In contrast, epigenetic changes might be targetable. Effector mechanisms, e.g., for metastatic spread, could be promising therapeutic targets. Metastasis development originates from subpopulations of tumor cells that acquire additional features for increased aggressiveness. These features depend on changes in gene expression, including that of ion channels, 6-8 presumably often by epigenetic mechanisms rather than metastasis-specific mutations. 9-11 Altered DNA methylation of specific loci as a stable epigenetic mark is a likely culprit in many instances. DNA hypermethylation and gene silencing occur in CpG islands and promoters of tumor suppressors. 9 DNA hypomethylation is thought to occur mainly in intergenic regions, with effects mainly on genome stability. 9 However, the potential pathophysiological and clinical relevance of DNA hypomethylation of specific

Orai channels have been associated with cell proliferation, survival and metastasis in several cancers. The present study investigates the expression and the role of Orai3 in cell proliferation in non-small cell lung cancer (NSCLC). We show that Orai3 is over-expressed in cancer tissues as compared to the non-tumoral ones. Furthermore, Orai3 staining is stronger in high grade tumors. Pharmacological inhibition or knockdown of Orai3 significantly reduced store operated calcium entry (SOCE), inhibited cell proliferation and arrested cells of two NSCLC cell lines in G0/G1 phase. These effects were concomitant with a down-regulation of cyclin D1, cyclin E, CDK4 and CDK2 expression. Moreover, Orai3 silencing decreased Akt phosphorylation levels. In conclusion, Orai3 constitutes a native SOCE pathway in NSCLC that controls cell proliferation and cell cycle progression likely via Akt pathway.

Intracellular Ca2+ levels are important regulators of cell cycle and proliferation. We, and others, have previously reported the role of KCa3.1 (KCNN4) channels in regulating the membrane potential and the Ca2+ entry in association with cell proliferation. However, the relevance of KC3.1 channels in cancer prognosis as well as the molecular mechanism of Ca2+ entry triggered by their activation remain undetermined. Here, we show that RNAi-mediated knockdown of KCa3.1 and/or TRPC1 leads to a significant decrease in cell proliferation due to cell cycle arrest in the G1 phase. These results are consistent with the observed upregulation of both channels in synchronized cells at the end of G1 phase. Additionally, knockdown of TRPC1 suppressed the Ca2+ entry induced by 1-EBIO-mediated KCa3.1 activation, suggesting a functional cooperation between TRPC1 and KCa3.1 in the regulation of Ca2+ entry, possibly within lipid raft microdomains where these two channels seem to co-localize. We also show significant correlations between KCa3.1 mRNA expression and poor patient prognosis and unfavorable clinical breast cancer parameters by mining large datasets in the public domain. Together, these results highlight the importance of KCa3.1 in regulating the proliferative mechanisms in breast cancer cells as well as in providing a promising novel target in prognosis and therapy.

ObjectiveHepatocellular carcinoma (HCC) development occurs with non-alcoholic fatty liver disease (NAFLD) in the absence of cirrhosis and with an increasing incidence due to the obesity pandemic. Mutations of tumour suppressor (TS) genes and oncogenes (ONC) have been widely characterised in HCC. However, mounting evidence indicates that non-genomic alterations of TS/ONC occur early with NAFLD, thereby potentially promoting hepatocarcinogenesis in an inflammatory/fibrotic context. The aim of this study was to identify and characterise these alterations.DesignThe proteome of steatotic liver tissues from mice spontaneously developing HCC was analysed. Alterations of TSs/ONCs were further investigated in various mouse models of NAFLD/HCC and in human samples. The inflammatory, fibrogenic and oncogenic functions of S100A11 were assessed through in vivo, in vitro and ex-vivo analyses.ResultsA whole set of TSs/ONCs, respectively, downregulated or upregulated was uncovered in mice and human with NAFLD. Alterations of these TSs/ONCs were preserved or even exacerbated in HCC. Among them, overexpression of S100A11 was associated with high-grade HCC and poor prognosis. S100A11 downregulation in vivo significantly restrains the development of inflammation and fibrosis in mice fed a choline/methionine-deficient diet. Finally, in vitro and ex-vivo analyses revealed that S100A11 is a marker of hepatocyte de-differentiation, secreted by cancer cells, and promoting cell proliferation and migration.ConclusionCellular stress associated with NAFLD triggers non-genomic alterations of a whole network of TSs/ONCs fostering hepatocarcinogenesis. Among those, overexpression of the oncogenic factor S100A11 promotes inflammation/fibrosis in vivo and is significantly associated with high-grade HCC with poor prognosis.

Activin, a member of the transforming growth factor B (TGFB) superfamily, regulates diverse processes, such as cellular growth and differentiation. There is increasing evidence that TGFB and its signaling effectors are key determinants of tumor cell behavior. Loss of sensitivity to TGFB-induced growth arrest is an important step toward malignancy. We previously characterized FLRG as an extracellular antagonist of activin. Here, we show that activin-induced growth inhibition is altered in FLRG-expressing breast cancer lines. Silencing FLRG induced growth inhibition, which is reversible upon addition of exogenous FLRG. We showed that FLRG silencing effects resulted from restoration of endogenous activin functions as shown by increased levels of phosphorylated smad2 and up-regulation of activin target gene transcripts. Furthermore, the growth inhibition induced by FLRG silencing was reversible by treatment with a soluble form of type II activin receptor. Finally, a strong expression of FLRG was observed in invasive breast carcinomas in contrast with the normal luminal epithelial cells in which FLRG was not detected. Our data provide strong evidence that endogenous FLRG contributes to tumor cell proliferation through antagonizing endogenous activin effects. [Cancer Res 2007;67(15):7223-9]