miR-193b/365a cluster controls progression of epidermal squamous cell carcinoma.

Incidence of cutaneous squamous cell carcinomas (cSCCs) constantly increases in the Caucasian population. Developing preferentially on precancerous lesions such as actinic keratoses due to chronic sunlight exposure, cSCCs result from the malignant transformation of keratinocytes. Although a resection of the primary tumor is usually curative, a subset of aggressive cSCCs shows a high risk of recurrence and metastases. The characterization of the molecular dysfunctions involved in cSCC development should help to identify new relevant targets against these aggressive cSCCs. In that context, we have used small RNA sequencing to identify 100 microRNAs (miRNAs) whose expression was altered during chemically induced mouse skin tumorigenesis. The decreased expression of the miR-193b/365a cluster during tumor progression suggests a tumor suppressor role. Ectopic expression of these miRNAs in tumor cells indeed inhibited their proliferation, clonogenic potential and migration, which were stimulated in normal keratinocytes when these miRNAs were blocked with antisense oligonucleotides. A combination of in silico predictions and transcriptome analyses identified several target genes of interest. We validated KRAS and MAX as direct targets of miR-193b and miR-365a. Repression of these targets using siRNAs mimicked the effects of miR-193b and miR-365a, suggesting that these genes might mediate, at least in part, the tumor-suppressive action of these miRNAs.

Tumor suppressor function of miR-483-3p on squamous cell carcinomas due to its pro-apoptotic properties.

The frequent alteration of miRNA expression in many cancers, together with our recent reports showing a robust accumulation of miR-483-3p at the final stage of skin wound healing, and targeting of CDC25A leading to an arrest of keratinocyte proliferation, led us to hypothesize that miR-483-3p could also be endowed with antitumoral properties. We tested that hypothesis by documenting the in vitro and in vivo impacts of miR-483-3p in squamous cell carcinoma (SCC) cells. miR-483-3p sensitized SCC cells to serum deprivation- and drug-induced apoptosis, thus exerting potent tumor suppressor activities. Its pro-apoptotic activity was mediated by a direct targeting of several anti-apoptotic genes, such as API5, BIRC5, and RAN. Interestingly, an in vivo delivery of miR-483-3p into subcutaneous SCC xenografts significantly hampered tumor growth. This effect was explained by an inhibition of cell proliferation and an increase of apoptosis. This argues for its further use as an adjuvant in the many instances of cancers characterized by a downregulation of miR-483-3p.

miR-483-3p controls proliferation in wounded epithelial cells.

The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up-regulation of miR-483-3p in 2 distinct models of wound healing: scratch-injured cultures of human keratinocytes and wounded skin in mice. miR-483-3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of "healing" progression. Using an in vitro wound-healing model, videomicroscopy, and 5-bromo-2'-uridine incorporation, we observed that overexpression of miR-483-3p inhibits keratinocyte migration and proliferation, whereas delivery of anti-miR-483-3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti-miR treatment. Expression profiling of keratinocytes transfected with miR-483-3p identified 39 transcripts that were both predicted targets of miR-483-3p and down-regulated after miR-483-3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR-483-3p that control keratinocyte proliferation. miR-483-3p-mediated down-regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.

Focal adhesion kinase pp125FAK interacts with the large conductance calcium-activated hSlo potassium channel in human osteoblasts: potential role in mechanotransduction.

UNLABELLED: Molecular events of mechanotransduction in osteoblasts are poorly defined. We show that the mechanosensitive BK channels open and recruit the focal adhesion kinase FAK in osteoblasts on hypotonic shock. This could convert mechanical signals in biochemical events, leading to osteoblast activation. INTRODUCTION: Mechanical strains applied to the skeleton influence bone remodeling and architecture mainly through the osteoblast lineage. The molecular mechanisms involved in osteoblastic mechanotransduction include opening of mechanosensitive cation channels and the activation of protein tyrosine kinases, notably FAK, but their interplay remains poorly characterized. The large conductance K+ channel (BK) seems likely as a bone mechanoreceptor candidate because of its high expression in osteoblasts and its ability to open in response to membrane stretch or hypotonic shock. Propagation of the signals issued from the mechanosensitivity of BK channels inside the cell likely implies complex interactions with molecular partners involved in mechanotransduction, notably FAK. METHODS: Interaction of FAK with the C terminus of the hSlo alpha-subunit of BK was investigated using the yeast two-hybrid system as well as immunofluorescence microscopy and coimmunoprecipitation experiments with a rabbit anti-hslo antibody on MG63 and CAL72 human osteosarcoma cell lines and on normal human osteoblasts. Mapping of the FAK region interacting with hSlo was approached by testing the ability of hSlo to recruit mutated ot truncated FAK proteins. RESULTS: To the best of our knowledge, we provide the first evidence of the physical association of FAK with the intracellular part of hslo. We show that FAK/hSlo interaction likely takes place through the Pro-1-rich domain situated in the C-terminal region of the kinase. FAK/hSlo association occurs constitutively at a low, but appreciable, level in human osteosarcoma cells and normal human osteoblasts that express endogenous FAK and hSlo. In addition, we found that application of an hypo-osmotic shock to these cells induced a sustained activation of BK channels associated to a marked increase in the recruitment of FAK on hSlo. CONCLUSIONS: Based on these data, we propose that BK channels might play a triggering role in the signaling cascade induced by mechanical strains in osteoblasts.

Prostaglandin E2 induces interaction between hSlo potassium channel and Syk tyrosine kinase in osteosarcoma cells.

Prostaglandins (PGs) are important mediators of bone response to growth factors, hormones, inflammation, or mechanical strains. In this study, we show that in MG63 osteosarcoma cells, prostaglandin E2 (PGE2) produces the opening of a large conductance Ca2+-dependent K+ channel (BK). This PGE2-mediated channel opening induces the recruitment of various tyrosine-phosphorylated proteins on the hSlo alpha-subunit of BK. Because the C-terminal domain of hSlo encompasses an immunoreceptor tyrosine-based activation motif (ITAM), we show that the Syk nonreceptor tyrosine kinase, reported yet to be expressed mainly in hematopoietic cells, is expressed also in osteoblastic cells, and recruited on this ITAM after a PGE2-induced docking/activation process. We show that Syk/hSlo association is dependent of an upstream Src-related tyrosine kinase activity, in accord with the classical two-step model described for immune receptors. Finally, we provide evidence that this Syk/hSlo interaction does not affect the electrical features of BK channels in osteosarcoma cells. With these data, we would like to suggest the new notion that besides its conductance function, hSlo channel can behave in bone cells, as a true transduction protein intervening in the bone remodeling induced by PGE2.