Hypoxia and MITF control metastatic behaviour in mouse and human melanoma cells.

Melanomas are very aggressive neoplasms with notorious resistance to therapeutics. It was recently proposed that the remarkable phenotypic plasticity of melanoma cells allows for the rapid development of both resistance to chemotherapeutic drugs and invasive properties. Indeed, the capacity of melanoma cells to form distant metastases is the main cause of mortality in melanoma patients. Therefore, the identification of the mechanism controlling melanoma phenotype is of paramount importance. In the present report, we show that deletion of microphthalmia-associated transcription factor (MITF), the master gene in melanocyte differentiation, is sufficient to increase the metastatic potential of mouse and human melanoma cells. MITF silencing also increases fibronectin and Snail, two mesenchymal markers that might explain the increased invasiveness in vitro and in vivo. Furthermore, ablation of this population by Forskolin-induced differentiation or MITF-forced expression significantly decreases tumour and metastasis formation, suggesting that eradication of low-MITF cells might improve melanoma treatment. Moreover, we demonstrate that a hypoxic microenvironment decreases MITF expression through an indirect, hypoxia-inducible factor 1 (HIF1)α-dependant transcriptional mechanism, and increases the tumourigenic and metastatic properties of melanoma cells. We identified Bhlhb2, a new factor in melanoma biology, as the mediator of hypoxia/HIF1α inhibitory effect on MITF expression. Our results reveal a hypoxia-HIF1α-BHLHB2-MITF cascade controlling the phenotypic plasticity in melanoma cells and favouring metastasis development. Targeting this pathway might be helpful in the design of new anti-melanoma therapies.

SPARC functions as an anti-stress factor by inactivating p53 through Akt-mediated MDM2 phosphorylation to promote melanoma cell survival.

Aberrant expression of Secreted Protein Acidic and Rich in Cysteine (SPARC)/osteonectin has been associated with an invasive tumor cell phenotype and poor outcome in human melanomas. Although it is known that SPARC controls melanoma tumorigenesis, the precise role of SPARC in melanoma cell survival is still unclear. Here, we show that SPARC has a cell-autonomous survival activity, which requires Akt-dependent regulation of p53. Suppression of SPARC by RNA interference in several human melanoma cells and xenografted A375 tumors triggers apoptotic cell death through the mitochondrial intrinsic pathway and activation of caspase-3. Cell death induced by depletion of SPARC is dependent on p53 and induction of Bax, and results in the generation of ROS. Stabilization of p53 in SPARC-depleted cells is associated with a decrease in Akt-mediated activating phosphorylation of MDM2. Inhibition of Akt signaling pathway is important for the observed changes as overexpression of constitutively active Akt protects cells against apoptosis induced by SPARC depletion. Conversely, increased expression of SPARC stimulates Akt and MDM2 phosphorylation, thus facilitating p53 degradation. Finally, we show that overexpression of SPARC renders cells more resistant to the p53-mediated cytotoxic effects of the DNA-damaging drug actinomycin-D. Our study indicates that SPARC functions through activation of Akt and MDM2 to limit p53 levels and that acquired expression of SPARC during melanoma development would confer survival advantages through suppression of p53-dependent apoptotic pathways.

Pharmacological targeting of NF-kappaB potentiates the effect of the topoisomerase inhibitor CPT-11 on colon cancer cells.

NF-kappaB interferes with the effect of most anti-cancer drugs through induction of anti-apoptotic genes. Targeting NF-kappaB is therefore expected to potentiate conventional treatments in adjuvant strategies. Here we used a pharmacological inhibitor of the IKK2 kinase (AS602868) to block NF-kappaB activation. In human colon cancer cells, inhibition of NF-kappaB using 10 microM AS602868 induced a 30-50% growth inhibitory effect and strongly enhanced the action of SN-38, the topoisomerase I inhibitor and CPT-11 active metabolite. AS602868 also potentiated the cytotoxic effect of two other antineoplasic drugs: 5-fluorouracil and etoposide. In xenografts experiments, inhibition of NF-kappaB potentiated the antitumoural effect of CPT-11 in a dose-dependent manner. Eighty-five and 75% decreases in tumour size were observed when mice were treated with, respectively, 20 or 5 mg kg(-1) AS602868 associated with 30 mg kg(-1) CPT-11 compared to 47% with CPT-11 alone. Ex vivo tumour analyses as well as in vitro studies showed that AS602868 impaired CPT-11-induced NF-kappaB activation, and enhanced tumour cell cycle arrest and apoptosis. AS602868 also enhanced the apoptotic potential of TNFalpha on HT-29 cells. This study is the first demonstration that a pharmacological inhibitor of the IKK2 kinase can potentiate the therapeutic efficiency of antineoplasic drugs on solid tumours.