TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis.

BACKGROUND: TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown. METHODS: miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC. RESULTS: miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels. CONCLUSION: We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.

Id-1B, an alternatively spliced isoform of the inhibitor of differentiation-1, impairs cancer cell malignancy through inhibition of proliferation and angiogenesis.

Id-1 is a member of the helix-loop-helix family of proteins that regulates the activity of transcription factors to suppress cellular differentiation and to promote cell growth. Overexpression of Id-1 in tumor cells correlates with increased malignancy and resistance to chemotherapy and radiotherapy. Id-1B is an isoform generated by alternative splicing that differs from the classical Id-1 in the 13-C-terminal amino acids, whose function is at present unknown. We have studied the role of Id-1B in cancer and its expression in healthy/malignant lung tissues. Overexpression of Id-1B in A549 lung and PC3 prostate cancer cells reduced anchorage-dependent and independent proliferation and clonogenic potential. Moreover, it increased the proportion of cells in the G0/G1 phase of the cell cycle and p27 levels, while reduced phospho-Erk and cyclin A levels. Through microarray analysis, we identified genes involved in cell growth and proliferation that are specifically deregulated as a consequence of Id-1B overexpression, including IGF2, BMP4, Id2, GATA3, EREG and AREG. Id-1B overexpressing cells that were treated with 4Gy irradiation dose were significantly less resistant to cell death. In vivo assays demonstrated that tumors with high Id-1B levels exhibited less growth (p<0.01), metabolic activity (glucose uptake) and angiogenesis (p<0.05) compared to tumors with low Id-1B expression; mice survival was significantly extended (p<0.05). Quantification by qRT-PCR revealed that expression of Id-1B was significantly lower (p<0.01) in human lung tumors compared to their matched nonmalignant counterparts. In conclusion, our results demonstrate that Id-1B decreases the malignancy of lung and prostate cancer cells, sensitizes them to radiotherapy-induced cell death, and counteracts the protumorigenic role of the classical form of Id-1.

The quinoline imidoselenocarbamate EI201 blocks the AKT/mTOR pathway and targets cancer stem cells leading to a strong antitumor activity.

Methylimidoselenocarbamates have previously proven to display potent antitumor activities. In the present study we show that these compounds act as multikinase inhibitors. We found that the most effective compound, quinoline imidoselenocarbamate EI201, inhibits the PI3K/AKT/mTOR pathway, which is persistently activated and contributes to malignant progression in various cancers. EI201 blocked the phosphorylation of AKT, mTOR and several of its downstream regulators (p70S6K and 4E-BP1) and ERK1/2 in PC-3, HT-29 and MCF-7 cells in vitro, inducing both autophagy and apoptosis. EI201 also contributes to the loss of maintenance of the selfrenewal and tumorigenic capacity of cancer stem cells (CSCs). 0.1 µmol/L EI201 triggered a reduction in size and number of tumorspheres in PC-3, HT-29 and MCF-7 cells and 4 µmol/L induced the elimination of almost all the tumorspheres in the three studied cell lines. In addition, EI201 suppressed almost 80% prostate tumor growth in vivo (p < 0.01) compared to controls at a relatively low dose (10 mg/kg) in a mouse xenograft model. There was a significant decrease in the subcutaneous primary tumor [18F]-FDG uptake (76.5% reduction, p < 0.05) and in the total tumor burden (76.8% reduction, p < 0.05) after EI201 treatment compared to vehicle control, without causing toxicity in mice. Taken together, our results support further development of EI201 as a novel multi-kinase inhibitor that may be useful against cancers with aberrant upregulation of PI3K/AKT and MAPK signaling pathways.

The novel Akt inhibitor Palomid 529 (P529) enhances the effect of radiotherapy in prostate cancer.

Radiotherapy (RT) is a common treatment for localised prostate cancer, but can cause important side effects. The therapeutic efficacy of RT can be enhanced by pharmacological compounds that target specific pathways involved in cell survival. This would elicit a similar therapeutic response using lower doses of RT and, in turn, reducing side effects. This study describes the antitumour activity of the novel Akt inhibitor 8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one (Palomid 529 or P529) as well as its ability to decrease radiation-activated phospho-Akt (p-Akt) signalling in a prostate cancer model. P529 showed a potent antiproliferative activity in the NCI-60 cell lines panel, with growth inhibitory 50 (GI50) <35 microM. In addition, P529 significantly enhanced the antiproliferative effect of radiation in prostate cancer cells (PC-3). Analysis of signalling pathways targeted by P529 exhibited a decrease in p-Akt, VEGF, MMP-2, MMP-9, and Id-1 levels after radiation treatment. Moreover, the Bcl-2/Bax ratio was also reduced. Treatment of PC-3 tumour-bearing mice with 20 mg kg(-1) P529 or 6 Gy radiation dose decreased tumour size by 42.9 and 53%, respectively. Combination of both treatments resulted in 77.4% tumour shrinkage. Decreased tumour growth was due to reduced proliferation and increased apoptosis (as assessed by PCNA and caspase-3 immunostaining). Our results show the antitumour efficacy of P529 alone, and as a radiosensitiser, and suggest that this compound could be used in the future to treat human prostate cancer.