Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis, which lacks effective targeted therapies. There is an urgent need to better understand the underlying molecular mechanisms of TNBC aggressiveness and identify novel, efficient targets for therapeutic intervention.MethodsmiRNA qRT-PCR was used to determine the expression of miR-1296 in cell lines. The miR-1296 overexpression effects in TNBC cell lines were investigated using assays of colony formation, cell cycle and apoptosis. Immunoblotting was performed to determine the expression of the miR-1296 target protein, and luciferase assays were performed to confirm the target of miR-1296 action.ResultsmiR-1296 expression was significantly suppressed in TNBC cell lines and tissues samples. Overexpression of miR-1296 significantly suppressed cell proliferation of two TNBC cell lines when compared to control miRNA-expressing cells. A significant decrease in the S-phase of the cell cycle was observed following miR-1296 overexpression, accompanied by induction of apoptosis in TNBC cells. Cyclin D1 (CCND1) was identified as a target of miR-1296 action. miR-1296 overexpression significantly suppressed the luciferase activity of reporter plasmid containing the 3′UTR of CCND1 and protein expression levels of CCND1 in TNBC cells. The effects of miR-1296 overexpression on TNBC cell growth were reversed by CCND1 overexpression. miR-1296 expression sensitized TNBC cells to cisplatin treatment.ConclusionOur results demonstrate a novel tumor suppressor role for miR-1296 in triple-negative breast cancer cell lines, identify CCND1 as its target of action, and demonstrate a potential role for miR-1296 in sensitizing breast cancer cells to cisplatin.
Microphthalmia-associated transcription factor (MITF) plays a critical and complex role in melanocyte transformation. Although several downstream targets of MITF action have been identified, the precise mechanisms by which MITF promotes melanocytic tumor progression are incompletely understood. Recent studies identified an oncogenic role for the bromodomain plant homeodomain finger transcription factor (BPTF) gene in melanoma progression, in part through activation of BCL2, a canonical target of MITF signaling. Analysis of the BPTF promoter identified a putative MITF-binding site, suggesting that MITF may regulate BPTF expression. Overexpression of MITF resulted in up-regulation of BPTF in a panel of melanoma and melanocyte cell lines. shRNA-mediated down-regulation of MITF in melanoma cells was accompanied by down-regulation of BPTF and BPTF-regulated genes (including BCL2) and resulted in reduced proliferative capacity of melanoma cells. The suppression of cell growth mediated by MITF silencing was rescued by overexpression of BPTF cDNA. Binding of MITF to the BPTF promoter was demonstrated using ChIP analysis. MITF overexpression resulted in direct transcriptional activation of BPTF, as evidenced by increased luciferase activity driven by the BPTF promoter. These results indicate that BPTF transduces key prosurvival signals driven by MITF, further supporting its important role in promoting melanoma cell survival and progression. melanoma | signaling cascade | oncogenes M icrophthalmia-associated transcription factor (MITF) is known to play a key role in melanocyte biology and progression. MITF performs these functions by activating transcription of many genes through interaction with the consensus DNA-binding sequence (CACGTG, termed E box) present on the promoters of target genes. MITF controls expression of several proteins required for melanin synthesis, including tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and dopachrome tautomerose (DCT) (1-3). In addition, MITF plays a critical role in melanocyte survival and serves as a lineagespecific oncogene in melanoma, as evidenced by its amplification in a subset of melanomas (4-6). MITF expression is regulated by several factors, including SOX-10, CREB, PAX3, LEF1, and ATF2 (7,8). MITF in turn regulates a plethora of genes, with its complex role in melanoma biology, as its overexpression has been shown to result in both proproliferative and antiproliferative stimuli (4, 5, 9, 10). Accordingly, verified targets of MITF include both genes that promote cell survival and block apoptosis, (e.g., CDK2, BCL2, DIAPH1, and TBX2), and genes that block cell cycle progression (e.g., CDKN2A and p21Cip1). Thus, although several downstream targets of MITF action have been identified, the precise mechanisms by which MITF promotes melanocytic tumor progression are still poorly understood.Recently, we identified a role for the bromodomain plant homeodomain finger transcription factor (BPTF) gene in melanoma progression (11). BPTF is the largest subunit of the nucleoso...
Talin is a force-sensing multidomain protein and a major player in cellular mechanotransduction. Here, we use single-molecule magnetic tweezers to investigate the mechanical response of the R8 rod domain of talin. We find that under various force cycles, the R8 domain of talin can display a memory-dependent behavior: At the same low force (<10 pN), the same protein molecule shows vastly different unfolding kinetics. This history-dependent behavior indicates the evolution of a unique force-induced native state. We measure through mechanical unfolding that talin R8 domain binds one of its ligands, DLC1, with much higher affinity than previously reported. This strong interaction can explain the antitumor response of DLC1 by regulating inside-out activation of integrins. Together, our results paint a complex picture for the mechanical unfolding of talin in the physiological range and a new mechanism of function of DLC1 to regulate inside-out activation of integrins.
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