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Late-stage breast cancer metastasis is driven by dysregulated TGF-β signaling, but the underlying molecular mechanisms have not been fully elucidated. We attempted to recapitulate tumor and metastatic microenvironments via the use of biomechanically compliant or rigid 3D organotypic cultures and combined them with global microRNA (miR) profiling analyses to identify miRs that were upregulated in metastatic breast cancer cells by TGF-β. Here we establish miR-181a as a TGF-β-regulated "metastamir" that enhanced the metastatic potential of breast cancers by promoting epithelial-mesenchymal transition, migratory, and invasive phenotypes. Mechanistically, inactivation of miR-181a elevated the expression of the proapoptotic molecule Bim, which sensitized metastatic cells to anoikis. Along these lines, miR-181a expression was essential in driving pulmonary micrometastatic outgrowth and enhancing the lethality of late-stage mammary tumors in mice. Finally, miR-181a expression was dramatically and selectively upregulated in metastatic breast tumors, particularly triple-negative breast cancers, and was highly predictive for decreased overall survival in human breast cancer patients. Collectively, our findings strongly implicate miR-181a as a predictive biomarker for breast cancer metastasis and patient survival, and consequently, as a potential therapeutic target in metastatic breast cancer. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide-inducible clone-5 (Hic-5, or androgen receptor-associated protein 55) is a transforming growth factor-β-inducible LIM protein whose deregulation is implicated in the progression of prostate cancer. Here, we report that Hic-5 binds to Smads 1, 5 and 8, and represses bone morphogenetic protein (BMP) signaling responses. Myc-Hic-5 but not Myc-paxillin was specifically immunoprecipitated with anti-FLAG IgG1 from lysates of HEK293 co-transfected with either Myc-Hic-5 or Myc-paxillin and FLAG-tagged Smads 1, 5 or 8. We showed that such interactions require the LIM3 domain of Hic-5 and the MH2 domain of those Smads. Anti-Hic-5 antibody specifically pulled down endogenous Smad1 in both the PC3 human prostate cell line and primary cultures of rat prostate fibroblasts, supporting that Hic-5 binds to Smad1 at the endogenous level. Bacterially expressed glutathione S-transferase (GST)-Smads 1, 5 or 8, but not GST alone, pulled down in vitro transcribed and translated Hic-5, implicating that Hic-5 binds directly to Smads 1, 5 and 8. Significantly, using Hic-5 small hairpin RNA silencing and overexpression systems, we show that Hic-5 (at both the endogenous and exogenous levels) represses the ability of BMP4 to induce expression of the inhibitor of differentiation-1 (Id1; a downstream target gene of BMP), activate the Id1 gene promoter and induce apoptosis in human and rat prostate epithelial cells. Moreover, silencing of Hic-5 in PC3 cells as well as in the WPMY-1 human prostate stroma cell line greatly enhances the levels of endogenous phospho-Smad1/5/8. Finally, we provide fluorescent microscopic imaging to support that Smad1 and Hic-5 mutually interact also at the level of their nuclear export mechanisms. Collectively, these results provide the first evidence for a physical and mutual functional interaction between Hic-5 and the BMP signaling pathway. [ABSTRACT FROM AUTHOR]

Earlier, we have reported that 70 kDa subunit of Ku protein heterodimer (Ku70) binds and inhibits Bax activity in the cytosol and that ubiquitin (Ub)-dependent proteolysis of cytosolic Ku70 facilitates Bax-mediated apoptosis. We found that Hdm2 (human homolog of murine double minute) has an ability to ubiquitinate Ku70 and that Hdm2 overexpression in cultured cells causes a decrease in Ku70 expression levels. An interaction between Ku70 and Hdm2 was shown by means of immunoprecipitation, whereas none could be shown between 80 kDa subunit of Ku protein heterodimer and Hdm2. Vascular endothelial growth factor (VEGF) is known to inhibit endothelial cell (EC) apoptosis through an Akt-mediated survival kinase signal; however, the mechanism underlying this inhibition of apoptosis has not been fully elucidated. We found that VEGF inhibited cytosolic Ku70 degradation induced by apoptotic stress. It is known that Akt-dependent phosphorylation of Hdm2 causes nuclear translocation of Hdm2 followed by Hdm2-mediated inactivation of p53. We found that VEGF stimulated nuclear translocation of Hdm2 in EC and efficiently inhibited Ku70 degradation. We also found that constitutively active Akt, but not kinase-dead Akt, inhibited Ku70 degradation in the cytosol. Furthermore, Ku70 knockdown diminished antiapoptotic activity of Akt. Taken together, we propose that Hdm2 is a Ku70 Ub ligase and that Akt inhibits Bax-mediated apoptosis, at least in part, by maintaining Ku70 levels through the promotion of Hdm2 nuclear translocation.Cell Death and Differentiation (2009) 16, 758–769; doi:10.1038/cdd.2009.6; published online 27 February 2009 [ABSTRACT FROM AUTHOR]

We recently reported that hydrogen peroxide-inducible clone-5 (Hic-5, also named androgen receptor-associated protein 55) can bind to the transforming growth factor-β (TGF-β)-signaling regulator Smad3, thereby inhibiting certain Smad3-dependent TGF-β responses. We now show that Hic-5 can also control TGF-β responses through an alternative mechanism involving Smad7, a key negative regulator of TGF-β signaling. Hic-5 binds directly to Smad7. This interaction requires the LIM3 domain of Hic-5, and enhances TGF-β signaling through causing loss of Smad7 protein but not mRNA. Enforced expression of Hic-5 reverses the ability of Smad7 to suppress TGF-β-induced phosphorylation of Smads 2 and 3 and activation of the plasminogen activator inhibitor-1 promoter (in NRP-154 and PC3 prostate carcinoma and WPMY-1 prostate myofibroblast cell lines). Lentiviral-mediated small-hairpin RNA silencing of endogenous Hic-5 reduced TGF-β responses in PC3 and WPMY-1 cells. Further work suggests that the level of Smad7 is modulated by its physical interaction with Hic-5 and targeted to a degradation pathway not likely to be proteasomal. Our findings support that Hic-5 functions as a cell-type-specific activator of TGF-β signaling through its ability to physically interact with and neutralize Smad7.Oncogene (2008) 27, 6791–6805; doi:10.1038/onc.2008.291; published online 1 September 2008 [ABSTRACT FROM AUTHOR]

Survivin is a prosurvival protein overexpressed in many cancers through mechanisms that remain poorly explored, and is implicated in control of tumor progression and resistance to cancer chemotherapeutics. Here, we report a critical role for survivin in the induction of apoptosis by transforming growth factor-β (TGF-β). We show that TGF-β rapidly downregulates survivin expression in prostate epithelial cells, through a unique mechanism of transcriptional suppression involving Smads 2 and 3, Rb/E2F4, and the cell-cycle repressor elements CDE and CHR. This TGF-β response is triggered through a Smad2/3-dependent hypophosphorylation of Rb and the subsequent association of the Rb/E2F4 repressive complex to CDE/CHR elements in the proximal region of the survivin promoter. Viral-mediated gene delivery experiments, involving overexpressing or silencing survivin, reveal critical roles of survivin in apoptosis induced by TGF-β alone or in cooperation with cancer therapeutic agents. We propose a novel TGF-β/Rb/survivin axis with a putative role in the functional switch of TGF-β from tumor suppressor to tumor promoter.Oncogene (2008) 27, 5326–5338; doi:10.1038/onc.2008.165; published online 26 May 2008 [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) elicits a potent growth inhibitory effect on many normal cells by binding to specific serine/threonine kinase receptors and activating specific Smad proteins, which regulate the expression of cell cycle genes, including the p21 cyclin-dependent kinase (CDK) inhibitor gene. Interestingly, cancer cells are often insensitive to the anti-mitogenic effects of TGF-β for which the molecular mechanisms are not well understood. In this study, we found that the candidate prostate cancer susceptibility gene ELAC2 potentiates TGF-β/Smad-induced transcriptional responses. ELAC2 associates with activated Smad2; the C-terminal MH2 domain of Smad2 interacts with the N-terminal region of ELAC2. Small interfering siRNA-mediated knock-down of ELAC2 in prostate cells suppressed TGF-β-induced growth arrest. Moreover, ELAC2 was shown to specifically associate with the nuclear Smad2 partner, FAST-1 and to potentiate the interaction of activated Smad2 with transcription factor Sp1. Furthermore, activation of the p21 CDK inhibitor promoter by TGF-β is potentiated by ELAC2. Taken together our data indicate an important transcriptional scaffold function for ELAC2 in TGF-β/Smad signaling mediated growth arrest.Oncogene (2006) 25, 5591–5600. doi:10.1038/sj.onc.1209571; published online 24 April 2006 [ABSTRACT FROM AUTHOR]

Cited (CBP/p300-interacting transactivators with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain) 2, which is a CBP/p300-binding transcription co-activator without typical DNA-binding domains, has been implicated in control of cell growth and malignant transformation in Rat1 cells. In this report, we provide evidence that Cited2 is an important regulator of transforming growth factor (TGF)-β signaling. Overexpression of Cited2 enhanced TGF-β-mediated transcription of a Smad-Binding Element-containing luciferase reporter construct, SBE4-Luc. This may occur through a direct physical association of Cited2 with Smads 2 and 3, as supported by co-immunoprecipitation, mammalian two-hybrid and glutathione S-transferase-pull down assays. The transcription factor p300, which binds to Smad3, was shown to further enhance the interaction between Cited2 and Smad3, and the transcriptional responses of Smad3 by Cited2 in reporter assays. Cited2 enhances TGF-β-mediated upregulation of matrix metalloproteinase 9 (MMP9) in Cited2 inducible mouse embryo fibroblasts. Overexpression of Cited2 enhanced TGF-β-mediated MMP9 promoter reporter activity. Moreover, knockdown of Cited2 in MDA-MB-231 cells attenuated TGF-β-mediated upregulation of MMP9 and TGF-β-mediated cell invasion. Chromatin immunoprecipitation showed that Cited2 and Smad3 were recruited to MMP9 promoter upon TGF-β stimulation. This is the first demonstration that Cited2 functions as a Smad3/p300-interacting transcriptional co-activator in modulating the expression of MMP9, which could affect tumor cell invasion mediated by TGF-β.Oncogene (2006) 25, 5547–5560. doi:10.1038/sj.onc.1209552; published online 17 April 2006 [ABSTRACT FROM AUTHOR]