Background: TGF-␤ promotes cell migration in advanced breast cancer. Results: TGF-␤ down-regulates miR-584, leading to a PHACTR1 overexpression, and both are involved in cell migration and actin reorganization. Conclusion:The regulation of miR-584 and regulation of its novel target PHACTR1 are necessary steps for breast cancer cell migration. Significance: MicroRNAs offer an interesting therapeutic target in the treatment of advanced breast malignancy.

IntroductionDeregulation of the cell cycle machinery is often found in human cancers. Modulations in the cell cycle regulator function and expression result not only in proliferative advantages, but also lead to tumor progression and invasiveness of the cancer. In particular, cyclin D1 and p21 are often over-expressed in human cancers, correlating with high tumor grade, poor prognosis and increased metastasis. This prompted us to investigate the role of the cyclin D1/p21 signaling axis downstream of transforming growth factor beta (TGFβ) in breast cancer progression.MethodsCyclins mRNA and protein expressions were assessed by quantitative real-time PCR and Western blot in triple negative breast cancer cell lines. Co-localization and interaction between cyclin D1 and p21 were performed by immunocytochemistry and co-immunoprecipitation, respectively. Cell migration was assessed by wound healing and quantitative time-lapse imaging assays. In addition, the effects of cyclin D1 on cellular structure and actin organization were examined by staining with F-actin marker phalloidin and mesenchymal intermediate filament vimentin. Finally, a mammary fat pad xenograft mouse model was used to assess mammary tumor growth and local invasion.ResultsWe found TGFβ to specifically up-regulate the expression of cyclin D1 in triple negative breast cancer cells. Induction of cyclin D1 is also required for TGFβ-mediated cell migration. Suppression of cyclin D1 expression not only resulted in a rounded and epithelial-like phenotype, but also prevented TGFβ-induced vimentin and F-actin co-localization at the cell edge as well as invadopodia formation. Furthermore, TGFβ promoted the nuclear co-localization and physical interaction between cyclin D1 and p21. The co-expression of cyclin D1 and p21 proteins are required for the initial steps of tumor development, as double knockdown of these two molecules prevented primary tumor formation in a Xenograft mouse model. Moreover, the in vivo studies indicated that locally advanced features of the invasive tumors, including skeletal muscle, mammary fat pad and lymphovascular invasion, as well as ulcerated skin, were attenuated in the absence of cyclin D1 and p21.ConclusionsThus, our findings highlight the cyclin D1/p21 signaling axis as a critical regulator of TGFβ-mediated tumor growth initiation and local tumor cell invasion, both in vitro and in vivo.

Triple negative breast cancers exhibit very aggressive features and poor patient outcomes. These tumors are enriched in cancer stem cells and exhibit resistance to most treatments and chemotherapy. In this study, we found the cyclin-dependent kinase (CDK4) to act as a cancer stem cell regulator and novel prognostic marker in triple negative breast cancers. We found CDK4 to be highly expressed in these tumors and its expression to correlate with poor overall and relapse free survival outcomes, high tumor grade and poor prognostic features of triple negative breast cancer patients. Moreover, we found that blocking CDK4 expression or kinase activity, using a pharmacological inhibitor prevented breast cancer stem cell self-renewal. Interestingly, suppression of CDK4 expression or kinase activity reversed the basal-B TNBC mesenchymal phenotype to an epithelial- and luminal-like phenotype which correlates with better clinical prognosis. Finally, blocking CDK4 activity efficiently eliminated both normal and chemotherapy-resistant cancer cells in triple negative breast cancers, highlighting CDK4 as a promising novel therapeutic target for these aggressive breast tumors.

IntroductionThis study helps to define the implications of breast cancer anti-estrogen resistance 3 (BCAR3) in breast cancer and extends the current understanding of its molecular mechanism of action. BCAR3 has been shown to promote cell proliferation, migration and attachment to extracellular matrix components. However, in a cohort of metastatic breast cancer patients who received tamoxifen treatment, high BCAR3 mRNA levels were associated with favorable progression-free survival outcome. These results suggest that, besides its established roles, BCAR3 may have additional mechanisms of action that regulate breast cancer aggressive phenotype. In this study, we investigated whether BCAR3 is a novel antagonist of the canonical transforming growth factor β (TGFβ) pathway, which induces potent migration and invasion responses in breast cancer cells.MethodsWe surveyed functional genomics databases for correlations between BCAR3 expression and disease outcomes of breast cancer patients. We also studied how BCAR3 could regulate the TGFβ/Smad signaling axis using Western blot analysis, coimmunoprecipitation and luciferase assays. In addition, we examined whether BCAR3 could modulate TGFβ-induced cell migration and invasion by using an automated imaging system and a confocal microscopy imaging–based matrix degradation assay, respectively.ResultsRelatively low levels of BCAR3 expression in primary breast tumors correlate with poor distant metastasis-free survival and relapse-free survival outcomes. We also found a strong correlation between the loss of heterozygosity at BCAR3 gene alleles and lymph node invasion in human breast cancer, further suggesting a role for BCAR3 in preventing disease progression. In addition, we found BCAR3 to inhibit Smad activation, Smad-mediated gene transcription, Smad-dependent cell migration and matrix digestion in breast cancer cells. Furthermore, we found BCAR3 to be downregulated by TGFβ through proteasome degradation, thus defining a novel positive feedback loop mechanism downstream of the TGFβ/Smad signaling pathway.ConclusionBCAR3 is considered to be associated with aggressive breast cancer phenotypes. However, our results indicate that BCAR3 acts as a putative suppressor of breast cancer progression by inhibiting the prometastatic TGFβ/Smad signaling pathway in invasive breast tumors. These data provide new insights into BCAR3’s molecular mechanism of action and highlight BCAR3 as a novel TGFβ/Smad antagonist in breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0476-9) contains supplementary material, which is available to authorized users.

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