KiSS1 gene as a novel mediator of TGFß-mediated cell invasion in triple negative breast cancer.

The invasive and metastatic phenotypes of breast cancer correlate with high recurrence rates and poor survival outcomes. Transforming growth factor-ß (TGFß) promotes tumor progression and metastasis in aggressive breast cancer. Here, we identified the kisspeptin KiSS1 as a downstream target of canonical TGFß/Smad2 pathway in triple negative breast cancer cells. We also found KiSS1 expression to be required for TGFß-induced cancer cell invasion. Indeed, knockdown expression of KiSS1 blocked TGFß-mediated cancer cell invasion as well as metalloproteinase (MMP9) expression and activity. Interestingly, Kisspeptin-10 (KP-10), the smallest active form of kisspeptin also stimulates cancer cell invasive behavior through activation of MAPK/Erk pathway. We described a positive feedback loop between KiSS1 and p21 downstream of TGFß, further contributing to TGFß-induced cancer cell invasion. Lastly, we explored both the clinical utility of KiSS1 as a lymph node involvement predictive tool and its potential as a therapeutic target. We found KiSS1 high expression to correlate with lymph node positive status. Furthermore, blocking KiSS1 using a specific small peptide antagonist (p234) impaired TGFß-mediated cell invasion and MMP9 induction. Together, our results define an essential role of KiSS1 in regulating TGFß pro-invasive effects and define KiSS1 as a therapeutic new target for triple negative breast cancer.

The Retinoblastoma Tumor Suppressor Protein (pRb)/E2 Promoter Binding Factor 1 (E2F1) Pathway as a Novel Mediator of TGFß-induced Autophagy.

TGFß is a multifunctional cytokine that regulates cell proliferation, cell immortalization, and cell death, acting as a key homeostatic mediator in various cell types and tissues. Autophagy is a programmed mechanism that plays a pivotal role in controlling cell fate and, consequently, many physiological and pathological processes, including carcinogenesis. Although autophagy is often considered a pro-survival mechanism that renders cells viable in stressful conditions and thus might promote tumor growth, emerging evidence suggests that autophagy is also a tumor suppressor pathway. The relationship between TGFß signaling and autophagy is context-dependent and remains unclear. TGFß-mediated activation of autophagy has recently been suggested to contribute to the growth inhibitory effect of TGFß in hepatocarcinoma cells. In the present study, we define a novel process of TGFß-mediated autophagy in cancer cell lines of various origins. We found that autophagosome initiation and maturation by TGFß is dependent on the retinoblastoma tumor suppressor protein/E2 promoter binding factor (pRb/E2F1) pathway, which we have previously established as a critical signaling axis leading to various TGFß tumor suppressive effects. We further determined that TGFß induces pRb/E2F1-dependent transcriptional activation of several autophagy-related genes. Together, our findings reveal that TGFß induces autophagy through the pRb/E2F1 pathway and transcriptional activation of autophagy-related genes and further highlight the central relevance of the pRb/E2F1 pathway downstream of TGFß signaling in tumor suppression.

Breast cancer anti-estrogen resistance 3 inhibits transforming growth factor ß/Smad signaling and associates with favorable breast cancer disease outcomes.

INTRODUCTION: This 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 ß (TGFß) pathway, which induces potent migration and invasion responses in breast cancer cells. METHODS: We surveyed functional genomics databases for correlations between BCAR3 expression and disease outcomes of breast cancer patients. We also studied how BCAR3 could regulate the TGFß/Smad signaling axis using Western blot analysis, coimmunoprecipitation and luciferase assays. In addition, we examined whether BCAR3 could modulate TGFß-induced cell migration and invasion by using an automated imaging system and a confocal microscopy imaging-based matrix degradation assay, respectively. RESULTS: Relatively 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 TGFß through proteasome degradation, thus defining a novel positive feedback loop mechanism downstream of the TGFß/Smad signaling pathway. CONCLUSION: BCAR3 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 TGFß/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 TGFß/Smad antagonist in breast cancer.

Development of buffers for fast semidry transfer of proteins.

Western blot is an extensively used method for protein detection in cell biology. To optimize this procedure, here we examined a panel of buffers for their ability to efficiently transfer proteins from SDS-polyacrylamide gels onto nitrocellulose membranes in a short 12-min period, designated here as fast semidry transfer. Our results show for the first time that HEPES- and HEPPS/EPPS-based buffers represent the most efficient buffers for fast semidry transfer.

Transforming growth factor-beta inhibits telomerase through SMAD3 and E2F transcription factors.

Cancer arises from multiple genetic changes within the cell, among which constitutive telomerase activity and attainment of immortality are central. Expression of hTERT, the protein component of telomerase, is increased in most cancer cells. Transforming growth factor-beta (TGFbeta), a potent tumor suppressor, has been reported to regulate hTERT expression. We found that TGFbeta represses hTERT expression in normal and cancer cells and that this effect is mediated through Smad3 but also requires Erk1/2, p38 kinase and histone deacetylase activity. Furthermore, we identified four critical E2F transcription factor binding sites within the hTERT gene promoter that confer the TGFbeta response. Finally, using the E2F-1 knockout model, we showed that loss of E2F-1 abolishes TGFbeta inhibition of telomerase expression. These findings highlight the prominent role of TGFbeta in regulating telomerase expression and identify Smad3 and E2F-1 as critical mediators of TGFbeta effects in both normal and cancer cells.