Tamoxifen decreases extracellular TGF-beta1 secreted from breast cancer cells--a post-translational regulation involving matrix metalloproteinase activity

Transforming growth factor-β1 (TGF-β1) promotes cancer progression by regulating tumor cell growth and angiogenesis and high levels of TGF-β1 have been associated with metastatic disease and poor prognosis in breast cancer patients. We have previously reported anti-angiogenic effects of the anti-estrogen tamoxifen in breast cancer, by increased matrix metalloproteinase-9 (MMP-9) activity and generation of endostatin. Here, we show that exposure of tamoxifen to ER-positive breast cancer cells for 7 days, decreased extracellular TGF-β1. Intracellular TGF-β1 levels were unaffected by tamoxifen treatment, indicating a post-translational regulation of TGF-β1. Inhibition of MMP activity restored TGF-β1 levels, suggesting an involvement of MMP activities in the down-regulation of TGF-β1 by tamoxifen. Moreover, using an in vivo model of solid MCF-7 tumors in nude mice, we analyzed tumor levels of TGF-β1 after in vivo treatment with estradiol and tamoxifen. Exposure of tumor-bearing mice to tamoxifen significantly decreased tumor TGF-β1 protein levels, tumor growth and angiogenesis. In conclusion, our findings suggest a novel mechanism of action of tamoxifen in breast cancer via sex steroid dependent modulation of the proteolytic tumor microenvironment resulting in reduced extracellular TGF-β1 levels.     

UV radiation resistance-associated gene (UVRAG) promotes cell proliferation,migration, invasion by regulating cyclin-dependent kinases (CDK) and integrin-β/Src signaling in breast cancer cells

Breast cancer is a highly heterogeneous group of human cancer with distinct genetic, biological and clinicopathological features. Triple-negative breast cancer (TNBC) is the most aggressive and metastatic type of breast cancer and associated with poor patient survival. However, the role of UV Radiation Resistance-Associated Gene (UVRAG) in TNBC remains unknown. Here, we report that UVRAG is highly upregulated in all TNBC cells and its knockdown leads to the inhibition of cell proliferation, colony formation and progression of cell cycle, which is associated with and reduced expression of cell cycle related protein expression, including Cyclin A2, B1, D1, cdc2 and cdk6 in TNBC cells. Inhibition of UVRAG also suppressed cell motility, migration and invasion of TNBC cells by inhibition of Integrin β1 and β3 and Src activity. Our findings suggest for the first time that UVRAG expression contributes to proliferation, cell cycle progression, motility/migration and invasion of TNBC cells. Thus, targeting UVRAG could be a potential strategy in breast cancer especially against TNBC.

     

TGFB1 polymorphisms and TGF-β1 plasma levels identify gastric adenocarcinoma patients with lower survival rate and disseminated disease

TGF-β1 is involved in tumour growth. Four TGFB1 SNPs and TGF-β1 production by stimulated PBMC were determined in seventy-eight gastric adenocarcinoma patients. In addition, TGF-β1 levels were measured in the plasma of further thirty patients. rs1800471-G/C genotype was prevalent in patients (20.7%) compared to controls (8.4%), as it also was the rs1800468 SNP-G/A genotype in stage IV patients (20.7%) compared to stage I, II and III patients, combined (10.3%). Conversely, the T/T rs1800469 SNP-T/T genotype was absent in the former group and present in 19.0% in the latter. Furthermore, the rs1800469-C/rs1800470-T (CT) haplotype was found in 15.0% of stage IV patients as compared to 3.0% of the remaining patients (3.0%) and also identifies patients with worse five-year life expectancy (P = .03). TGF-β1 synthesis by stimulated PBMCs was significantly lower in patients with the risk SNPs or haplotype, compared to the alternative genotype. Finally, TGF-β1 plasma levels were lower in patients with worse life expectancy. Analysis of TGFB1 SNPs and measurement of plasma TGF-β1 levels serves to identify patients at risk of developing a more aggressive disease.     

Adipose derived stem cells (ASCs) isolated from breast cancer tissue express IL-4, IL-10 and TGF-β1 and upregulate expression of regulatory molecules on T cells: do they protect breast cancer cells from the immune response?

Immunomodulatory function of bone marrow derived mesenchymal stem cells in cancer has recently been investigated. But the resident mesenchymal stem cells as whole in cancer and in the breast cancer tissue have not been studied well. In the present work we isolated adipose derived stem cells (ASCs) from breast cancer and normal breast tissues to investigate the expressions of IL-4, IL-10 and transforming growth factor (TGF)-β1 in ASCs and to see if ASCs isolated from patients can modulate the regulatory molecules on peripheral blood lymphocytes. Our results showed that IL-10 and TGF-β1 have significantly higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals (P value <0.05). The culture supernatant of ASCs isolated from breast cancer patients with pathological stage III induced upregulation of the mRNA expression levels of IL-4, TGF-β1, IL-10, CCR4 and CD25 in PBLs. In addition, the percentage of CD4+CD25^highFoxp3⁺ T regulatory cells was increased in vitro. When the same culture supernatant was added to ASCs isolated from normal subjects augmentation of the mRNA expressions of IL-4, IL-10, IL-8, MMP2, VEGF and SDF-1 in normal ASCs was also observed. These data collectively conclude that resident ASCs in breast cancer tissue may have crucial roles in breast tumor growth and progression by inducing regulatory molecules and promoting anti-inflammatory reaction within the tumor microenvironment. Further investigation is required to see if the immune suppression induced by ASCs is an independent property from tumor cells or ASCs gain their immunosuppressive potential from malignant cells.     

Plasma levels of TGF-β1 in homeostasis of the inflammation in sickle cell disease

Sickle cell disease (SCD) represents a chronic inflammatory condition with complications triggered by the polymerization of hemoglobin S (Hb S), resulting in a series of cellular interactions mediated by inflammatory cytokines, as the transforming growth factor beta (TGF-β), which plays an important role in inflammation resolution. This study assessed the relation between SCD inflammation and the plasma concentration of TGF-β1, and also checked the influence of the presence of −509C/T polymorphism in TGFB1 gene on TGF-β1 plasma values. The plasma levels of TGF-β1 were quantified by ELISA in 115 patients with SCD (genotypes SS, SD-Los Angeles, Sβ-thalassemia and SC) and in 58 individuals with no hemoglobinopathies (Hb AA), as the control group. The −509C/T polymorphism in TGFB1 gene was screened by PCR-RFLP. The correlation between TGF-β1 plasma levels and the inflammation was based on its association with the count of platelets, total white blood cells (WBC) and neutrophils in the peripheral blood. Patients with SCD showed plasma levels of TGF-β1 higher than the control group, especially the Hb SS genotype, followed by the group with Hb SD. Polymorphism investigation showed no interference in the values obtained for the cytokine in the groups evaluated. All SCD groups showed TGF-β1 levels positively correlated to the platelets and WBC counts. The original data obtained in this study for SCD support the involvement of TGF-β1 in regulating of the inflammatory response and suggest that this marker possibly may become a potential therapeutic target in the treatment of the disease.     

Erythropoietin suppresses epithelial to mesenchymal transition and intercepts Smad signal transduction through a MEK-dependent mechanism in pig kidney (LLC-PK1) cell lines

Purpose

Tumor growth factor-β1 (TGF-β1) plays a pivotal role in processes like kidney epithelial-mesenchymal transition (EMT) and interstitial fibrosis, which correlate well with progression of renal disease. Little is known about underlying mechanisms that regulate EMT. Based on the anatomical relationship between erythropoietin (EPO)-producing interstitial fibroblasts and adjacent tubular cells, we investigated the role of EPO in TGF-β1-mediated EMT and fibrosis in kidney injury.

Methods

We examined apoptosis and EMT in TGF-β1-treated LLC-PK1 cells in the presence or absence of EPO. We examined the effect of EPO on TGF-β1-mediated Smad signaling. Apoptosis and cell proliferation were assessed with flow cytometry and hemocytometry. We used Western blotting and indirect immunofluorescence to evaluate expression levels of TGF-β1 signal pathway proteins and EMT markers.

Results

We demonstrated that ZVAD-FMK (a caspase inhibitor) inhibited TGF-β1-induced apoptosis but did not inhibit EMT. In contrast, EPO reversed TGF-β1-mediated apoptosis and also partially inhibited TGF-β1-mediated EMT. We showed that EPO treatment suppressed TGF-β1-mediated signaling by inhibiting the phosphorylation and nuclear translocation of Smad 3. Inhibition of mitogen-activated protein kinase kinase 1 (MEK 1) either directly with PD98059 or with MEK 1 siRNA resulted in inhibition of EPO-mediated suppression of EMT and Smad signal transduction in TGF-β1-treated cells.

Conclusions

EPO inhibited apoptosis and EMT in TGF-β1-treated LLC-PK1 cells. This effect of EPO was partially mediated by a mitogen-activated protein kinase-dependent inhibition of Smad signal transduction.     

Expression analysis of selected miR-206 targets from the transforming growth factor-β signaling pathway in breast cancer

Breast cancer as a molecularly heterogeneous malignancy is associated with dysregulation of several signaling pathways, including transforming growth factor-β (TGF-β) signaling. On the other hand, several recent studies have demonstrated the role of microRNAs (miRNAs) in breast cancer pathogenesis. In the current study, we performed a computerized search to find miR-206 target genes that are functionally linked to the TGF-β signaling pathway. We selected LEF1, Smad2, and Snail2 genes to assess their expression in 65 breast cancer samples and their adjacent noncancerous tissues (ANCTs) in correlation with expression levels of miR-206 as well as clinicopathological characteristics of patients. miR-206 was significantly downregulated in (Estrogen receptor) ER-positive breast cancer samples compared with their corresponding ANCTs. Association analysis between expression levels of genes and demographic features of patients showed significant association between expressions of SMAD2 and LEF1 genes and body mass index ( P values of 0.03 and 0.02, respectively). miR-206 low-expression levels were associated with TNM stage, mitotic rate, and lymph node involvement ( P values of 0.02, 0.01, and 0.01 respectively). In addition, SMAD2 high-expression levels were associated with HER2 status ( P = 0.02). Consequently, our data highlight the role of TGF-β signaling dysregulation in the pathogenesis of breast cancer and warrant further evaluation of miRNAs and messenger RNA coding genes in this pathway to facilitate detection of cancer biomarkers and therapeutic targets.