Determination of TGFβ1 protein level in human primary breast cancers and its relationship with survival

Transforming growth factor-beta (TGFβ)1 is thought to be implicated in breast cancer progression. However, data about the influence of TGFβ1 on breast cancer development are conflicting. To clarify the clinical relevance of TGFβ1, TGFβ1 protein level has been measured by enzyme-immoassay in 193 breast tumour samples. We found that 94.3% of patients expressed TGFβ1 with a range of 0–684 pg mg−1 protein. In the overall population, an increase of tumoral TGFβ1 was observed in premenopausal patients when compared to postmenopausal subgroup (P=0.0006). When patients were subdivided according to nodal status, TGFβ1 was correlated to type-1 plasminogen activator inhibitor in the node-negative subgroup (P=0.040). Multivariate analysis revealed that, after lymph node status (P=0.0002) and urokinase-type plasminogen activator (P=0.004), TGFβ1 was an independent prognostic marker for DFS (P=0.005) in the overall population. In the node-negative population, TGFβ1 was the prominent prognostic factor (P=0.010). In the same population, Kaplan–Meier curves demonstrated that high TGFβ1 level was correlated with a shorter disease-free survival (P=0.020). These data suggest that the measurement of tumoral TGFβ1 protein level, especially for node-negative patients, might help to identify a high-risk population early in tumour progression

Enhanced progression of human prostate cancer PC3 cells induced by the microenvironment of the seminal vesicle

The objective of this study was to characterise the mechanism mediating the prostate cancer progression induced by the microenvironment of seminal vesicle (SV). The invasive potential of PC3 cells significantly increased after treatment with extract from SV of NOD/SCID mouse. Among several growth factors and cytokines that were present in the SV extract, transforming growth factor-β1 (TGF-β1) significantly enhanced the invasive potential of PC3 cells; however, the additional treatment with neutralising antibody against TGF-β1 suppressed the enhanced invasive potential induced by the SV extract. Changes in the invasive potential in PC3 cells after treatment with the SV extract and/or TGF-β1 were in proportion to those in the production of urokinase-type plasminogen activator (uPA) by PC3 cells. Tumour growth as well as the incidence of lymph node metastasis in NOD/SCID mice after the injection of PC3 cells into the SV were significantly greater than those after the injection into the prostate. These findings suggest that the microenvironment of SV enhances the progression of prostate cancer through a stimulated invasive potential, and that enhanced uPA production in prostate cancer cells induced by TGF-β1 could therefore be one of the most important mechanisms involved in the progression of prostate cancer after SV invasion

Tissue level, activation and cellular localisation of TGF-β1 and association with survival in gastric cancer patients

Transforming growth factor-β1 (TGF-β1), a tumour suppressing as well as tumour-promoting cytokine, is stored as an extracellular matrix-bound latent complex. We examined TGF-β1 activation and localisation of TGF-β1 activity in gastric cancer. Gastric tumours showed increased stromal and epithelial total TGF-β1 staining by immunohistochemistry. Active TGF-β1 was present in malignant epithelial cells, but most strongly in smooth muscle actin expressing fibroblasts. Normal gastric mucosa from the same patient showed some staining for total, and little for active TGF-β1. Active TGF-β1 levels were determined by ELISA on tissue homogenates, confirming a strong increase in active TGF-β1 in tumours compared to corresponding normal mucosa. Moreover, high tumour TGF-β1 activity levels were significantly associated with clinical parameters, including worse survival of the patients. Total and active TGF-β1 levels were not correlated, suggesting a specific activation process. Of the different proteases tested, active TGF-β1 levels were only correlated with urokinase activity levels. The correlation with urokinase activity suggests a role for plasmin in TGF-β1 activation in the tumour microenvironment, resulting in transformation of resident fibroblasts to tumour promoting myofibroblasts. In conclusion we have shown localisation and clinical relevance of TGF-β1 activity levels in gastric cancer

Transforming Growth Factor β Signaling Pathway Associated Gene Polymorphisms May Explain Lower Breast Cancer Risk in Western Indian Women

Transforming growth factor β1 (TGFB1) T29C and TGF β receptor type 1 (TGFBR1) 6A/9A polymorphisms have been implicated in the modulation of risk for breast cancer in Caucasian women. We analyzed these polymorphisms and combinations of their genotypes, in pre menopausal breast cancer patients (N = 182) and healthy women (N = 236) from western India as well as in breast cancer patients and healthy women from the Parsi community (N = 48 & 171, respectively). Western Indian women were characterized by a higher frequency of TGFB1*C allele of the TGF β T29C polymorphism (0.48 vs 0.44) and a significantly lower frequency of TGFBR1*6A allele of the TGFBR1 6A/9A polymorphism (0.02 vs 0.068, p<0.01) as compared to healthy Parsi women. A strong protective effect of TGFB1*29C allele was seen in younger western Indian women (<40 yrs; OR = 0.45, 95% CI 0.25–0.81). Compared to healthy women, the strikingly higher frequencies of low or intermediate TGF β signalers in patients suggested a strong influence of the combination of these genotypes on the risk for breast cancer in Parsi women (for intermediate signalers, OR = 4.47 95%CI 1.01–19.69). The frequency of low signalers in Parsi healthy women, while comparable to that reported in Europeans and Americans, was three times higher than that in healthy women from western India (10.6% vs 3.3%, p<0.01). These observations, in conjunction with the low incidence rate of breast cancer in Indian women compared to White women, raise a possibility that the higher frequency of TGFB1*29C allele and lower frequency of TGFBR1*6A allele may represent important genetic determinants that together contribute to a lower risk of breast cancer in western Indian women

TGF-β1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models.</p> <p>Methods</p> <p>The mRNA expression levels of TGF-β isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-β1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays.</p> <p>Results</p> <p>In general, TGF-β2, TβRI and TβRII are over-expressed in more aggressive cells, except for TβRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-β1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-β1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-β1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-β1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-β1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors.</p> <p>Conclusion</p> <p>Altogether, our results support that TGF-β1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-β1 still remains a promising target for breast cancer treatment.</p

TGF-β1 genotype and phenotype in breast cancer and their associations with IGFs and patient survival

Transforming growth factor-β (TGF-β)-mediated signals play complicated roles in the development and progression of breast tumour. The purposes of this study were to analyse the genotype of TGF-β1 at T29C and TGF-β1 phenotype in breast tumours, and to evaluate their associations with IGFs and clinical characteristics of breast cancer. Fresh tumour samples were collected from 348 breast cancer patients. TGF-β1 genotype and phenotype were analysed with TaqMan® and ELISA, respectively. Members of the IGF family in tumour tissue were measured with ELISA. Cox proportional hazards regression analysis was performed to assess the association of TGF-β1 and disease outcomes. Patients with the T/T (29%) genotype at T29C had the highest TGF-β1, 707.9 pg mg−1, followed by the T/C (49%), 657.8 pg mg−1, and C/C (22%) genotypes, 640.8 pg mg−1, (P=0.210, T/T vs C/C and C/T). TGF-β1 concentrations were positively correlated with levels of oestrogen receptor, IGF-I, IGF-II and IGFBP-3. Survival analysis showed TGF-β1 associated with disease progression, but the association differed by disease stage. For early-stage disease, patients with the T/T genotype or high TGF-β1 had shorter overall survival compared to those without T/T or with low TGF-β1; the hazard ratios (HR) were 3.54 (95% CI: 1.21–10.40) for genotype and 2.54 (95% CI: 1.10–5.89) for phenotype after adjusting for age, grade, histotype and receptor status. For late-stage disease, however, the association was different. The T/T genotype was associated with lower risk of disease recurrence (HR=0.13, 95% CI: 0.02–1.00), whereas no association was found between TGF-β1 phenotype and survival outcomes. The study suggests a complex role of TGF-β1 in breast cancer progression, which supports the finding of in vitro studies that TGF-β1 has conflicting effects on tumour growth and metastasis

Evolution of a Distinct Genomic Domain in Drosophila: Comparative Analysis of the Dot Chromosome in Drosophila melanogaster and Drosophila virilis

The distal arm of the fourth (“dot”) chromosome of Drosophila melanogaster is unusual in that it exhibits an amalgamation of heterochromatic properties (e.g., dense packaging, late replication) and euchromatic properties (e.g., gene density similar to euchromatic domains, replication during polytenization). To examine the evolution of this unusual domain, we undertook a comparative study by generating high-quality sequence data and manually curating gene models for the dot chromosome of D. virilis (Tucson strain 15010–1051.88). Our analysis shows that the dot chromosomes of D. melanogaster and D. virilis have higher repeat density, larger gene size, lower codon bias, and a higher rate of gene rearrangement compared to a reference euchromatic domain. Analysis of eight “wanderer” genes (present in a euchromatic chromosome arm in one species and on the dot chromosome in the other) shows that their characteristics are similar to other genes in the same domain, which suggests that these characteristics are features of the domain and are not required for these genes to function. Comparison of this strain of D. virilis with the strain sequenced by the Drosophila 12 Genomes Consortium (Tucson strain 15010–1051.87) indicates that most genes on the dot are under weak purifying selection. Collectively, despite the heterochromatin-like properties of this domain, genes on the dot evolve to maintain function while being responsive to changes in their local environment