Role of TGF-β1 and its Receptors in Breast Carcinogenesis： Evaluation of Gene Expression Pattems and Clinical Implications

OBJECTIVE Transforming growth factor β1 （TGF-β1） is a multifunc- tional cytokine that may play an important role in tumor development and progression. METHODS We evaluated gene expression patterns of TGF-β1 and its receptors [transforming growth factor β type Ⅰ receptor （TβR- Ⅰ ） and transforming growth factor β type Ⅱ receptor （TβR- Ⅱ ）] in tumor tissue from patients with breast cancer or with benign breast diseases （BBD） and adjacent normal tissue from the patients with breast cancer. Included in the study were 527 breast cancer patients and 213 BBD patients who participated in the Shanghai Breast Cancer Study. RESULTS The expression levels of the TGF-β1, TβR- Ⅰ and TβR-Ⅱ genes in breast tissue were quantified using real-time PCR. TIER- Ⅱ expression in cancer tissue was decreased by over 50% as compared to either adjacent normal tissue from the same patients or benign tumor tissue from BBD patients （p〈0.001）. TGF-β1 expression was lower by approximately 20% in cancer tissue compared to adjacent normal tissue （p=0.14） or to benign tumor tissue （p=0.002）. Although TβR-Ⅰ expression was also reduced in cancer tissue compared to adjacent normal tissue, or benign tumor tissue, the magnitude of the reduction was less apparent than that for TβR- Ⅱ. Compared to patients with the lowest tertile value for TβR- Ⅱ, patients with median tertile value for TβR- Ⅱ had more favorable overall survival （HR 0.47, 95% CI 0.27-0.85） and disease-free survival （HR 0.65, 95% CI 0.39-1.06）. No apparent associations, however, were observed between TGF-β1 or TβR- Ⅰ expression and overall or disease-free survival. CONCLUSION The results from this study support the hypothesis that a decreased level of TβR-Ⅱ gene expression, and thus reduced TGF-β1 sensitivity, is related to breast tumor progression.     

TGF-β1 alters microRNA profile in human gastric cancer cells

Objective: MicroRNAs (miRNAs) are important regulators that play a key role in tumorigenesis and tumor progression. Transforming growth factor-β1 (TGF-β1) is involved in invasion and metastasis in many tumors. In this study, we investigated the microRNAs (miRNA) profiles altered by TGF-β1 in gastric cancer (GC) cells. Methods: We detected the expression profiles of miRNA by miRNA microarray and quantitative real-time polymerase chain reaction. Migration and invasion, wound-healing assay, prediction of miRNA targets, Western blot and qRT-PCR analysis were carried out to determine the role of one selected miRNA, namely miR-193b, in affecting the biological behaviors of GC BGC823 cells. Results: Among 847 human miRNAs in the microarray, three miRNAs (miR-27a, miR-29b-1 and miR-194) were up-regulated and three (miR-574-3p, miR-193b and miR-130b) were down-regulated in BGC823 cells treated with TGF-β1 compared with control. miR-193b suppressed the invasion and metastasis of GC cells in vivo and in vitro, and down-regulated urokinase-type plasminogen activator (uPA) protein in GC cells. Conclusions: TGF-β1 altered miRNA expression profile in BGC823 cells. Among the altered miRNAs, TGF-β1 induced the down-regulation of miR-193b, which inhibited cell invasion and metastasis in vivo and in vitro, and down-regulated uPA protein in GC cells.     

Expression of TGF-β1, Snail, E-cadherin and N-cadherin in Gastric Cancer and Its Significance

OBJECTIVE To investigate the expression of TGF-β1,Snail,E-cad-herin and N-cadherin in gastric cancer(GC),and to examine its relationship to malignant features of the tumors. METHODS The expression of TGF-β1,Snail,E-cadherin and N-cadherin proteins was detected in GC and adjacent tissues by immunohistochemical staining,and compared with the clinico-pathological data. RESULTS Positive rates of expression for TGF-β1,Snail,E-cadherin and N-cadherin were 63.5%,83.3%,37.5%and 44.8%in GC,and 28.8%, 41.3%,100%,11.3%in adjacent tissues,respectively.The expression of all four proteins showed a significant difference between the GCs and adjacent tissues(P<0.05).The positive rate of TGF-β1,Snail and N-cadherin,or the negative rate of E-cadherin expression was significantly related to the differentiated degree,histological type,invasion and metastasis of GC.In addition,the expression of N-cadherin was positively related to that of TGF-β1, but negatively related to that of E-cadherin.There was negative correlation between expression of E-cadherin and TGF-β1 and Snail in GC(P<0.05). CONCLUSION The over-expression of TGF-β1 and Snail and decreased expression of E-cadherin and the abnormal expression of N-cad-herin were involved in the process of invasion and metastasis of GC.The data showed that E-cadherin might switch to N-cadherin.TGF-β1 and Snail might play a fundamental role in the process.     

The Role of Expression of Mismatch Repair Proteins hMSH2 and hMLH1 in Gastric Carcinogenesis and its Clinical Significance

OBJECTIVE To investigate the expression of the mismatch repair proteins hMSH2 and hMLH1,and to examine the clinical significance of the intracellular expression site(ICES)in gastric carcinogenesis. METHODS Specimens from 172 cases of gastric cancer,151 tissues from paraneoplastic gastric mucosa and 34 from noncancerous gastric mucosa were collected in Dalain,China.An immunohistochemical method was used to determine the expression of the hMSH2,hMLH1 proteins and their ICES in the gastric mucosas. RESULTS The rate of hMSH2 expression in gastric cancers,paraneoplastic gastric mucosas and noncancerous gastric mucosas were respectively 69.8%,49.7%and 32.4%.The rate was significantly higher in gastric cancer compared to the latter two groups(P=0.000),but there was no obvious difference in the expression between the two latter groups(P=0.067). The hMLH1 protein expression rates were respectively 73.3%,57.6%and 41.2%in the above three groups.The expression was significantly higher in the gastric cancer group compared to the two latter groups(P=0.000),while there was no significant difference between the latter groups(P=0.082). There was no obvious correlation between the hMSH2 and hMLH1 protein expression rates and related factors,such as gender,age and differentiated level of gastric cancer etc.The cell-nuclear expression of the hMSH2 protein was respectively 70.0%,58.7%and 36.4%in the gastric cancer,paraneoplastic gastric mucosa and noncancerous gastric mucosa groups.The cytoplasmic expression rates were 30.0%,41.3%and 63.6%in the three groups. The cell-nuclear expression rate of the hMSH2 protein gradually decreased in the gastric mucosas in the fol owing order:cancer,paraneoplastic and noncancerous but cytoplasmic expression only increased slightly in these groups(r=0.161,P=0.020).There was no significant difference in the ICES of the hMLH1 protein among the three different gastric mucosas(P=0.659). CONCLUSION Simultaneous determination of the expression and ICES of the mismatch repair proteins hMSH2 and hMLH1 in the gastric mucosa may be helpful in detecting early gastric cancer.     

Sex hormone-induced mammary carcinogenesis in female Noble rats: Expression of TGF-β1 and its receptors, TGF-α, and EGF-R in mammary carcinogenesis

We have established a Noble rat model to explore the mechanisms of hormonal mammary carcinogenesis, in which the role of androgen in promoting mammary carcinogenesis was highlighted. We have also established that stromal–epithelial interactions may be responsible for the promotional effects of testosterone in mammary carcinogenesis. Based on these understandings, in the present study we examined the expression of transforming growth factor beta-1 (TGF-₁) and its receptors (TGF- RI, TGF- RII), transforming growth factor alpha (TGF-), and epidermal growth factor receptor (EGF-R) in 'pre-malignant' mammary glands treated with different protocols of sex hormones, as well as in mammary cancers. We observed that TGF-₁ was strongly expressed in most mammary tumors, whereas TGF- RI and TGF- RII were negative in most mammary tumor cells. The results from comparative study of 'pre-malignant' glands further showed that when the animals were treated with testosterone, either alone or in combination with 17-estradiol, the mammary gland epithelial cells expressed high levels of TGF-₁. This over-expression of TGF-₁ can be blocked by flutamide, indicating that testosterone may be responsible for the expression of TGF-₁ in mammary glands. TGF- RI and TGF- RII were also expressed strongly in testosterone-treated mammary epithelial cells and only weakly detectable in 17-estradiol treated and control mammary epithelial cells. Furthermore, TGF- RI and TGF- RII were also expressed in stromal cells, both in mammary tumors and in hormone-treated mammary glands. These observations indicate that the mechanism of testosterone in mammary carcinogenesis may be through its regulation of expression of TGF-₁ and its receptors. On the other hand, TGF- was also expressed in all 39 mammary cancers, while only 81% of the cancers were EGF-R positive. TGF- was also strongly expressed in stromal cells in all three experimental groups, but only moderately expressed in epithelial cells when treated with a combination of testosterone and 17-estradiol. By contrast, EGF-R was strongly expressed in epithelial cells in the three experimental groups but negative in stromal cells. Flutamide or tamoxifen was unable to block the expression of TGF- induced by the combined sex hormone treatment. However, they were effective in blocking the expression of TGF- when the animals were treated with testosterone or 17-estradiol alone, respectively. These results suggest that both testosterone and 17-estradiol may be required for the over-expression of TGF- in the mammary carcinogenesis induced by sex hormones. To our knowledge, this is the first experimental study to explore the regulation of TGF-₁, TGF-, and their receptors by testosterone and 17-estradiol in mammary carcinogenesis.     

Sex hormone-induced mammary carcinogenesis in female Noble rats: Expression of TGF-β1 and its receptors, TGF-α, and EGF-R in mammary carcinogenesis

We have established a Noble rat model to explore the mechanisms of hormonal mammary carcinogenesis, in which the role of androgen in promoting mammary carcinogenesis was highlighted. We have also established that stromal-epithelial interactions may be responsible for the promotional effects of testosterone in mammary carcinogenesis. Based on these understandings, in the present study we examined the expression of transforming growth factor beta-1 (TGF-beta1) and its receptors (TGF-beta RI, TGF-beta RII), transforming growth factor alpha (TGF-alpha), and epidermal growth factor receptor (EGF-R) in 'pre-malignant' mammary glands treated with different protocols of sex hormones, as well as in mammary cancers. We observed that TGF-beta1 was strongly expressed in most mammary tumors, whereas TGF-beta RI and TGF-beta RII were negative in most mammary tumor cells. The results from comparative study of 'pre-malignant' glands further showed that when the animals were treated with testosterone, either alone or in combination with 17beta-estradiol, the mammary gland epithelial cells expressed high levels of TGF-beta1. This over-expression of TGF-beta1 can be blocked by flutamide, indicating that testosterone may be responsible for the expression of TGF-beta1 in mammary glands. TGF-beta RI and TGF-beta RII were also expressed strongly in testosterone-treated mammary epithelial cells and only weakly detectable in 17beta-estradiol treated and control mammary epithelial cells. Furthermore, TGF-beta RI and TGF-beta RII were also expressed in stromal cells, both in mammary tumors and in hormone-treated mammary glands. These observations indicate that the mechanism of testosterone in mammary carcinogenesis may be through its regulation of expression of TGF-beta1 and its receptors. On the other hand, TGF-alpha was also expressed in all 39 mammary cancers, while only 81% of the cancers were EGF-R positive. TGF-alpha was also strongly expressed in stromal cells in all three experimental groups, but only moderately expressed in epithelial cells when treated with a combination of testosterone and 17beta-estradiol. By contrast, EGF-R was strongly expressed in epithelial cells in the three experimental groups but negative in stromal cells. Flutamide or tamoxifen was unable to block the expression of TGF-alpha induced by the combined sex hormone treatment. However, they were effective in blocking the expression of TGF-alpha when the animals were treated with testosterone or 17beta-estradiol alone, respectively. These results suggest that both testosterone and 17beta-estradiol may be required for the over-expression of TGF-alpha in the mammary carcinogenesis induced by sex hormones. To our knowledge, this is the first experimental study to explore the regulation of TGF-beta1, TGF-alpha, and their receptors by testosterone and 17beta-estradiol in mammary carcinogenesis.     

Influence of PPAR -γligand on radiation - induced PAI - 1 and TGF -βmRNA in fibroblast cells

Objective To observe the influence of peroxisome proliferator activated receptor-γligand (PPAR-γ, pioglatazone) on expression of PAI-1 and TGF-β mRNA and proliferation in fibroblast cells before and after X-ray radiation, and to study the effect of PPAR-γon normal cells during radiation induced fibrosis process. Methods RT-PCR method was used to measure PPAR-γgene expression in L929 cells.After X-ray irradiation of 10 Gy,4 Gy or 2 Gy, the expressions of PAI-1 and TGF-β mRNA in mouse lung fibroblast cells (L929) were measured using RT-PCR. After X-ray irradiation and pioglatazone treatment,the influence of pioglatazone on PAI-1 and TGF-β was measured using RT-PCR method. MTT method was used to test cell proliferation after the treatment of irradiation and pioglatazone. Results PPAR-γ mRNA expression was observed in L929 cells. Expression of PAI-1 and TGF-β mRNA reached the highest level 483.40,P =0. 090) ). At 48 h after the treatment of pioglatazone and 10 Gy radiation, pioglatazone decreased 0. 36, 0. 34 and 0. 32( F = 3.90, P = 0. 040) ). The inhibitory effect was significantly increased when L9292. 50,P =0. 005)). Conclusions X-ray irradiation can increase the expression of PAI-1 and TGF-β in L929 cells. Pioglatazone can decrease the expression of radiation-induced PAI-1 and TGF-β, and restrain the fibroblast proliferation.