Overexpression Of Transforming Growth Factor β1 In Malignant Prostate Cells Is Partly Caused By Runaway Of TGF-β1 Auto-Induction Mediated Through A Defective Recruitment Of Protein Phosphatase 2A By TGF-β Type I Receptor

Background:Many cancer cells, including prostate cancer, are able to over-express TGF-β1. Aside from the growth inhibitory effect, TGF-β1 can also stimulate extracellular matrix production, promote angiogenesis, facilitate invasion, and suppress the host immune system. Thus, cancer cells may circumvent the suppressive effects of TGF-β1, especially for aggressive cancer cells lack of normal core components of TGF-βsignal pathway. For these cancer cells, it is possible that approaches to disrupt the TGF-β1 overexpression may offer a strategy to suppress their aggressive phenotype. To achieve this aim, firstly we should understand the mechanism why these tumor cells produce more TGF-β1 than their benign counterparts do. Yet, little is known about the regulation of TGF-β1 expression. The most potent inducer of TGF-β1 is itself. It was showed TGF-β1 can induce its own mRNA transcription and protein synthesis in various cells.The exact mechanism of TGF-β1 auto-induction is still not clear. Previous investigations showed that ERK and JNK are involved in TGF-β-induced AP-1 complex, which contributes to TGF-β1 auto-induction. Further, Smad3/Smad4 takes part in the TGF-β1 mRNA transcription, while p38 pathway influences de novo synthesis of TGF-β1. Among these pathways, ERK activation by TGF-β1 was proved to be essential for TGF-β1 auto-induction. However, the effect of TGF-β1 on ERK activation remains controversial and seems to be dependent on cellular context. Many studies described that TGF-β1 could activate ERK; while others reported that TGF-β1 inactivated or had no effect on ERK. Therefore it is reasonable to deduce that TGF-β1 auto-induction through ERK activation is also cellular context dependent. The aim of the present study is to elucidate whether there is a difference in TGF-β1 auto-induction in malignant and benign prostate epithelial cells and whether TGF-β1 auto-induction contributes to TGF-β1 overexpression in malignant cells.METHODS:Human prostate cancer cell lines, PC3, DU145, and the benign human prostate epithelial cell line, RWPE1, benign prostate hyperplastic epithelial cell line, BPH1 were used in this study. TGFβlevel of these four cell lines was assayed by using ELISA. TGFβ1 mRNA was assayed by real time qRT-PCR, where total RNA was extracted from cells upon stimulation with low dose (0.1ng/ml) or high dose (10 ng/ml) of exogenous TGFβ1 for 12h in the absence or present of Phospho-Mek inhibitor UO126, or cells cultured with serum free medium for 2 days and then 100ug/ml of TGFβneutralizing antibody 1D11 added for 12h. In some indicated experiments, cells were infected with a dominant negative TGF-βtypeⅡreceptor (TβRIIDN) retroviral vector. Extracellular signal-regulated kinase (ERK) activation was detected by using western-blot. Activities of serine/threonine phosphatase were performed by using commercial kit according to established procedures. Immunoprecipitation and following western-blot were carried out to evaluate the physical interaction of TGFβreceptorⅠand the regulatory subunit, Ba, of protein phosphatase 2A (PP2A-Bα).RESULTS:The basal level of TGFβin DU145 and PC3 cells was higher (P＜0.01) than in BPH and RWPE1. Once cells endogenous TGFβwas neutralized, its mRNA level decreased in Du145 (P<0.05) and PC3 (P<0.05), but not BPH or RWPE1. Upon the low dose of exogenous TGFβstimulation, TGFβ1 mRNA level increased in all these four cell lines, however the high dose of exogenous TGFβcan only autoinduce TGFβin DU145 and PC3 cells but not in BPH1 or RWPE1 cells. TGFβautoinduction was blocked after inhibition of Erk activation by using UO126. This differential TGFβautoinduction in malignant and benign cells correlates with ERK activation change upon TGFβstimulation:at the low dose of TGF-β1, all cells showed a rapid and transient activation of Erk. With the high dose of TGF-β1 malignant cells continued to display a rapid activation of Erk, but benign cells showed a transient inactivation of ERK. Investigation of phosphatase activities showed that, the serine/threonine phosphatases activity increased in a dose related manner in benign cells, but not in malignant cells. Results of immunoprecipitation experiments demonstrated that, with TGF-β1 treatment, antibody to TGF-βtypeⅠreceptor (TβRI) was able to pull down PP2A-Ba in benign cell but not in malignant cells, and the physical interaction of PP2A-Bαwith TGFβreceptorⅠin benign cells increased in a dose related manner upon TGFβstimulation. Inhibition of PP2A by using okadaic acid (10 nM) abrogated the ERK deactivation and then retrieved TGFβautoinduction in benign cells upon the high dose of TGFβstimulation.CONCLUSION:The results suggest that a dose dependent increase of physical interaction of TGFβreceptor I and PP2A Ba deactivates ERK and then the positive-feedback of TGFβautoinduction can be terminated upon high dose of TGFβstimulation in benign cells. However, TGFβautoinduction becomes runaway,, which may partly contributes to the high level of TGF P, in malignant cells due to lack of this kind of negative feedback mechanism.