| PTEN (phosphatase and tensin homolog deleted on chromosome 10) or what is also known as MMAC1 (mutated in multiple advanced cancers) is a tumor suppressor gene localized to chromosome 10q23 in which it encodes a lipid phosphatase protein of PTEN. The deletion of the tumor suppressor gene of PTEN has been found in a number of human cancers. In gastric cancer PTEN was found to be lost, though mutations in the gene were not reported at high frequencies. SMAD-4, on the other hand, which is the only co-SMAD, is considered by some researchers as a tumor suppressor gene that mediates signals through TGF-β(transforming growth factor beta). SMAD-4 is found to be lost or mutated in many cancers including gastric cancer.Here, we studied the role of SMAD-4 and TGF-βon the expression of the tumor suppressor gene of PTEN in both human embryonic kidney cells of 293T and in malignant gastric carcinoma cells of MGC-803.Primarily results of promoter activity of PTEN have shown a significant increase in activity when SMAD-4 protein was over expressed inside 293T cells using dual luciferase reporter assay, indicating some interaction between the promoter region of PTEN and SMAD-4 protein.Transfection of human embryonic kidney cells of 293T with SMAD-4 expressional plasmid in the presence or absence of TGF-β1 up-regulated the expression of PTEN mRNA and protein levels whereas the ultimate expression was achieved when cells were treated with TGF-β1 and transfected with SMAD-4. While, knocking down SMAD-4 expression via SiRNA down-regulated the expression of PTEN on both mRNA and protein levels.Data from chromatin immuno-precipitation (ChIP) revealed a positive interaction between the SMAD-4 protein and three sites at PTEN promoter with variable binding affinities. However, in malignancies the role of both SMAD-4 and TGF-βin tumorsuppression seems to be different and far away from that behavior found in normal cells, as many studies mentioned controversies of either TGF-βor SMAD-4 over PTEN regulation in different cell lines. In human malignant gastric carcinoma cells of MGC-803, the transfection of SMAD-4 expressional plasmid in the presence or absence of TGF-β1 resulted in down-regulated PTEN expression on both mRNA and protein levels. The less PTEN expression was found in cells treated with TGF-βand transfected with SMAD-4.Many reports have mentioned the role of active RAS oncogene and its down stream in gastric tumorigenesis. Here, we treated MGC-803 cells with the MEK inhibitor PD98059, and then cells were transfected with SMAD-4 and signaled by TGF-β1. Results obtained from Western blot and RT-PCR showed that PTEN expression was turned on once again, while the maximum expression was noticed in cells transfected with SMAD-4 and signaled through TGF-β.Our data showed that RAS/ERK pathway was active in tumor cells and inhibited by the use of MEK inhibitor PD98059. Furthermore, checking SMAD-4 protein abundance in cytosol and nuclear fractions of MGC-803 cells respectively revealed that SMAD-4 protein was translocated into nucleus when the cells were treated with the MEK inhibitor and signaled with TGF-β1. Hence, SMAD-4 was restored from facilitating the suppression of PTEN expression to be an up-regulator of PTEN with the inhibition of RAS/ERK which indeed restored the signaling of TGF-β.Finally, SMAD-4 was found to induce PTEN expression through TGF-βsignaling in normal 293T cells. While this up-regulation was lost and turned to be suppression for PTEN expression in MGC-803 cells whereas the active RAS/ERK pathway might prevente SMAD-4 to translocate inside the nucleus and induce PTEN expression. Hence, this expression was restored by inhibiting RAS/ERK pathway. |
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