Inherent Antagonism Of Tumor Suppressors P53 With RASSF1A Regulate Apoptosis

It is widely accepted that oncogenes and tumor suppressors coordinately control cell growth or death. Here we show that, in addition, the p53 tumor suppressor antagonizes another tumor suppressor RASSFIA (RAS-association domain family 1A) by binding to promoter of RASSFIA and significantly inhibiting its expression. The suppression of RASSFIA by p53 regulates apoptosis. Protein interaction analysis showed that RASSFIA interacts with IXL (Intersex-like), a new subunit of mediator complex, which also binds to a-tubulin as RASSFIA does. Moreover, the interaction RASSFIA with IXL elicits diffusion of IXL protein within cancer cell, suggesting a role of RASSFIA as a cargo vehicle to exert function of protein IXL transportation along the microtubules. Besides, GC-rich region of RASSFIA promoter has the maximum activity and is hypermethylated, and the hypermethylation is associated RASSF1A silencing in human testis tumor. Coordinative action of the p53-RASSF1A-IXL was further revealed on tissue chips by co-localization and mislocalization of the three proteins in human testis tumors. These results suggest a crosstalk within tumor suppressors p53 and RASSFIA, and that RASSF1A-IXL pathway may mediate cancer development through action of p53 against RASSFIA. Our results provide a new start to conquer cancer inherent antagonism by tumor suppressors. 1. RASSF1A is hypermethylated and silenced in testis tumorsGenital system tumors are hackneyed malignancy which disserve health severely and testis tumor is most frequently appeared in young man with high case fatality and bad prognosis. Inactivation of RASSFIA is closely related to the genesis and development of tumors. Recent articles have reported that RASSFIA is rarely mutant in cancers and the main mechanism of inactivation is due to the high methylation of it’s promoter. To investigate the methylation state and experssion pattern of RASSF1A in testis tumor, we made a study of RASSFIA promoter. Several promoter regions of different length were cloned and transfected into Siha cell. By flow cytometry, we showed that the-301bp region which within the CpG island possess the highest activation. By MSP and sequencing, we demonstrated the high methylation of RASSFIA promoter and the detailed methylated CpG residues. Further, immunohistochemistry stain showed that methylation of promoter resulted in the decreased level of RASSF1A expression.2. p53 binds to RASSFIA and induces expression decrease of RASSF1ABy promoter analysis, we forecasted that in-2781bp of RASSFIA promoter, there existed a p53 binding site. We prokaryotically expressed and purified HIS-p53 fusion protein. Gel shift and EMSA (electrophoretic mobility-shift assay) showed that p53 binds to RASSFIA promoter. Further, transient cotransfection of p53 and RASSF1A-EGFP in Cos7 cell revealed that overexpression of p53 induced conspicuous decrease expression of EGFP. In Siha cells which endogenous express RASSFIA, when overexpressed p53, we also found that endogenous RASSFIA expression was depressed. From above,we concluded that p53 has a DNA-binding ability to RASSFIA and down regulated the expression of RASSFIA.3. HPV16-E6 modulates RASSFIA expression through p53HPV infection is the most predisposing factor of reproductive system tumors including testis、cervical and prostate cancers. Among them, high-risk human papillomavirus (HPV), usually HPV type 16 (HPV 16) or HPV 18 play an important role in the genesis、development and transference of tumors. HPV16 and HPV18 mainly encode E6 and E7 proteins which involved in the tumorigenesis through p53 and Rb proteins. The E6 protein binds to and degradates p53 protein and targets it for the further destruction of p53 function such as responding to DNA damage, G1/S checkpoint transition and apoptosis. It’s unknown that whether there exists a relationship between HPV16-E6 and RASSFIA. For this,we constructed HPV16-E6 RNAi vector and transfected it into Siha cell which endogenous HPV16 expression was detected. Real-time PCR and western blot showed that RNAi resulted in the down regulation of HPV16-E6 and RASSFIA, and the up regulation of p53 expression. The results indicated that HPV16-E6 could modulate RASSF1A expression through p53.4. p53 induced apoptosis through RASSF1Ap53 and RASSFIA are tumor suppressors. Decreasing of RASSFIA expression by p53 arises an interesting question:what will happen if p53 was transfected into Siha cells which endogenously expressed RASSFIA. We transfected p53 into Siha cells and performed apoptosis analysis. By flow cytometry, we detected the obvious apoptosis. It indicated that the total apoptosis proportion was increased because of p53 overexpression, but the decreased part of RASSFIA expression could partly counteracts the apoptosis effect induced by p53 and maintain a dynamic equilibrium of cell growth to avoid excessive suppressor phenomenon.5. RASSFIA acts with Intersex for its subcellular transportationTo analyze the RASSF1 function, we constructed the Intersex-dsRED vector and co-transfected it into the Cos7 cells with RASSF1A-EGFP vector. By confocal microscope, we observed the change of Intersex subcellular location. The distribution of Intesex-dsRED conversed from congregation around the cytoplasm surface of nucleus to dispersive state in plasma indicating. Co-immunoprecipitation indicated the interaction of Intersex and RASSF1A. These results indicated that microtubule-binding RASSFIA exerts role of protein transportation for IXL along microtubules after translation. On the other hand, IXL interacted with a-tubulin, which was confirmed by co-immunoprecipitation, and Intersex-dsRED was also distributed within cytoplasm similar to a-tubulin after transfected into RASSF1A-expressing HeLa cells. When microtubules were demolished by the nocodazole, IXL protein was showed mainly in nucleus. These data suggests that RASSF1A may work as an engine to transport IXL protein along microtubules.6. HPV16-E6-p53-RASSFIA-IntersexFrom above mentioned, we suggested a new pathway for tumorigenesis of genital system:p53-RASSF1A-Intersex. p53 acts with RASSF1A which resulted in the downregulation of RASSF1A expression and controls apoptosis together. HPV16-E6 may function in the pathway through p53. RASSF1A interacts with Intersex and microtubulin, which may be involved in the protien transportation of Intersex. p53 targets Intersex through RASSF1A and induces apoptosis in the end.