The Regulation Mechanism Of RB-P53 Network In The Development And Metastasis Of Breast Cancer

Objective: To assess the effect of HU and ADM on the induction of DNA damage in breast cancer cells, probe into the effect of RB and P53 on DNA damage and EMT in breast cancer, and then explore the regulation mechanism of RB-P53 network in the development and metastasis of breast cancer.Methods: DNA damage was induced by HU and ADM treatment in breast cancer cells. Western blot was performed to detect the level of RB and P53, and flow cytometry and MTT were used to respectively detect the cell cycle and cell proliferation. Then transfection was adopted to suppress the expression of RB in MCF-7, followed by HU and ADM treatment, and detect the level of RB and P53. cell cycle and cell proliferation. In the end, RB knockdown and P53 knockdown were respectively or both performed in MCF-7(P53wt), MCF10A(P53wt) and SKBR3(P53mt), then RT-PCR and western blot were used to detect the level of RB, P53 and protein biomarkers of EMT, such as E-cadherin and snail.Results: We found that HU and ADM could induce breast cancer cells(including MCF-7, SKBR3 and MDA-MB-468) to be arrested at G1/S effectively, and if mutated, P53 will lose control of G1-S transition. Compared to MCF-7 and SKBR3, MDA-MB-468 got a higher G2/M arrest after HU was washed out, similar to RB KD MCF-7. What's more, RB KD could suppress the inhibitory effect of HU and ADM on MCF-7 and make it get a higher G2/M arrest induced by ADM. RB KD could down-regulate P53 in all the three cell lines above. Finally, RB KD or P53 KD could result in EMT in MCF-7, down-regulating E-cadherin and up-regulating snail, which is similar in MCF10 A. Otherwise, both RB KD and P53 KD could make a lower expression level of E-cadherin and higher snail. RB KD could up-regulate snail in SKBR3, with E-cadherin deficient and P53 mutant.Conclusion: These results suggest that both RB and P53 could interdependently regulate DNA damage induced by HU and ADM in breast cancer cells. And when RB was down-regulated, P53 wt would be down-regulated accordingly, then EMT occurred to breast cancer cells. Furthermore, breast cancer cells' sensibility to HU and ADM would decrease. EMT could be induced by P53 wt down-regulation. However, down-regulating RB and P53 simultaneously could make a more evident EMT. In conclusion, these results point we could estimate the sensibility to DNA damage drugs and the risk of metastasis by detecting tumor's expression level of RB and P53 in clinical breast cancer patients. Therefore, further study of RB-P53 network will provide more supports to diagonosis and treatment of breast cancer.