Experimental Study On The Anti-TNBC Mechanism Of Hypophosphorylation Of Retinoic Acid Receptor Alpha

Objective:Retinoic acid receptor alpha(RARα)is a transcription factor regulating cell growth and differentiation,which is expressed in many tissues and organs.The study show that the phosphorylation of RARα is related to the malignant proliferation of acute promyelocytic leukemia(APL).In APL,cyclin dependent kinase activated kinase(CAK)phosphorylates serine(RARαS77)at the 77 th position of RARα,which inhibits its transcriptional activation of downstream target genes and promotes cell proliferation in vitro and in vivo.However,ATRA can reduce the phosphorylation of RARα by CAK,and then block the cells in G1 phase and induce differentiation.However,the role of RARα in ATRA resistant triple negative breast cancer(TNBC)is not clear.The main purposes of this study are:1.To explore the expression of RARα(p-Ser77)in TNBC;2.To explore the effect of hypophosphorylated RARα on the malignant proliferation of TNBC in vitro and in vivo;3.To explore the mechanism of hypophosphorylated RARαon the malignant proliferation of TNBC.Methods:1.Immunohistochemistry was performed to detect the expression of RARα(p-Ser77)in cancerous and adjacent tissues of TNBC collected from Zhejiang Cancer Hospital and tissue microarray for triple negative and non-TNBC.2.The expression of RARα(p-Ser77)in ATRA-sensitive breast cancer cell lines and TNBC cell lines was detected by Western blotting experiments.3.MDA-MB-231,MDA-MB453 overexpressing hypophosphorylated RARα(RARαS77A)cell lines and wild-type RARα cell lines were constructed by lentiviral transfection and the proliferation inhibition of TNBC cell lines with or without RARs agonist were determined by MTT and plate colony assays.4.The effects of RARαS77A in apoptosis and cycle arrest of TNBC cell lines were analyzed by flow cytometry and Western blotting with or without low-concentration RARs agonists.5.AO staining and Western blotting were performed to study the autophagy changes of RARαS77A TNBC cell lines with or without lowconcentration RARs agonists.6.The xenograft model in nude mice was used to explore the effect of RARαS77A on the growth of breast cancer.Results:1.The expression of RARα(p-Ser77)in TNBC was higher than that in adjacent breast cancer and non-TNBC.2.The results of Western blotting showed that the expression of RARα(p-Ser77)in ATRA sensitive breast cancer cells was lower than that in TNBC cells but no significant change in RARα expression.3.MTT and plate colony experiments showed that the RARαS77A was able to inhibit the proliferation of TNBC but cannot further inhibit when combined with low concentration RARs agonist.Meanwhile,the wild type had no inhibition effect.4.Flow cytometry showed that the RARαS77A induced apoptosis and similarly there was no concentration-dependent manner.Western blotting results showed that it increased the cleavage of Caspase-3 and PARP protein;at the same time,it arrested G0/G1 phase and up-regulated the expression of p27,Cyclin D1 and CDK4.5.AO staining showed that the RARαS77A increased the acid vesicles and Western blotting showed that the proportion of LC3II type increased,the expression of ATG7 increased and p62 decreased.6.Xenograft study demonstrated that hypophosphorylation RARα could suppress the tumor growth in vlvo.Conclusion:Compared with other breast cancer types that are sensitive to ATRA,TNBC has higher phosphorylation levels at RARαS77,suggesting that hyperphosphorylation of RARαS77 may be related to TNBC resistance to ATRA.RARαS77A overexpressed in TNBC mimics hypophosphorylated RARα and can significantly inhibit the proliferation of TNBC in vitro and in vivo in a ligandindependent manner,whereas overexpression of wild-type RARα does not.Further research indicates that the inhibitory effect on proliferation is caused by the induction of apoptosis,cycle arrest and lethal autophagy.This study provides theoretical basis for the development of anti-TNBC drugs based on the regulation of RARα phosphorylation and new ideas for clinical treatment of TNBC.