Molecular Mechanism By Which Inhibition Of RAC1 Expression By YAP Promotes Multi-Drug Resistance Of Hepatocellular Carcinoma

Background:Hepatocellular carcinoma（HCC）is one of the malignant tumors in digestive system with a high malignancy and a poor prognosis.At present,the clinical treatment for HCC is mainly based on surgical resection and liver transplantation,but a considerable proportion of patients lost the opportunity of surgical treatment when they are diagnosed.Therefore,systemic therapy based on chemotherapy and molecular targeted therapy is particularly important in the clinical treatment for patients who suffer from HCC.Currently,however,the effect of commonly used chemotherapy agent,such as doxorubicin,fluorouracil,and cisplatin,are limited in clinical treatment.The multi-drug resistance in HCC cells is an important reason for the failure of chemotherapy,and the molecular mechanism of multidrug resistance in HCC has not been clarified.Therefore,a deep study on molecular mechanism of multi-drug resistance is able to provide new therapeutic strategies for hepatocellular carcinoma.YAP（Yes-associated Protein）plays an important role in progression of hepatocellular carcinoma and is involved in drug resistance of HCC.However,the molecular mechanism of YAP promoting multi-drug resistance in HCC cells is not clear.Objective:The purpose of the research is to investigate the mechanism of YAP promoting multi-drug resistance in HCC.First,the expression of YAP was detected in HCC and adjacent normal tissue,the correlation with clinical pathological characteristics,such as tumor size and disease stage were analyzed.In order to clarify molecular mechanism of YAP promoting drug resistance,we study the role of YAP in multi-drug resistance of HCC cells through Xenograft model and cell functional experiments,and deeply explore the downstream molecules,biological processes and signaling pathways regulated by YAP by Co-immunoprecipitation and other methods.Material and methods:1.93 paired HCC tissue samples were collected.The expression level of YAP in HCC and adjacent normal tissues was detected by immunohistochemical staining.Pearson correlation analysis and survival analysis was conducted to study the correlation between the expression level of YAP and clinical pathological characteristic and clinical prognosis in HCC patients.2.In order to analyze the expression levels of YAP in HCC sample and cell lines,the profile data of HCC tissue and HCC cell lines from Oncomine database were collected.3.The expression level of YAP in drug resistant cell line BEL/FU and seven HCC cell lines were detected by RT-q PCR and Western-blot.4.The cell lines with YAP stably knockdown and overexpression were established.The small molecular inhibitor which targets YAP were used to inhibit the function of YAP.Through CCK-8,colony formation,flow cytometry and Xenograft model,we verified the effect of YAP on multi-drug resistance in HCC.5.Through Cell ROX probe,detection of NADPH/NADP+,western-blot,flow cytometry and colony formation,we explore the effects of YAP on generation of reactive oxygen species and NADP+and the regulation of RAC1 expression.Meanwhile,we verified the role of RAC1 in YAP promoting multi-drug resistance in HCC.6.By Co-immunoprecipitation and western-blot,we investigated the role of YAP on ubiquitination degradation of RAC1.The E3 ubiquitination ligase HACE1 was verified as key regulator in degradation of RAC1.7.The effects of YAP,RAC1 and reactive oxygen species on regulation of m TOR signaling and autophagy were studied by LC3-m RFP-GFP adenovirus and western-blot.Based on analysis of apoptosis,we verified that YAP promote multi-drug resistance by regulating m TOR and inhibition of autophagy through downregulation of RAC1 and reactive oxygen species in HCC.Results:1. Based on analysis of profile data of HCC tissues and cell lines in Oncomine database,it was found that YAP was highly expressed in HCC tissues and HCC cell lines.Furthermore,we verified that YAP was highly expressed in 93 paired HCC samples,HCC cell lines and drug-resistant cell line BEL/FU.Moreover,the expression of YAP in HCC tissues is correlated to the number of tumors and poor prognosis of HCC patients significantly.2. In vitro study has demonstrated that overexpression of YAP was able to significantly reduce the proportion of apoptosis in BEL/7402 cells treated with fluorouracil or doxorubicin.After knockdown or inhibition of YAP,the IC₅₀,colony formation and cell proliferation of BEL/FU cells significantly reduced under treatment of fluorouracil or doxorubicin,while its apoptosis level increased.3. Through Xenograft model,we verified that Verteporfin（a small molecule inhibitor target YAP）effectively inhibited the growth of subcutaneous tumor under treatment of fluorouracil or doxorubicin and the number of TUNEL+cells increased significantly.4. The overexpression of YAP in HCC cell lines inhibited the production of reactive oxygen species and NADP+.Under treatment of fluorouracil or doxorubicin,RAC1 is the critical gene that product reactive oxygen species in HCC cell lines.Meanwhile,we found that YAP promoted drug resistance in HCC cells mainly through downregulation of RAC1 expression by apoptotic analysis of flow cytometry and colony formation.5. By Co-immunoprecipitation and western-blot,we demonstrated that YAP downregulate expression of YAP mainly by promoting ubiquitination degradation of RAC1 protein.HACE1 is the critical ubiquitination ligase which mediates the degradation of RAC1,and its expression was regulated by YAP.6. The knockdown of YAP in HCC cells was able to downregulate the phosphorylation level of m TOR protein and its downstream molecules（S6,4E-BP1）.In contrast,the phosphorylation level of m TOR,S6,and 4E-BP1 protein significantly increased after overexpression of YAP.Meanwhile,knockdown or overexpression of RAC1 and neutralization of intracellular reactive oxygen species reversed the impact on the activation of m TOR signaling by YAP.7. The treatment of rapamycin（m TOR inhibitor）combined with fluorouracil or doxorubicin promoted the expression of cleaved-PARP and cleaved-caspase3 and autophagy influx in BEL/FU cells.After downregulation of YAP,the autophagy influx was promoted in BEL/FU cells,meanwhile,the sensitivity to fluorouracil and proportion of apoptotic cells were significantly increased.In addition,the apoptotic proportion of BEL/FU cells was reduced significantly after blockade of autophagy influx by treatment of chloroquine,3-MA and si ATG5,si BECN1.Conclusion:1.YAP was upregulated in HCC tissues.In addition,the upregulation of YAP was highly correlated to the poor prognosis of HCC patients.2.Inhibition or downregulation of YAP was able to increase the sensitivity of BEL/FU cells to fluorouracil or doxorubicin obviously.And YAP could promote drug resistance in HCC cells by downregulation of RAC1 through ubiquitination degradation and inhibiting generation of reactive oxygen species.3.In HCC cells,under treatment of chemo-drugs,YAP improved the resistance to chemotherapy mainly based on promoting and maintaining activation of m TOR and ultimately inhibition of excessive autophagy influx through downregulation of RAC1and reactive oxygen species generation.