Synergistic Lethal Effects And Mechanism Of Combined Inhibition Of WWP1 And SHP2 In Colorectal Cancer

1.BackgroundColorectal cancer(CRC)is the third most common cancer and the second cause of cancer deaths worldwide,with approximately 50% of patients developing distant metastases.Although improved surgical techniques and the development of anticancer drugs have greatly improved the prognosis of patients with metastatic CRC,resistance to targeted drugs remains a challenge.Therefore,it is urgent to explore the resistance mechanism of targeted therapy for CRC and screen new therapeutic targets.SHP2 is a protein tyrosine phosphatase,acts as a key molecule mediating the activation of RAS-RAF-MEK-ERK pathway and PI3K-AKT pathway by RKTs molecules such as EGFR and HER2.Double inhibition of the MAPK signaling pathway is achieved when combined with kinase inhibitors,thereby reducing the occurrence of adaptive resistance.However,SHP2 allosteric inhibitor SHP099 has poor efficacy and extensive resistance when used alone,which requires a combination of drugs to achieve the goal of low toxicity and high efficiency.As an E3 ubiquitin ligase,WWP1 can ubiquitinate and degrade some tumor suppressor proteins,such as TP53,TGFBR1 and Smad2.In breast cancer,WWP1 can activate the PI3K-AKT pathway signaling by inhibiting the function of the suppressor oncogene PTEN.A natural compound 3-indolemethanol(I3C),which was discovered in cruciferous plants,can efficiently inhibit WWP1 and reactivate PTEN thus exerting anticancer activity.To overcome resistance to targeted therapy for CRC,new strategies for treatment are being searched.This study intends to explore the biological functions of combined inhibition of WWP1 and SHP2 in CRC.To screen and validate the downstream signaling that mediates the interference of combined inhibition of WWP1 and SHP2 in CRC.To evaluate the prognostic predictive value of WWP1 and SHP2 expression patterns in patients undergoing CRC surgery.2.MethodsPartⅠ:(1)The basic characteristics of WWP1 molecule were investigated by searching Gene Card,HPA,TGCA,and GEO databases.(2)The basal expression of WWP1 in different CRC cells was detected by q RT-PCR and Western Blot.(3)CRC cell lines with stable knockdown and overexpression of WWP1 were constructed by lentiviral infection.(4)The effects of WWP1 expression on the proliferative ability of CRC cells,the effects of WWP1 expression on the antitumor effect of SHP099,and the effects of combined inhibition of WWP1 and SHP2 on the proliferation of CRC cells in vitro were detected by CCK-8 and clone formation assays.(5)The effects of combined inhibition of WWP1 and SHP2 on apoptosis and cell cycle of CRC cells were examined by flow cytometry.(6)The cell line-derived xenograft was constructed in nude mice using CRC cells.The effects of combined inhibition of WWP1 and SHP2 on CRC cell growth in vivo were tested after gavage administration.(7)Detection of proliferation and apoptosis of CRC cells in vivo by Ki-67 staining and TUNEL staining.PartⅡ:(1)The differentially expressed genes were screened by RNA sequencing of I3 C and SHP099-treated multiple CRC cells,and GO,KEGG,and GSEA enrichment analyses were performed to search for signaling pathways affected by WWP1 and SHP2 inhibition.(2)The key molecules of downstream signaling pathways were further verified by plasmid transfection,immunoprecipitation,plasma membrane separation and protein immunoblotting.PartⅢ:(1)Based on immunohistochemical staining of a large sample of CRC tissue microarrays and the corresponding clinical data,the expression of WWP1 and its correlation with the clinical characteristics of patients undergoing CRC surgery were analyzed.(2)Prognostic risk factors were screened by COX regression modeling,predictive models were constructed using nomogram models,and the effects of WWP1 and SHP2 expression on the predictive models were evaluated in terms of discrimination,calibration,and clinical applicability.The prognosis of CRC patients was assessed based on the risk stratification of the nomogram models.3.Results3.1 Synergistic lethal effects of combined inhibition of WWP1 and SHP2 in colorectal cancerPublic databases suggested that the E3 ubiquitin ligase WWP1 is mainly localized in the cytoplasm,and its expression in colorectal tissues is at an intermediate level throughout the body,and that high expression of WWP1 in colorectal cancer is associated with a poor prognosis.By lentiviral infection of CRC cells,we successfully constructed cell lines with stable low expression of WWP1 in the KRAS mutant cell line SW480,the BRAF mutant cell line RKO,and the wild-type cell line Caco2,and successfully constructed cell lines with stable high expression of WWP1 in the KRAS mutant cell line HCT116,the BRAF mutant cell line HT29,and the wild-type cell line CW2.For in vitro functional assays,knockdown of WWP1 significantly inhibited the proliferation and clone formation ability of SW480,RKO and Caco2 cells,and overexpression of WWP1 led to opposite results in HCT116,HT29 and CW2 cells.In SW480,RKO and Caco2 cells,the combined inhibition of WWP1 and SHP2 synergistically suppressed the proliferative ability of tumor cells,promoted apoptosis and G1-phase cell cycle arrest.For in vivo functional experiments,SW620,RKO and Caco2 cells were successfully utilized to construct cell line-derived xenograft models in nude mice.Combined treatment with WWP1 and SHP2 inhibitors significantly inhibited the in vivo growth of tumor cells and promoted apoptosis under the condition of no obvious toxic side effects.3.2 Mechanism of synergistic lethality of combined inhibition of WWP1 and SHP2 in colorectal cancerRNA sequencing results achieved after treatment of SW620 and RKO cells with WWP1 and SHP2 inhibitors for 48 h suggested that combined inhibition of WWP1 and SHP2 affects the activity of cyclin-dependent protein kinase,serine/threonine protein kinase,protease inhibitor,which are involved in proteasomal protein catabolic process,endoplasmic reticulum stress,apoptosis,and other biological processes.Estrogen signaling pathway,p53 signaling pathway,MAPK signaling pathway,TNF signaling pathway,TGF-β signaling pathway,Hippo signaling pathway,especially PI3K-AKT signaling pathway,and ubiquitin mediated proteolysis may play important roles.Western blotting showed that combined inhibition of WWP1 and SHP2 deeply suppressed the PI3K-AKT signaling pathway in RKO and Caco2 cells.Combined with plasma membrane separation,trends in WWP1/PTEN levels and p AKT in the cytosol were correlated after SHP2 inhibition.Further immunoprecipitation of WWP1 confirmed the interaction between WWP1 and PTEN,and immunoprecipitation of PTEN showed that WWP1 could ubiquitinate modification of PTEN,and the ubiquitination level of PTEN was increased after SHP2 inhibition.Taken together,the WWP1-PTEN axis was involved in the feedback activation of p AKT after SHP2 inhibition,and the combined inhibition of WWP1 and SHP2 deeply suppressed the PI3K-AKT signaling pathway.3.3 Prognostic predictive value of WWP1 and SHP2 expression in patients undergoing surgery for colorectal cancerImmunohistochemical results of a microarray containing 180 colorectal tissues showed that WWP1 was mainly localized in the cytoplasm of intestinal mucosal epithelial cells and was significantly overexpressed in tumors and metastatic tissues compared with normal tissues,adenomas,and paraneoplastic tissues.Immunohistochemical results of microarrays containing 1049 CRC tissues showed that WWP1 was significantly overexpressed in patients with TNM stageⅡ-Ⅲand poorly differentiated pathology,and that high WWP1 expression was an independent risk factor for overall survival(OS)and disease-free survival(DFS)in patients undergoing surgery for CRC.The prognostic factors of CRC surgery patients were screened by COX regression,and the risk factors for OS included preoperative CEA level,postoperative pathological TNM stage,WWP1 level and SHP2 level,and the risk factors for DFS included preoperative CEA level,preoperative CA199 level,postoperative pathological TNM stage,degree of pathological differentiation,WWP1 level and SHP2 level.These prognostic factors were further used to construct nomogram models.Compared with the conventional models,the modified models added levels of WWP1 and SHP2.Results showed that the area under the curve(AUC)values for predicting 3-year OS and DFS in the modified model and the conventional model were 0.887,0.894,and 0.483,0.635,respectively,and the net reclassification indexes(NRI)for predicting 3-year OS and DFS were 0.978 and 0.767,respectively,and all these differences were statistically significant(p<0.05).It indicates that the modified models significantly improved the discrimination of the prognostic prediction models for patients undergoing CRC surgery.In addition,the calibration curves and decision curve analysis confirmed the advantages of the modified models in evaluating calibration and clinical applicability.Finally,risk stratification based on nomogram models scores confirmed that the modified models more accurately predicted OS and DFS in patients undergoing CRC surgery.4.ConclusionsThrough cell function assays and cell line-derived xenograft models,our study confirmed that WWP1 could promote the proliferative capacity of CRC cells in vitro and in vivo.Combined inhibition of WWP1 and SHP2 synergistically inhibited CRC cell proliferation,promoted apoptosis and cell cycle arrest in vitro and in vivo.In terms of mechanism,combined with RNA sequencing analysis and protein immunoblotting assays,we confirmed that the WWP1-PTEN axis was involved in the feedback activation of p AKT after SHP2 inhibition,and the combined inhibition of WWP1 and SHP2 deeply suppressed the PI3K-AKT signaling pathway.In terms of clinical application,it was verified in large-sample tissue microarrays that high expression of WWP1 predicted poor prognosis.Nomogram models combining the expression of WWP1 and SHP2 accurately predicted the long-term prognosis of patients undergoing CRC surgery.