SHP2 Inhibits Colorectal Cancer Cell Proliferation And Migration Through Regulating STAT3 Phosphorylation

BackgroundThe Src homology-2 domain-containing phosphatase SHP2,encoded by PTPN11,is composed of a single phosphotyrosine phosphatase(PTP)domain and two N-terminal SH2 domains.The C-terminal region of SHP2 contains sites of tyrosine phosphorylation and a proline-rich region.SHP2 could facilitate cell survival and proliferation elicited by cytokines,growth factors and hormones.And it is identified as a bona fide proto-oncogene in solid tumors and hematologic malignancies.Germline PTPN11 mutations are reported in nearly half of patients with Noonan Syndrome(NS),and somatic gain of function mutations in PTPN11 have been frequently detected in different types of leukemia,gastric carcinoma and breast cancer.In gastric cancer,the phosphorylation of Cag A(cytotoxin-associated antigen A)can bind with SHP2,and stimulates the phosphatase activity of SHP2,activating the ERK/MAPK signaling pathway which could induce a growth factor-like response in gastric epithelial cells;However,in liver cancer cells,SHP2 could promote the senescence of cells.The knockdown of SHP2 sensitizes liver to diethylnitrosamine(DEN)-induced hepatocellular carcinoma(HCC).These results show SHP2 functions specifically in different tissues.The incidence and mortality of colorectal cancer(CRC)are in the forefront of all cancers in China and western developed countries.Data from the course of recent studies show that ablation of SHP2 in intestine epithelial cells(IECs)results in the spontaneous development of colitis.Genetic studies have identified SHP2-encoding PTPN11 gene as an inflammation bowel disease(IBD)susceptibility gene.And SHP2 gene transcripts are significantly reduced in patients with ulcerative colitis.The epidemiological study in clinical data shows the low expression of SHP2 in CRC is associated with high mortality and other poor prognosis,implying the suppressing role of SHP2 in colorectal cancer.Meanwhile,there are articles reporting several of chemicals such as non steroidal anti-inflammatory drug amino salicylic acid,NS-398(a selective COX-2 inhibitor)and X-linked inhibitor of apoptosis protein(XIAP)inhibitor embelin,showing anti-tumor activity by promoting the activation or expression of SHP2 in CRC cells.But,currently there is no articles researching the direct role of SHP2 in the malignant phenotype of CRC,such as proliferation,migration,and apoptosis.So more extend researches are needed to focus on the role of SHP2 in CRC.Objective Explore the role of SHP2 in proliferation and migration of CRC cells;explore the molecular mechanism of SHP2 in CRC cells.Methods 1.The expression levels of SHP2 in different CRC cells(SW480? HCT116? RKO?Lo Vo)were checked by Western Blotting assay and Real-time PCR analysis.2.We identified the effect of SHP2 si RNA and plasmid overexpressing SHP2 WT by Western Blotting assay and Real-time analysis;the effects of SHP2 knockdown,overexpression and phosphatase activity inhibition on cell proliferation,migration were studied in CRC cells.MTT assay and plate colony formation were used to check the proliferation.Transwell was used to check the migration.3.The SHP2 effect on STAT3 phosphorylation was checked by Western Blotting assay.4.The expression levels in other parts of JAK2/STAT3 pathway(gp130,IL-6R,JAK2,SOCS3)were assessed by Real-time PCR.5.The SHP2 effect on p-STAT3 location in cell was checked by cell immunofluorescence.Results 1.SHP2 inhibited the proliferation and migration of CRC cells 1.1.We showed that the expression levels of SHP2 were different in CRC cell SW480,HCT116,RKO and Lo Vo: high in HCT116 and RKO;low in SW480 and Lo Vo.So we chose HCT116 and SW480 for subsequence researching.1.2.Knockdown of SHP2 promoted CRC cell SW480 and HCT116 proliferation and migration,whereas over expression of SHP2 significantly inhibited cell proliferation and migration of SW480 cell,in which SHP2 was low expressed.Meanwhile in control experiment,knockdown of SHP2 inhibited breast cancer cell proliferation.1.3.Inhibition of SHP2 phosphatase activity by PHPS1 facilitated SW480 and HCT116 proliferation and migration.2.In CRC cell SHP2 functions as a tumor suppressor by regulating the phosphorylation of STAT3 2.1.The phosphorylation of STAT3,a known oncogene in CRC,was regulated by SHP2 negatively: knockdown of SHP2 promoted the phosphorylation of STAT3;the phosphorylation of STAT3 was decreased after overexpression of SHP2;meanwhile the phosphorylation of STAT3 was increased after functional inhibition of SHP2 catalysis by PHPS1.2.2.Rescue assay demonstrated CRC cell proliferation affected by SHP2 was mediated by SHP2 functional regulation on STAT3 phosphorylation.2.3.si RNA mediating silencing of SHP2 promoted SW480 proliferation induced by IL-6.Conclusion Our findings reveal that SHP2 directly inhibits the proliferation and migration of CRC cells,and the molecular mechanism is the inhibition of STAT3 phosphorylation.