Molecular Mechanism Of MAP4Regulating Cell Invasion And Migration In Esophageal Squamous Carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies in the world. Cell invasion and migration are crucial steps in carcinogenesis progression that significantly contribute to tumor metastasis. We have previously reported that CTTN (cortactin) is an oncogene and exerts functions in tumor metastasis in ESCC. With GST-Pull Down and LC-MS/MS, we found that MAP4is in the CTTN-complex.The molecular mechanism underlying the role of MAP4in esophageal squamous cells remain unclear. We detected that high expression of MAP4is228(62.6%) in the364ESCCs, The high expression of MAP4was significantly associated with tumor stage (P=0.0010) and lymph node metastasis (P=0.0003). Kaplan-Meier analysis showed that patients with high MAP4expression had significantly poor overall survival (P=0.026). Multivariate Cox regression analysis indicated that MAP4high expression (P=0.007) was an independent prognostic factor.In order to characterize the role of MAP4in ESCC, we performed functional analysis by siRNA-mediated silencing. The results showed that MAP4did not influence cell proliferation, cell cycle and adhesion. Whereas MAP4RNAi decreased the ability of wound healing, haptotactic migration and matrigel invasion in Eca109and KYSE150cells.In vivo assay showed that inhibition of MAP4expression decreased tumor growth and metastasis in mouse models.At the molecular level, Real-time PCR and Western blot analysis showed that VEGFA were significantly reduced in MAP4-siRNA transfected Eca109and KYSE150cells and also in their culture supernatants. We then detected the levels of VEGFR1and VEGFR2expression in Eca109and KYSE150cells. The results indicated that VEGFR1expression was much higher than VEGFR2. Silencing VEGFA and VEGFR1expression also markedly inhibited the invasion and migration of Eca109and KYSE150cells. IHC showed a positive correlation between MAP4, VEGFA and VEGFR1expressions in the ESCC tissues.Futher analysis showed that the phosphorylation levels of ERK1/2were dramatically decreased in MAP4silenced cells, Knockdown of ERK1/2not only inhibited VEGFA expression but also suppressed the invasion and migration of Eca109and KYSE150cells. Co-immunoprecipitation (Co-Ip) assays showed that MAP4was co-immunoprecipitated with ERK1/2(Fig.4h), Knockdown of VEGFA or VEGFR1decreased the levels of phosphorylated ERK1/2. Collectively, these data suggest that there is a reciprocal activation between VEGFA/VEGFR1and ERK1/2, which contributes to the invasion and migration by MAP4in esophageal cancer cells regulated.An injection of MAP4-siRNA directly into the induced tumors, led to a decrease of the volume of tumor by86%as compared with the control. Injection of Bevacizumab via tail vein into nude mice suppressed the increase of the tumor volume. When combined intratumoral-injection of MAP4-siRNA with injection of Bevacizumab via tail vein, the inhibition to tumor growth was superior to that of Bevacizumab or MAP4-siRNA alone.In conclusion, We show that MAP4is of prognostic and therapeutic relevance. Elevated expression of MAP4protein is an independent indicator for shorter survival of the patients with ESCC. As an important regulator of ERK1/2-VEGFA/VEGFR1signaling, MAP4may serve as a candidate molecular target for ESCC therapy.