Functional Characterization Of Calreticulin In The Metastasis Of Esophageal Squamous Cell Carcinoma

Esophageal cancer is the fourth common malignancy in China, and squamous cell carcinoma (ESCC) is the most prevalent type. In order to reveal the molecular alterations underlying the development and progression, we have previously investigated the proteins dysregulated in ESCC tissues by proteomic approach. Calreticulin overexpression was observed in the diseases and correlated with poor prognosis. Calreticulin (CRT) is a multi-functional calcium binding protein and plays a critical role in protein synthesis and folding, regulation of intracellular Ca2+homeostasis and endoplasmic reticulum Ca2+storage. RNAi-mediated repression of CRT in ESCC cell lines led to a remarkable reduction of haptotactic migration and matrigel chemoinvasion.In the present study, we further identified the downstream genes of CRT by microarray analysis to elucidate the molecular mechanism of CRT regulating cell motility and aggressiveness. Realtime PCR and Western blot results showed that knock down of CRT reduced PTP1B mRNA and protein level in esophageal KYSE150cells. And enforced CRT expression in CRT-silenced cells can rescue PTP1B expression.PTP1B has been reported to influence cell motility in breast cancer cells. Here we demonstrated that reduced PTP1B expression can inhibit cell invasion and migration in KYSE150cells. We hypothesized that CRT may regulate cell movement through PTP1B. We observed that infection of Lenti-PTP1B to CRT-silenced cells restored PTP1B expression and increased the ability of cell movement. In vivo assay showed that inhibition of CRT or PTP1B expression decreased ESCC lung metastases.Bioinformatic analysis revealed that several conservative sequences of Stat5a binding in the upstream of PTPIB gene promoter. CRT repression inactivated the phosphorylation of Stat5a (Tyr694). Chromatin immunoprecipitation demonstrated that p-Stat5a combined to TT(N)5AA sequence located in-676to-595of PTP1B promoter, suggesting possible regulation of PTP1B transcription by CRT.Extracellular matrix (ECM) is an important structure within tissues. Changes in ECM composition are associated with cancer cell invasion. Matrix metalloproteinases (MMPs) are a superfamily of zinc-binding endopeptidases that degrade the ECM. We found that Erk1/2can regulate the expression of MMP-9, a member of MMPs and that CRT modulated Erkl/2activity through PTP1B and influenced MMP-9expression.Taken together, our findings showed that CRT regulated PTP1B expression through Stat5a. PTP1B played an important role in cell motility and CRT-signal pathway. CRT modulated Erk1/2activity by PTPIB, and the expression of MMP-9was driven by Erk1/2at the transcription level. These mechanisms were involved in motility of esophageal squamous carcinoma cells.