Expression Alteration And Function Analysis Of Calreticulin In Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC) is one of the leading causes of cancer death in China.Tumors and adjacent normal esophageal tissues from 41 patients with ESCC were previously analyzed by proteomic approach.A total of 22 proteins were identified to be differentially expressed between ESCC and normal esophageal tissues.In the present study,we further investigated five candidate proteins,western blot and immunohistochemical staining confirmed overexpression of calreticulin,GRP78,annexin V,and M2-PK in ESCC tissues,whereas donwregulation of Hsp27 in tumors.High expression of calreticulin and GRP78 was correlated with poor prognosis.Calreticulin(CALR) is a multi-functional calcium binding protein and plays a critical role in quality control processes during protein synthesis and folding and regulation of intracellular Ca²⁺ homeostasis and endoplasmic reticulum Ca²⁺ storage capacity.siRNA-mediated repression in ESCC cell lines revealed that CALR did not influence proliferation and cell cycle progression,but reduced wound healing, haptotactic migration,Matrigel chemoinvasion,colony formation in soft agar and anoikis resistance.CALR-overexpressing ESCC cells showed increase of anoikis resistance.In vivo assay showed that inhibition of CALR expression decreased ESCC tumor growth.Molecular analysis revealed that CALR repression decreased the assembling of F-actin,as well as the expression of p63 and paxillin protein.We previously reported that Cortactin(CTTN),as a candidate oncogene, promoted cell invasion and metastasis by enhancing the cellular motility and resistance to anoikis in ESCC.Here we found that CALR repression lowered the expression level of CTTN mRNA and protein,but downregulation of CTTN did not alter CALR expression.Bioinformatic analysis showed that several conservative STAT3 binding sequences exist in the upstream of CTTN gene promoter.Repression of CALR inactivated the phosphorylation of STAT3(Tyr⁷⁰⁵).CTTN(but not CALR) presented repression in JAK specific inhibitor AG490-treat tumor cells.Chromatin immunoprecipitation analysis demonstrated that p-STAT3 combined to TTATGAAA sequence located in-1003 to-996 of CTTN promoter,suggesting possible regulation of CTTN transcription by CALR.Immunofluorescence confocal and co-immunoprecipitation assay showed a potential association of CALR and CTTN protein,whereas CALR was not found to affect CTTN degradation.The present study suggests for the first time that CALR repression decreases the malignant phenotype of tumor cells both in vitro and in vivo.Our results shed light on a series of unreported functions of CALR in enhancing ESCC migration/invasion and promoting resistance to anoikis.In the molecular level,CALR regulates CTTN transcription through STAT3,participates in PI3K/Akt pathway and influences the expression of paxillin and p63.