Molecular studies of oncogenes JK-1 and GAEC1, cisplatin-resistance and tumor invasion in esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma accounts for 80% of esophageal cancers worldwide. It was characterized by great geographical variation in incidence and high mortality rate. Although many therapeutic strategies have been adopted, the survival rate is still poor. Therefore, better understanding of pathogenesis, development of chemo-resistance as well as discovery of novel therapeutic approaches are essential. In the study of a novel oncogene GAEC1, reduction in proliferation rate and increase in apoptotic population were observed with effective suppression of GAEC1 expression on ESCC cell line KYSE150 by GAEC1 targeting siRNA expression vector. Calpain 10 (CAPN10) was identified as the downstream regulatory gene of GAEC1. Immunohistochemical (IHC) staining of CAPN10 on tissue microarray (TMA) showed that low expression of CAPN10 predicted poor survival in ESCC patients. Target-suppression of another novel oncogene JK-1 on ESCC cell line KYSE70 was demonstrated using a JK-1 targeting siRNA expression vector. KYSE70 cells with suppressed JK-1 expression showed reduction in cell proliferation rate, decrease in cell migration rate, loss of anchorage-independent growth and acquisition of growth inhibition. For the IHC study of JK-1 expression, 14 out of 16 (87.5%) cases with pre-malignant lesions belonged to JK-1 high expression group which was less commonly found in tumor (7/26; 26.9%), suggesting that JK-1 expression may be more crucial at the pre-malignant stage during the early phase of tumor formation. For the study about the changes in gene expression profile in cisplatin (CDDP)-resistant ESCC tumor cells, a CDDP-resistant ESCC cell line, SLMT-1/CDDP1R, with 2.8-fold increase in resistance to cisplatin compared with the parental cells was established from SLMT-1 cells. cDNA microarray analysis revealed that IGFBP5 showed the highest level of down-regulation in SLMT-1/CDDP1R cells compared to SLMT-1 parent cells. Suppression of IGFBP5 mediated by IGFBP5-targeting siRNA in parental SLMT-1 cells confirmed that IGFBP5 suppression was one of the mechanisms for ESCC cells to acquire CDDP-resistance. More importantly, up-regulation of IGFBP5 using IGFBP5 expression vector sensitized SLMT-1/CDDP1R cells to cisplatin. For the study about the anti-cancer effect of a novel synthetic quinoline-based compound 91b1, cytotoxic effect on the five ESCC cell lines (KYSE150, KYSE450, KYSE510, KYSE30 and HKESC-4) was detected using MTS cytotoxicity assay with the MTS50 ranging from 1.80 -10.23 ug/mL. The anti-cancer effect of 91b1 was also comparable to the front-line chemotherapeutic drug CDDP and showed 1.6 and 2.1 times lesser cytotoxic effect to non-tumor cell lines NE-3 and HEK293 than CDDP. cDNA microarray analysis on the 91b1-treated (9.5 ug/mL) KYSE150 cells compared with the parental cells identified Chemokine (C-C motif) Ligand 5 (CCL5) as the most significantly downregulated target. By qPCR analysis, IHC staining of CCL5 and CCL5 specific ELISA, CCL5 was shown to be suppressed in mRNA and protein expression with 91b1 treatment in a dose-dependent manner. Up-regulation of CCL5 was also detected in 87.5% (7/8) of untreated ESCC cell lines by IHC analysis compared to non-tumor esophageal epithelial cell line NE-3. IHC analysis on ESCC specimens also revealed that upregulation of CCL5 was more commonly found in ESCC tumor and pre-malignant tissues, of which 20 out of 26 (76.9%) tumor specimens and 10 out of 16 (62.5%) specimens with pre-malignant tissues belonged to CCL5 high expression group. The trans-well matrigel invasion assay also demonstrated that CCL5 protein could enhance the invasion ability of KYSE30 cells compared with the untreated cells. The results implied that 91b1 could effectively induce cytotoxicity on ESCC cells and the mechanisms may involve the downregulation of CCL5 expression and suppression of tumor invasion. The overall results of the present study thus provided the possible novel therapeutic directions for the treatment of ESCC at molecular and pharmaceutical levels. The findings on the anti-cancer actions of the novel compound 91b1 and the approach for reversing the CDDP-resistance phenotype hopefully can be further extended to other tumor types in future.