To investigate the molecular mechanism by which AJUBA promoted MMP10 and MMP13 expression in ESCC cells, we examined the effects of AJUBA on ERK1/2 activation

To investigate the molecular mechanism by which AJUBA promoted MMP10 and MMP13 expression in ESCC cells, we examined the effects of AJUBA on ERK1/2 activation. of AJUBA. Thus, AJUBA upregulates the levels of MMP10 and MMP13 by activating ERK1/2. Taken together, these findings revealed that AJUBA serves as oncogenic gene in ESCC and may serve as a new target for ESCC therapy. homolog of AJUBA [6, 7, 15], and the role of AJUBA in human cancer development has been controversially reported [10, 16]. In the present study, we detected the expression levels of AJUBA by IHC and performed both and functional assays to characterize the biological effects of AJUBA on ESCC tumorigenicity and metastasis. The oncogenic mechanism of AJUBA was also investigated. RESULTS AJUBA was frequently overexpressed in ESCC Previously, through exome sequencing, we identified AJUBA somatic mutations in ESCC [11]. Here, we analyzed the mRNA levels of AJUBA and two other AJUBA family members, WTIP and LIMD1, in ESCC tumor tissues and in their matched adjacent non-tumor tissues. From 179 paired samples, we found that AJUBA was significantly overexpressed in tumor tissues than in adjacent non-tumor tissues (mean, 2.15-fold; < 0.001, paired Student's < 0.001, 2 test). When comparing the staining result of tumor tissues with their paired non-tumor tissues, 62% (37/60) of the tumor tissues exhibited increased AJUBA expression (Physique WIF1 ?(Physique1C).1C). These results indicated that AJUBA was frequently overexpressed in ESCC tumor tissues. Moreover, the results showed that in non-tumor Ethylmalonic acid tissues, 38% AJUBA positive cases showed nucleus staining, 62% AJUBA positive cases showed cytoplasm staining. While in tumor tissues, only 2% AJUBA positive cases had nucleus staining, 86% AJUBA positive cases had cytoplasm staining, and the remaining 12% cases had both nucleus and cytoplasm staining. Open in a separate window Physique 1 AJUBA was frequently upregulated in ESCC tissues compared with non-tumor tissues(A) Analysis of AJUBA mRNA level according to our previous microarray data (= 179). < 0.001, paired Student's < 0.001, 2 test. AJUBA expression in 60 paired ESCC tissues (right panel). T: tumor tissue; N: non-tumor tissue. Next, the relationships between AJUBA expression in ESCC tissues and clinicopathological characteristics were analyzed in 81 patients with ESCC. In this cohort, expression level of AJUBA was associated with tumor cell differentiation (= 0.043, 2 test) and invasion depth (T stage, = Ethylmalonic acid 0.005, Fisher's exact test). Furthermore, patients with high AJUBA expression had poorer differentiation and a higher tumor grade (Table ?(Table11). Table 1 The associations between AJUBA levels and clinicopathological characteristics in ESCC tissues = 81)valueand inoculated into the left or right dorsal flanks of female BALB/c-nu mice, respectively. The size (Physique 2E and 2F) and weight (Physique ?(Figure2G)2G) of tumors were significantly reduced in AJUBA knockdown mice compared with the control group (< 0.05, paired and and values were obtained using two-way ANOVA. (D) Ethylmalonic acid Representative inhibition of clone formation in 6-well plates by shAJUBA compared with control cells. The columns Ethylmalonic acid show the mean number of clones formed in three impartial experiments. *< 0.05; **< 0.01 based on Student's values were obtained using paired < 0.05; **< 0.01, Student's and < 0.05; **< 0.01; ***< 0.001, Student's and < 0.05; **< 0.01, Student's < 0.05, Student's < 0.05, Student's < 0.01, FDR < 0.1) by AJUBA knockdown in three cell lines were selected for Gene Ontology (GO) analysis. The GO analysis revealed that a number of genes involved in cell motility, cell adhesion and cell junctions were significantly dysregulated following AJUBA knockdown (Supplementary Physique S4). Among these genes, the mRNA levels of MMP10 and MMP13 were downregulated by 5.6-fold and 5.5-fold, respectively, in AJUBA-depleted cells compared with the control cells (Supplementary Table S1). The positive correlation between AJUBA and MMP10 and MMP13 expression was confirmed by RT-PCR (Physique ?(Figure6A)6A) and Western blot (Figure ?(Figure6B)6B) after AJUBA knockdown or overexpression in KYSE450 and KYSE510 cells. In addition, Ethylmalonic acid AJUBA mRNA levels were significantly associated with elevated MMP10 and MMP13 expression in 179 ESCC tumor tissues (= 0.441, < 0.001 and = 0.404, < 0.001, respectively,.