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We have previously reported the role of miR-217 in anti-cancer drug-resistance. miRNA array and miRNA hybridization analysis predicted miR-30a-3p as a target of miR-217. miR-30a-3p and miR-217 formed a negative feedback loop and regulated the expression of each other. Ago1 immunoprecipitation and co-localization analysis revealed a possible interaction between miR-30a-3p and miR-217. miR-30a-3p conferred resistance to anti-cancer drugs and enhanced the invasion, migration, angiogenic, tumorigenic, and metastatic potential of cancer cells in CAGE-dependent manner. CAGE increased the expression of miR-30a-3p by binding to the promoter sequences of miR-30a-3p, suggesting a positive feedback loop between CAGE and miR-30a-3p. miR-30a-3p decreased the expression of p53, which showed the binding to the promoter sequences of miR-30a-3p and CAGE in anti-cancer drug-sensitive cancer cells. Luciferase activity assays showed that p53 serves as a target of miR-30a. Thus, the miR-30a-3p-CAGE-p53 feedback loop serves as a target for overcoming resistance to anti-cancer drugs.

We have previously reported the role of miR-326-HDAC3 loop in anti-cancer drug-resistance. CAGE, a cancer/testis antigen, regulates the response to anti-cancer drug-resistance by forming a negative feedback loop with miR-200b. Studies investigating the relationship between CAGE and HDAC3 revealed that HDAC3 negatively regulated the expression of CAGE. ChIP assays demonstrated the binding of HDAC3 to the promoter sequences of CAGE. However, CAGE did not affect the expression of HDAC3. We also found that EGFR signaling regulated the expressions of HDAC3 and CAGE. Anti-cancer drug-resistant cancer cell lines show an increased expression of pEGFR(Y845). HDAC3 was found to negatively regulate the expression of pEGFR(Y845). CAGE showed an interaction and co-localization with EGFR. It was seen that miR-326, a negative regulator of HDAC3, regulated the expression of CAGE, pEGFR(Y845), and the interaction between CAGE and EGFR. miR-326 inhibitor induced the binding of HDAC3 to the promoter sequences in anti-cancer drug-resistant Malme3M(R) cells, decreasing the tumorigenic potential of Malme3M(R) cells in a manner associated with its effect on the expression of HDAC3, CAGE and pEGFR(Y845). The down-regulation of HDAC3 enhanced the tumorigenic, angiogenic and invasion potential of the anti-cancer drug-sensitive Malme3M cells in CAGE-dependent manner. Studies revealed that PKCδ was responsible for the increased expression of pEGFR(Y845) and CAGE in Malme3M(R) cells. CAGE showed an interaction with PKCδ in Malme3M(R) cells. Our results show that HDAC3-CAGE axis can be employed as a target for overcoming resistance to EGFR inhibitors.

We examined the role of c-FLIP in the motility of HeLa cells. A small interfering RNA (siRNA) directed against c-FLIP inhibited the adhesion and motility of the cells without affecting their growth rate. The long form of c-FLIP (c-FLIPL), but not the short form (c-FLIPS), enhanced adhesion and motility. Downregulation of c-FLIPL with siRNA decreased phosphorylation of FAK and ERK, while overexpression of c-FLIPL increased their phosphorylation. Overexpression of FAK activated ERK, and enhanced the motility of HeLa cells. FRNK, an inhibitory fragment of FAK, inhibited ERK and decreased motility. Inhibition of ERK also significantly suppressed c-FLIPL-promoted motility. Inhibition of ROCK by Y27632 suppressed the c-FLIPL-promoted motility by reducing phosphorylation of FAK and ERK. Overexpression of c-FLIPL increased the expression and secretion of MMP-9, and inhibition of MMP-9 by Ilomastat reduced c-FLIPL- promoted cell motility. A caspase-like domain (amino acids 222-376) was found to be necessary for the c-FLIPL-promoted cell motility. We conclude that c-FLIPL promotes the motility of HeLa cells by activating FAK and ERK, and increasing MMP-9 expression.

We previously identified a novel cancer/testis antigen gene CAGE by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera of gastric cancer patients. CAGE is expressed in many cancers and cancer cell lines, but not in normal tissues apart from the testis. In the present study, we investigated its role in the motility of cells of two human cancer cell lines: HeLa and the human hepatic cancer cell line, SNU387. Induction of CAGE by tetracycline or transient transfection enhanced the migration and invasiveness of HeLa cells, but not the adhesiveness of either cell line. Overexpression of CAGE led to activation of ERK and p38 MAPK but not Akt, and inhibition of ERK by PD98059 or p38 MAPK by SB203580 counteracted the CAGE-promoted increase in motility in both cell lines. Overexpression of CAGE also resulted in a reduction of ROS and an increase of ROS scavenging, associated with induction of catalase activity. Inhibition of ERK and p38 MAPK increased ROS levels in cells transfected with CAGE, suggesting that ROS reduce the motility of both cell lines. Inhibition of ERK and p38 MAPK reduced the induction of catalase activity resulting from overexpression of CAGE, and inhibition of catalase reduced CAGE-promoted motility. We conclude that CAGE enhances the motility of cancer cells by activating ERK and p38 MAPK, inducing catalase activity, and reducing ROS levels.