EC is one of the most common female genital malignant tumours, whose morbidity and mortality are increasing across the world, especially in China[19, 20]. The primary reasons for its treatment failure are distant metastasis and local recurrence. Therefore, discovering the molecular mechanisms underlying the cell proliferation and apoptosis of EC will be particularly helpful in providing an experimental basis for the development of valid targeted curative drugs. As far as we know, this study is the first report that DJ-1 regulates the apoptosis and proliferation of Ishikawa cels by activating the Cezanne/noncanonical NF-κB pathway.
Recently, Shu K showed that DJ-1 is an oncogene with abnormally high expression in EC and is closely associated with lymph node metastasis, the degree of differentiation and the invasion depth of EC. Notably, silencing DJ-1 expression by RNAi in EC inhibited cell viability and promoted cell apoptosis. These results demonstrated that DJ-1 regulates the apoptosis and proliferation of EC cells. Consistent with above-mentioned reports, we also revealed here that DJ-1 overexpression produced a lower percentage of apoptosis as well as higher proliferation in Ishikawa cells, whereas these effects disappeared in DJ-1-knockdown Ishikawa cells. The results again indicated that DJ-1 protein can inhibit cell apoptosis and promote cell proliferation in Ishikawa cells. However, its underlying mechanism remains unclear. Hence, in this study, we focus on investigating the signalling pathway by which DJ-1 suppresses the apoptosis and promotes the proliferation of Ishikawa cells.
It was known that the non-canonical NF-κB pathway is a key cell survival pathway, whose activation enhances cell survival by stimulating cell proliferation and inhibiting apoptosis[22, 11]. Several studies have observed that noncanonical NF-κB is aberrantly activated in many carcinomas, including EC, and affects hallmarks of cancer via the transcriptional activation of genes related to cell proliferation and apoptosis suppression [10, 9]. Moreover, it has been reported that Cezanne, which is classified as a member of the A20 protein family and possesses K48-specific deubiquitinase activity, is associated with the occurrence and progression of malignant tumours as a negative regulator of the noncanonical NF-κB signalling pathway[13–15]. Notably, an elegant study has shown that DJ-1 protein can interact with Cezanne and negatively regulate its activity in HEK293T cells. On this basis, we speculated that DJ-1 could inhibit the apoptosis and promote the proliferation of Ishikawa cells through inhibition of Cezanne and subsequent activation of noncanonical NF-κB signalling pathway. To clarify the question, we first investigated the impact of altered expression of DJ-1 on Cezanne activity and the key modulators of noncanonical NF-κB signalling pathway in Ishikawa cells. Interestingly, our study discovered that the association DJ-1 with Cezanne was increased, accompanied by the decreased Cezanne activity in DJ-1-overexpression Ishikawa cells. Meanwhile, TRAF3 expression was decreased, whereas NIK level was elevated and p52 expression and its nuclear translocation were increased. However, the aforementioned effects were reversed in DJ-1-knockdown Ishikawa cells. These findings suggested that DJ-1 not only interacts with and inhibits Cezanne, but also activates the noncanonical NF-κB signalling pathway in Ishikawa cells.
Moreover, to clarify the causal roles of Cezanne inhibition and noncanonical NF-κB pathway activation in the proliferation-promoting and apoptosis-inhibitory effects of DJ-1 and their upstream and downstream relationship, we further observed the effect of Cezanne or NIK knockdown on DJ-1 and Cezanne expression, noncanonical NF-κB signalling pathway activation, and cell apoptosis and proliferation. As expected, we observed that Cezanne knockdown reversed DJ-1 knockdown-induced proliferation inhibition and apoptosis increase, whereas the inhibition of the noncanonical NF-κB signalling pathway by NIK siRNA abrogated the proliferation-promoting and apoptosis-inhibitory effects of DJ-1 overexpression in Ishikawa cells. The above data convincingly demonstrated that the inhibition of Cezanne and the activation of noncanonical NF-κB signalling pathway are the critical mechanisms by which DJ-1 inhibits the apoptosis and promotes the proliferation of Ishikawa cells. Importantly, we found that Cezanne knockdown activated noncanonical NF-κB signalling pathway in Ishikawa cells and reversed noncanonical NF-κB inactivation in DJ-1-knockdown Ishikawa cells, but had no effect on DJ-1 expression. Moreover, NIK knockdown did not affect DJ-1 and Cezanne expression in Ishikawa cells but abolished the activation effect of DJ-1 overexpression on the noncanonical NF-κB signalling pathway. The above results indicated that DJ-1 associates with and negatively regulates Cezanne to activate the noncanonical NF-κB signalling pathway, resulting in inhibiting cell apoptosis and promoting cell proliferation in Ishikawa cells. Nevertheless, it is worth noting that the precise mechanism by which DJ-1-mediated the activation of noncanonical NF-κB pathway inhibits the apoptosis and promotes the proliferation of Ishikawa cells has yet to be determined. It is now documented that the noncanonical NF-κB activation can regulate transcriptional expression of multiple proliferation-promoting genes (eg, Cyclin D1, VCAM1, ICAM1, E Selectin, etc.) and anti-apoptotic genes (eg, Bcl-xL, Bcl2, IAP, XIAP, etc.)[11, 12, 23, 24]. Obviously, after activation of NF-κB signaling pathway by DJ-1, which specific proliferation- and apoptosis-related effectors are regulated thereby affecting Ishikawa cell proliferation and apoptosis is an important question for future research.
In conclusion, this study uncovered the potential mechanism by which DJ-1 regulates Ishikawa cell apoptosis and proliferation and found that DJ-1 associates with and inhibits Cezanne to activate the noncanonical NF-κB signalling pathway, thereby affecting the biological behaviours of Ishikawa cell proliferation and apoptosis. These findings may contribute to illuminate the mechanisms of the occurrence and progression of EC and provide important insights in the search for biomarkers for the development of EC targeted drugs.