Colon cancer is one of the most difficult digestive cancers to treat with increasing incidence and mortality rates worldwide, urging for more effective therapeutic targets. Previously, we found that CacyBP/SIP plays a potent role in colon cancer proliferation, showing significant research value[22]. In the present study, we further screened the downstream target proteins of CacyBP/SIP in the nucleus. We confirmed that CDK8 was the critical protein of cell cycle regulation after CacyBP/SIP nuclear translocation. CacyBP/SIP and CDK8 were highly expressed in primary colon cancer tissues, and down-regulation of CacyBP/SIP could suppress cell cycle progression by inhibiting the CDK8/ β-catenin signaling pathway. Furthermore, the CacyBP/SIP-formed E3 ligase can modulate the degradation of CDK8 and β-catenin. These findings may shed a light into the exploration in pathogenesis and treatment of colon cancer.
CacyBP/SIP, a binding partner of S100A6, is a critical component of E3 ubiquitin ligase and exhibits phosphatase activity, participating in various cellular processes[25–27]. CacyBP/SIP expression varies among tumors and its oncogenic or tumor-suppressive role depends on the cell types[14]. For instance, CacyBP/SIP was overexpressed in prostate cancer and promoted proliferation via p53[28]. In pancreatic cancer, CacyBP/SIP was significantly up-regulated and enhanced proliferation and G1/S transition by modulating Cyclin E, CDK2, p27 and Rb[29]. Moreover, CacyBP/SIP was reported to increase the expression of P-gp and Bcl-2, which enhanced the multidrug resistance of pancreatic cancer[30]. In the above tumors, CacyBP/SIP act as an oncogene, in some other tumors it can exert both oncogenic and tumor-suppressive role. CacyBP/SIP was detected accumulated in gastric cancer cell nuclear and stimulated ubiquitin-mediated degradation of p27Kip1, which contributed to cell proliferation[18]. On the other hand, CacyBP/SIP nuclear translocation can inhibit gastric cancer cell proliferation by degrading β-catenin and dephosphorylating REK1/2 during the G2 phase[19, 31]. Similarly, the function of CacyBP/SIP in breast cancer is duplex, CacyBP/SIP knockout enhanced apoptosis by regulating β-catenin signaling[32]. Conversely, down-regulation of CacyBP/SIP enhanced the expression of COX-2 in breast cancer, which facilitated cancer progression[33]. While in renal cancer, CacyBP/SIP expression was deficient and mainly acted as a tumor suppressor[34]. The exact role of CacyBP/SIP in tumors may depend on its phosphatase activity and CacyBP/SIP-mediated ubiquitin degradation. Besides, its intracellular target proteins typically have different functions. In our study, CacyBP/SIP was overexpressed in colon cancer and acted as a tumor promoter. Moreover, CacyBP/SIP knockdown can inhibit cell cycle progression, constituting a novel treatment target for colon cancer.
Cyclin-dependent kinases 8 (CDK8), a core member of CDKs, regulates transcriptional processes and cell cycle, constituting a valuable therapeutic target for cancers[35]. CDK8 acts as an oncogene and is overexpressed in various tumors, especially in colon cancer[36]. Indeed, our study showed that CDK8 was significantly up-regulated in colon cancer tissues and positively correlated with CacyBP/SIP. In addition, CDK8 can activate the Wnt/β-catenin signaling and inhibit the inhibitory activity of E2F1 for β-catenin, promoting cell cycle progression and proliferation of colon cancer[37, 38]. Our data also revealed that CDK8 knockdown inhibited the expression of β-catenin, and the cell cycle was arrested at the G1 phase in colon cancer. Furthermore, Our study confirmed that CDK8 was the downstream target protein of CacyBP/SIP after nuclear translocation by the cell cycle PCR chip. The expression levels of CacyBP/SIP, CDK8 and β-catenin were down-regulated in the SW480-CacyBP/SIPsh-1 cell line. These findings indicated that CacyBP/SIP knockdown arrested the cell cycle at the G0/1 phase by inhibiting CDK8 in colon cancer. Thus, our results supported that CacyBP/SIP nuclear translocation promoted the cell cycle process of colon cancer by activating the CDK8-mediated Wnt/ β-catenin signaling pathway.
E3 ubiquitin ligase, the key player in the ubiquitin-proteasome system (UPS), plays a potent role in recruiting substrates for ubiquitination[39]. The structure of E3 ligase was mainly composed of the Siah-1, Skp1, and F-box proteins[24]. CacyBP/SIP can bind to Siah-1 and Skp1 through the N-terminal region, jointly forming an E3 enzyme complex (CacyBP/SIP-Siah-1-Skp1-Cullin-1-Skp2)[40, 41]. Previous studies indicated that the CacyBP/SIP-formed E3 ligase regulated cell cycle via Wnt/β-catenin signaling in gastric cancer, glioma and leukemia[19, 42, 43]. To explore whether the CacyBP/SIP-formed E3 ligase regulates CDK8 degradation, we inhibit the activity of E3 ligases with MG132 and found that CDK8 expression was up-regulated in the SW480-CacyBPsh-1 cell line. This data indicated that MG132 did not synergistically inhibit CDK8 expression with CacyBP/SIP knockdown, but upregulated CDK8 expression. Thus, we speculated that MG132 not only inhibited the activity of the CacyBP/SIP-E3 ubiquitination ligase but also inhibited the direct degradation of CDK8 by other E3 ubiquitination ligases, enhancing the accumulation of CDK8 in colon cancer cells. Later, we further knockdown the the substrate recognition structure Skp2 of the CacyBP/SIP-formed E3 ubiquitination ligase. Notably, the F-box protein Skp2 plays a pivotal role in various cancer-associated signaling pathways[44]. Our data showed that the expression of CDK8 was down-regulated in the SW480-Skp2sh-3 cell line, indicating that the CacyBP/SIP-formed E3 ligase did not directly degrade CDK8. Previous studies revealed that Skp2 can promote the expression of CDK8 by promoting the ubiquitination degradation of mH2A1, which is a substrate of Skp2 in cancers[45, 46]. Inhibition of mH2A1 was involved in promoting tumorigenesis and cancer progression[47]. According to the above evidence, the CacyBP/SIP-formed E3 ligase may promote CDK8 expression by degrading mH2A1. In other words, when CacyBP/SIP and Skp2 were knock downed, the degradation function of CacyBP/SIP-Siah-1-Skp1-Cullin-1-Skp2 on mH2A1 was inhibited, which facilitated mH2A1 accumulation-mediated CDK8 degradation in colon cancer.
Several limitations should be considered when interpreting the results in the present study. The first limitation of this study is that we mainly used knockdown experiments to confirm that CacyBP/SIP promotes the cell cycle progression of colon cancer by activating the CDK8/Wnt/β-catenin signaling pathway. Therefore, overexpression validation experiments are needed in our future research. In addition, the regulatory effect of CacyBP/SIP on the Skp2-mH2A1-CDK8 axis has not yet been verified in colon cancer. Further study is necessary to detect the regulatory effect of CacyBP/SIP on Skp2 and mH2A1. Moreover, the role of the CacyBP/SIP(Skp2)-mH2A1-CDK8 axis in colon cancer cell cycle, cell proliferation, and apoptosis is worth further exploration in future research.