PCP4, which was initially detected in Purkinje cells and stellate neurons, plays a critical role in ventricular arrhythmias associated with heritable and acquired syndromes 16. Increasing research shows that PCP4, as an anti-apoptotic factor in neural cells, enhances cell migration, proliferation, and invasion and inhibits cell apoptosis in a number of human carcinoma cell lines 10,17. In this study, we found that the PCP4 OE induces PCa cell growth and metastasis in vitro and in vivo. Moreover, in clinicopathological patients, the expression of PCP4 is higher in androgen-resistant samples than in androgen-sensitive samples. In the CRPC tumor xenograft model, AR levels decreased in the first few days following castration and then returned to OE levels thereafter. PCP4 levels showed a trend similar to that of AR levels in CRPC tumors. Interestingly, the variation trend of xenograft tumor size was similar to the trend of AR and PCP4 expression in CRPC development. Accumulated evidence suggests that interconnected molecular mechanisms are related to dysregulated persistent AR signaling and alter androgen biosynthesis and metabolism 18. Intraprostatic conversion of adrenal androgens into testosterone and intratumoral androgen biosynthesis resulting in AR reactivation in CRPC has received special attention, and persistent AR-axis signaling is regarded a critical therapeutic target. In Fig. 3C, PCP4 and CaMKK2 decreased after treatment with EPI, an AR inhibitor. We also found that PCP4 expression is clearly regulated by AR. The clinicopathological data further showed that PCP4 is positively correlated with AR expression (Fig. 4D). These results demonstrate that PCP4, as the downstream target of AR, may contribute to promote CRPC growth and PCa hormone resistance.
In the CRPC xenograft tumor model, the tumor size decreased with hormone deprivation shortly after castration. As AR expression recovered, however, the tumor volume and PCP4 and CaMKK2 levels increased once more. In castration-resistant patients, AR signaling is often reactivated in the absence of androgens 19,20. Previous articles demonstrated that targeting CaMKK, the downstream target of AR, provides an attractive strategy to combat advanced PCa 21,22. Inhibition of AMPK, the target pathway of CaMKK, decreases tumor hormone resistance 12,23.
In Purkinje cells, CaMKK2, which binds to calmodulin and phosphorylates CaMKI, is activated by PCP4 5,24. We initially confirmed the interaction of CaMKK2 and PCP4 to induce the proliferation of PCa cells. Although various upstream signaling pathways regulate the phosphorylation of CaMKK2, the AR’s regulation is the first exhibited one of CaMKK2 expression by any signaling pathway. Our results show that the OE of mRNA and protein levels of CaMKK2 in CRPC tumors could be decreased by AR. This finding demonstrates the critical role of genomic androgens in cellular migration and proliferation. Other researchers have suggested that androgens alter cytoskeletal reorganization 25,26. Moreover, we found that the exogenous overexpression of PCP4 blocks the signaling pathway of AR-CaMKK2 and that AR inhibition is invalid in hormone-sensitive cells. In clinical patients, the expression of PCP4 and CaMKK2 is positively correlated with the sensibility of androgen-deprivation treatment.
In conclusion, we found that PCP4-CaMKK2, a molecule necessary for tumorigenesis, plays an important role of transmitting signaling in the mechanism of AR regulation CaMKK2. In future research, we will focus on developing a PCP4 inhibitor and illustrate the complete regulatory mechanism of AR in CRPC.