Increasing evidence supports a major role for PKD in cancer progression. In this report, we have established an essential role for PKD3, the least-studied of the PKD family members, in the progression of prostate cancer. We have demonstrated, using both cellular and in vivo models, that inhibition of PKD activity or expression reduces proliferation, motility, and secretion of key cancer-promoting factors. These data indicate that PKD, and in particular PKD3, may be a viable target for the development of novel chemotherapeutics in prostate cancer treatment.
Using stable inducible PKD3 knockdown prostate cancer cell lines, we showed that tet-induced PKD3 knockdown led to reduced cell proliferation and motility. The effect on proliferation was in line with our previous findings obtained using a transient knockdown approach (26
). Importantly, we have now provided the first evidence demonstrating that targeting PKD3 in vivo
causes a significant reduction in tumor growth in a subcutaneous xenograft mouse model. The effects of PKD3 knockdown on cell proliferation and motility are in contrast to those of PKD1 reported by Balaji and colleagues, showing an almost tumor-suppressor-like function of PKD1 in prostate cancer (30
). As the first-discovered and most intensely studied PKD isoform, PKD1's role in cell proliferation and motility has been somewhat controversial. This is particularly evident with regard to the role of PKD1 in cell migration. Although earlier studies demonstrated that PKD1 promotes cell movement (29
), more recent studies have suggested that PKD1 may have an inhibitory effect on cell migration and invasion (33
). Notably, studies in prostate cancer cells have shown that overexpression of PKD1 promotes cell aggregation and inhibits cell migration (31
), and other studies conducted in breast cancer and gastric cancer have shown that alteration of PKD1 expression or activity suppresses cell motility (22
). Several downstream targets of PKD1 have been implicated in this process, including slingshot phosphatase SSH1L (34
), RIN1 (33
), and MMPs (22
). Taken together, these findings highlight the complexity of PKD signaling and the importance of cellular context in shaping the biological functions of PKD in cells. The current study on PKD3, the least-studied member of the PKD family, further suggests that PKD isoforms may play specific and distinct roles in carcinogenesis and tumor progression. Accordingly, the PKD1 gene has been shown to be epigenetically silenced in primary breast and gastric cancer tissues and tumor cell lines (22
), while such regulation of PKD3 has not been demonstrated. Thus, despite belonging to the same kinase family and sharing high sequence homology, the PKD isoforms may have unique roles in different cancers or at different stages of cancer development.
It is noteworthy that even with incomplete knockdown (50–65%) of PKD3, potent effects on prostate cancer cell proliferation and motility were observed, implying that PKD3 may affect multiple tumor-promoting pathways. In this study, when conditioned medium from PKD3 knockdown cells was applied to normal PC3 cells, migration was significantly inhibited. Moreover, the PKD3 knockdown phenotype was rescued by applying conditioned medium collected from parental PC3 cells, indicating that PKD3 promoted the secretion of a motility-stimulating factor into the conditioned medium. Upon further investigation, we found that knockdown of PKD3 caused a global reduction in cytokine secretion in PC3 cells. These results were confirmed by ELISA for several significant targets, including IL-6, IL-8, and GROα, and were not a result of changes in the transcript levels of these proteins, suggesting that the reduced detection of cytokines was due to inhibition of secretion rather than gene expression. Thus, our data indicate that the robust effects of this partial PKD3 knockdown may be due to inhibition of the secretion of key cancer-promoting factors, thereby mediating several signaling pathways that affect proliferation and motility through reduction of autocrine and paracrine signaling. Nonetheless, our data do not exclude the possibility that PKD3 may regulate other cellular processes that impact cell growth and motility, such as cell adhesion, integrin expression, and cytoskeleton remodeling, and further studies are required to investigate these potential mechanisms.
Pro-inflammatory cytokines such as IL-6, IL-8, and GROα have well-documented, significant roles in cancer progression (37
). IL-8 in particular has been demonstrated to have various functions in cancer cells that promote angiogenesis, migration, metastasis, and proliferation, and is significantly upregulated in human prostate tumors (38
). Besides signaling in an autocrine fashion to stimulate cell growth and motility, these factors are also regulators of the tumor microenvironment (41
) and are potent stimulators of angiogenesis (42
). We have demonstrated that there is reduced secretion of multiple critical angiogenic cytokines with PKD3 knockdown, and this may account for the potent effects on prostate cancer cell proliferation and motility seen in our cellular studies as well as the significant reduction in tumor growth in our xenograft model, where we observed reduced levels of GROα.
A recent report showed that VEGF treatment of endothelial cells stimulated PKD1-dependent secretion of multiple cytokines, possibly mediating angiogenesis and inflammation (20
). In this study, researchers found that silencing PKD1 by siRNA caused reduced VEGF-stimulated expression and secretion of IL-6, IL-8, and GROα. However, they found no effects of PKD3 knockdown on cytokine secretion, which is contrary to our findings. This discrepancy may be explained by the relatively low expression of PKD3 in endothelial cells, which is in contrast to the high expression and likely aberrant hyperactivity of PKD3 in our PC3 and DU145 cells. This may contribute to constitutive hyperactivity of the secretory pathway in these cells, leading to increased secretion of key tumor-promoting factors and enhancing autocrine growth/motility signals.
The chemokine GROα has been studied in a variety of cancers, and has been found to promote proliferation, invasion, and angiogenesis in many different contexts (43
). In prostate cancer, GROα was shown to promote invasion and chemotaxis (44
). Thus, the significant PKD3-dependent reduction in intratumoral GROα is an important observation that may explain in part the reduced tumor growth observed in shPKD3-1 C7-derived tumors. Though we do not yet know the precise mechanisms through which PKD3 mediates GROα expression in vivo
, our cellular studies suggest that PKD3 knockdown impairs the secretory pathway rather than transcription of this important tumor-promoting factor. It is noteworthy that in the in vivo
study, reduced GROα expression was observed in tumor tissue lysates, which suggested possible transcriptional regulation. This phenomenon may be in part explained by the very different cellular environments in culture versus in vivo
, where cells must grow into tumors and become vascularized, and by the heterogeneous nature of tumor tissue. It is also possible that the duration of the in vivo
experiment, with tumors experiencing PKD3 knockdown for approximately 20 days, may have triggered a feedback or autoregulatory mechanism that resulted in the reduction of GROα expression. Further studies are necessary to elucidate these mechanisms.
In conclusion, our data support a tumor-promoting function of PKD3 in prostate cancer progression. We have shown that the cumulative effects of reduced cytokine secretion can cause significant changes in cancer cell proliferation and motility. Furthermore, we have reported, for the first time, that inhibition of PKD3 expression suppresses prostate tumor growth in vivo, validating PKD as a promising target in the development of novel therapies for prostate cancer.