Downregulation of Dickkopf-3 disrupts prostate acinar morphogenesis through TGF-β/Smad signaling

Abstract

SummaryLoss of tissue organization is a hallmark of the early stages of cancer, and there is considerable interest in proteins that maintain normal tissue architecture. Prostate epithelial cells cultured in Matrigel form three-dimensional acini that mimic aspects of prostate gland development. The organization of these structures requires the tumor suppressor Dickkopf-3 (Dkk-3), a divergent member of the Dkk family of secreted Wnt signalling antagonists that is frequently downregulated in prostate cancer. To gain further insight into the function of Dkk-3 in the prostate, we compared the prostates of Dkk3-null mice with those of control littermates. We found increased proliferation of prostate epithelial cells in the mutant mice and changes in prostate tissue organization. Consistent with these observations, cell proliferation was elevated in acini formed by human prostate epithelial cells stably silenced for Dkk-3. Silencing of Dkk-3 increased TGF-b/Smad signalling, and inhibitors of TGF-b/Smad signalling rescued the defective acinar phenotype caused by loss of Dkk-3. These findings suggest that Dkk-3 maintains the structural integrity of the prostate gland by limiting TGF-b/Smad signalling.

Prostate organogenesis: tissue induction, hormonal regulation and cell type specification

“…Notably, studies of the rat prostate have shown that apoptosis occurs in the center of prostate epithelial cords during early canalization (Bruni-Cardoso and Carvalho, 2007). This process might be regulated by genes such as Dkk3 and Mmp2, which have been previously shown to play a role in normal prostate lumen formation (Table 1) (Bruni-Cardoso et al, 2010b;Kawano et al, 2006;Romero et al, 2013). Further studies are required to determine whether prostate ductal canalization occurs through anoikis and to elucidate the regulatory genes that drive lumen formation.…”

Dickkopf-3 function in the prostate

“…32 We therefore investigated the effects of silencing Dkk-3 on TGF-β signaling, finding elevated Smad2 phosphorylation and TGF-β-dependent gene reporter activity in Dkk-3-silenced prostate epithelial cells. 25 Importantly, inhibition of TGF-β/Smad signaling in 3D cultures of Dkk-3-depleted cells rescued the acinar morphogenesis phenotype, indicating the relevance of the link between Dkk-3 and TGF-β to the function of Dkk-3. These results raise a quandary: TGF-β is known to inhibit proliferation of non-transformed cells, something that we and others 33 have also observed in RWPE-1 cells.…”

“…14,25 Using the acinar morphogenesis assay described above, we found that depletion of Dkk-3 using siRNA or microRNA-adapted shRNA resulted in the formation of irregularly shaped and non-polarized acinar structures, suggesting Dkk-3 might play a role in the architectural organization of the prostate gland. This would be consistent with its significant downregulation in high Gleason grade prostate cancer, where the tumor tissue is very disorganized.…”

“…To address this, we examined the effect of inhibiting endogenous TGF-β signaling using the TGF-β receptor inhibitor SB431542 and found that this reduced proliferation in 3D cultures. 25 Thus, the 3D context not only reveals a function for Dkk-3 not observed in monolayer cultures, but also alters the proliferative response to TGF-β. To try to address how Dkk-3 affects the proliferative response to TGF-β, we measured the expression of TGF-β/Smad target genes.…”

“…25 The case for a link between Dkk-3 and TGF-β signaling is stronger, however, with reported effects of Dkk-3 on Smad4 in Xenopus and zebrafish embryos, 30,31 in both cases enabling differentiation processes to proceed. Moreover, TGF-β has been observed to inhibit acinar morphogenesis.…”