Calcipotriol Targets LRP6 to Inhibit Wnt Signaling in Pancreatic Cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy in need of more effective treatment approaches. One potential therapeutic target is Wnt/β-catenin signaling, which plays important roles in PDAC tumor initiation and progression. Among Wnt inhibitors with suitable in vivo biologic activity is vitamin D, which is known to antagonize Wnt/β-catenin signaling in colorectal cancer and have antitumor activity in PDAC. For this study, the relationship between vitamin D signaling, Wnt/β-catenin activity, and tumor cell growth in PDAC was investigated through the use of calcipotriol, a potent non-hypercalcemic vitamin D analogue. PDAC tumor cell growth inhibition by calcipotriol was positively correlated with vitamin D receptor expression and Wnt/β-catenin activity. Furthermore, vitamin D and Wnt signaling activity were found to be reciprocally linked through feedback regulation. Calcipotriol inhibited autocrine Wnt/β-catenin signaling in PDAC cell lines in parallel with decreased protein levels of the low-density lipoprotein receptor-related protein 6 (LRP6), a requisite coreceptor for ligand-dependent canonical Wnt signaling. Decrease in LRP6 protein seen with calcipotriol was mediated through a novel mechanism involving transcriptional upregulation of low-density lipoprotein receptor adaptor protein 1 (LDLRAP1). Finally, changes in LRP6 or LDLRAP1 expression directly altered Wnt reporter activity, supporting their roles as regulators of ligand-dependent Wnt/β-catenin signaling.

Implications: This study provides a novel biochemical target through which vitamin D signaling exerts inhibitory effects on Wnt/β-catenin signaling, as well as potential biomarkers for predicting and following tumor response to vitamin D-based therapy.

Figure 1

Inhibition of pancreatic cancer growth by calcipotriol correlates with levels of VDR expression and autocrine Wnt/β-catenin signaling. (A) Western blot for VDR and tubulin (loading control) and mean Wnt reporter activity as indicated by BAR/fuBAR ratios and measured by dual luciferase assays 48 hours after co-transfection with control Renilla and either BAR-luciferase or fuBAR-luciferase reporter constructs in indicated PDAC cell lines (n=3 biological replicates). (B) MTT growth assays (measured at 72 hours) and (C) soft agar colony formation at indicated concentrations of calcipotriol. Representative images of AsPC-1 soft agar colony formation are also shown. Data are normalized to vehicle only and reported as mean ± standard deviation (SD) with one representative experiment of 3-5 biological repeats shown. *P<0.05, **P<0.01, ***P<0.001.

Figure 2

Calcipotriol mediates inhibition of Wnt signaling through VDR. (A) Dose response curve for calcipotriol on Wnt signaling as measured at 24 hours in AsPC-1 cells with stable BAR-luciferase reporter. (B) AXIN2 expression measured by qPCR at 24 hours following treatment with vehicle or 100 nM (AsPC-1), 5 μM (YAPC) or 1 μM (HPAF-2 and Suit2) calcipotriol. (C) VDR, LRP6 and tubulin (loading control) western blots and (D) BAR-luciferase activity for AsPC-1 transfected with either control or VDR siRNA for 48 hours and an additional 24 hour treatment with vehicle or 100 nM calcipotriol. All values were normalized to respective controls, with BAR reported as mean ± SD and qPCR reported as mean ± SEM. One representative experiment from at least three biological repeats is shown. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Calcipotriol treatment reduces LRP6 expression in PDAC. (A-B) Western blots for LRP6 or tubulin (loading control) were performed on whole cell lysates at indicated time points after cell line treatment with vehicle or 100 nM (AsPC-1), 5 μM (YAPC) or 1 μM (HPAF-2 and Suit2) calcipotriol. (C) BAR-luciferase activity for AsPC-1 transfected with full-length LRP6 expression vector or control GFP vector for 48 hours and measured after a further 24 hour treatment with vehicle or 100 nM calcipotriol. (D) RXRα, RXRβ, LRP6 and tubulin western blots on AsPC-1 whole cell lysates after control or tandem RXRA and RXRB siRNA transfection for 48 hours and further treatment with vehicle or 100 nM calcipotriol for 24 hours. (E-F) LRP6 gene expression measured by qPCR after 100 nM calcipotriol treatment for indicated times. Cells were synchronized by serum-starvation prior to calcipotriol treatment. All values were normalized to respective controls, with BAR reported as mean ± SD and qPCR reported as mean ± SEM. One representative experiment of 2-3 biological repeats is shown. *P<0.05, **P<0.01, ***P<0.001, ns = not significant.

Figure 4

Calcipotriol induces LDLRAP1 to regulate LRP6 protein levels. (A) Heat map of selected genes from RNA-seq after 2 or 6 hours treatment of AsPC-1 cells with vehicle or 100 nM calcipotriol. (B) Western blot for LC3B, LRP6 and tubulin (loading control) after AsPC-1 cells were treated with vehicle, 100 nM Calcipotriol, 100 nM Bafilomycin A1 or a combination of the two for 6 hours. (C) LDLRAP1 expression by qPCR after treatments described in Figure 3E. (D) Western blot for LDLRAP1, LRP6 and tubulin (loading control). AsPC-1 cells were transfected with control or LDLRAP1 siRNA for 48 hours and then treated with vehicle or 100 nM calcipotriol for 24 hours. Relative densitometry values for LDLRAP1 and LRP6 expression normalized to tubulin loading control are shown beneath corresponding blots. (E) BAR-luciferase activity in AsPC-1 cells treated as in (D). Values are shown normalized to respective controls, with BAR reported as mean ± SD and qPCR reported as mean ± SEM. One representative experiment of 3 biological repeats is shown. *P<0.05, **P<0.01, ***P<0.001.

Figure 5

VDR is regulated by Wnt signaling in PDAC. (A) Gene set enrichment analysis(27) enrichment plot for PDAC-specific Wnt/β-catenin target gene set determined relative to VDR expression phenotype using a published gene expression microarray dataset of 25 primary PDAC tumor samples. The top curve shows a running enrichment score for the target gene set, while the middle portion shows a heatmap of VDR expression (red – high, blue – low) and bottom portion shows the value of the ranking metric down the list of genes. (B) BAR luciferase activity and VDR expression by qPCR48 hours after transfection of AsPC-1 cells with control or WNT7B siRNA. (C) BAR luciferase activity and VDR expression by qPCR 24 hours after treatment of indicated PDAC cell lines with vehicle or 5 μM CHIR99021 (CHIR). Values are shown normalized to respective controls, with BAR reported as mean ± SD and qPCR reported as mean ± SEM. One representative experiment of 3 biological repeats is shown. *P<0.05, **P<0.01, ***P<0.001.