PORCN moonlights in a Wnt-independent pathway that regulates cancer cell proliferation

Abstract

Porcupine (PORCN) is a membrane-bound O-acyl transferase that is required for the palmitoylation of Wnt proteins, and that is essential in diverse Wnt pathways for Wnt-Wntless (WLS) binding, Wnt secretion, and Wnt signaling activity. We tested if PORCN was required for the proliferation of transformed cells. Knockdown of PORCN by multiple independent siRNAs results in a cell growth defect in a subset of epithelial cancer cell lines. The growth defect is transformation-dependent in human mammary epithelial (HMEC) cells. Additionally, inducible PORCN knockdown by two independent shRNAs markedly reduces the growth of established MDA-MB-231 cancers in orthotopic xenografts in immunodeficient mice. Unexpectedly, the proliferation defect resulting from loss of PORCN occurs in a Wnt-independent manner, as it is rescued by re-expression of catalytically inactive PORCN, and is not seen after RNAi-mediated knockdown of the Wnt carrier protein WLS, nor after treatment with the PORCN inhibitor IWP. Consistent with a role in a Wnt-independent pathway, knockdown of PORCN regulates a distinct set of genes that are not altered by other inhibitors of Wnt signaling. PORCN protein thus appears to moonlight in a novel signaling pathway that is rate-limiting for cancer cell growth and tumorigenesis independent of its enzymatic function in Wnt biosynthesis and secretion.

Figure 1. Knockdown of PORCN affects breast cancer cell proliferation.

A. STF3A cells were transfected with 100 nM of the indicated siRNA and PORCN message was analyzed 48 hrs later by quantitative real time PCR. Histogram represents relative PORCN mRNA normalized to Actin mRNA. NT, not transfected; siC, siRNA control; siP7 and siP8, specific PORCN siRNAs. B. PORCN knockdown blocks Wnt/β-catenin signaling. Relative Wnt/β-catenin signaling was measured in STF3A cells transfected with 100 nM of siC, siP7, or siP8 siRNA. C. PORCN knockdown inhibits WNT3A secretion. STF3A cells were transfected with 100 nM of the indicated siRNA. Media was changed to 1% FCS media 48 hours after transfection, and collected 16 hrs later. The abundance of WNT3A was assessed in 30 µL conditioned media (Media) and 15 µg of whole cell lysates (WC) by SDS-PAGE and immunoblotting with the indicated antibodies. Actin immunoblotting demonstrates equal loading of whole cell lysates. D. Relative numbers of MDA-MB-231 breast cancer cells is decreased after PORCN knockdown. Cells were transfected with the indicated siRNAs and proliferation assessed as in Experimental Procedures. Error bars indicate standard deviation. E. PORCN knockdown slows the growth of multiple breast cancer cell lines. Cell number was assessed 4 days post-transfection of siP7 siRNA and plotted as % of proliferation of control siRNA-transfected cells. *, p<0.05; **, p<0.01 for difference from control. F. PORCN knockdown selectively slows the growth of transformed hMECs. The effect of PORCN knockdown on the growth of hMECs immortalized with hTERT or hMEC-hTERT cells transformed by expression of H-RasV12 and stable knockdown of p53 was assessed 6 days after transfection with control or siP7 siRNA. Transfection efficiency in both cell types was >80% as assessed by GFP expression. *, p<0.05 compared with control siRNA. G. PORCN knockdown alters expression of Wnt/β-catenin target genes. Relative message of PORCN and other Wnt/β-catenin target genes in MDA-MB-231 cells (top) and SK-BR-3 cells (bottom) was assessed by qRT-PCR 72-hrs after transfection with 100 nM of the indicated siRNA.

Figure 2. Inducible knockdown of PORCN delays cancer growth in an orthotopic xenograft model.

A. Establishment of PORCN inducible knockdown. MDA-MB-231 cells with stable integration of pTRIPZ- shC (control), -shP1, or -shP2 shRNAmir were treated with 5 ng/mL Doxycycline (right) or vehicle control (left). PORCN and Actin mRNA were assessed by RT-PCR, and RFP expression was assessed by fluorescence microscopy. B. Inducible knockdown of PORCN inhibits Wnt/β-catenin signaling. The stable MDA-MB-231 cell lines were transiently transfected with WNT3A expression vector and SuperTOPflash and Renilla luciferase reporter plasmids, and then treated with 5 ng/mL Dox for 48 hrs before assessment of Wnt/β-catenin signaling as described. Histogram represents SuperTOPflash signaling relative to Renilla luciferase expression. C. Inducible knockdown of PORCN mRNA in orthotopic xenografts. The stable cell lines were injected orthotopically into the 4^th mammary fat pad of BALB/c nude mice. After establishment of palpable tumors (15 days), Dox was added to the water for a 7 day period, tumors were harvested and mRNA abundance was assessed by qRT-PCR. Histogram represents relative PORCN mRNA normalized to Actin mRNA. D. PORCN knockdown slows cancer growth. Tumors were harvested and weighed 19 days after the start of doxycycline treatment. E. Cancer growth is slowed by inducible PORCN knockdown. Tumor volume was measured by calipers at the indicated times.

Figure 3. PORCN knockdown has Wnt/β-catenin-independent effects.

A. WLS, β-catenin, and PORCN knockdown all inhibit Wnt/β-catenin signaling in STF3A cells. The following siRNAs were used at 100 nM: for PORCN, siP7; for WLS, siW5; for β-catenin, siβC11. B. IWP-1 inhibits WNT3A secretion. Conditioned medium from STF3A cells was assessed for WNT3A as described after cells were incubated for 16 hours in the presence of the indicated concentration of IWP-1. C. IWP-1 inhibits WNT3A-driven signaling in MDA-MB-231 cells. MDA-MB-231 cells were co-transfected with WNT3A and STF, and treated overnight with vehicle (DMSO) or indicated doses of IWP-1. D. The indicated cell lines were either transfected with siRNAs as above, or treated with IWP-1 or vehicle control. Total cell count at day 6 was assessed as described and compared with untreated cells. IWP-1 was used at 2 µM and refreshed every 24 h. E. Western blot of WLS in MDA-MB-231 cells stably expressing shC, shW1, and shW5 shRNAs. F. WLS knockdown does not slow tumor growth. MDA-MB-231 cells stably expressing shC, shW1, and shW5 shRNAs were injected orthotopically into BALB/C nude mice and tumor growth monitored as described. G. WLS message levels in tumors extracted from (E), assessed by qRT-PCR and normalized to Actin.

Figure 4. Wild type and mutant PORCN both rescue the slow growth phenotype.

A. Western blot analysis of MDA-MB-231 cells transfected with P7 immune constructs of HA-tagged WT and H341A PORCN. The PORCN construct contains 2 additional silent mutations that render it immune to P7 siRNA knockdown. EV, empty vector; WT, wildtype PORCN-HA; MT, H341A-PORCN-HA. B. Wildtype but not mutant PORCN stimulates Wnt/β-catenin signaling. MDA-MB-231 cells were co-transfected with plasmids encoding for WT- and H341A-PORCN, WNT3A, and the SuperTOPflash reporter. H341A-PORCN has a significant dominant negative effect on Wnt/β-catenin signaling. C. Wildtype and mutant PORCN are appropriately localized. Indirect immunofluorescence microscopy was performed with anti-HA antibody and the endoplasmic reticulum marker calnexin. D. Wildtype and dominant negative H341A PORCN are equally effective at rescuing the growth defect caused by PORCN knockdown. MDA-MB-231 were co-transfected with P7 siRNA and PORCN expression constructs as indicated. Cell numbers were measured as described. Histogram represents cell number at day 5. *, p<0.01 for ability of constructs to rescue slowed proliferation; **, p<0.01 for ability of PORCN knockdown to slow proliferation relative to control. E. WT but not H341A-PORCN is able to rescue Wnt/β-catenin signaling. PORCN null ES cells were co-transfected with plasmids encoding WT- and H341A-PORCN, WNT3A, and the SuperTOPflash reporter. In the absence of ectopic PORCN in these ES cells, there is no WNT3A-driven signaling.

Figure 5. PORCN regulates a subset of genes in a Wnt-independent manner.

A. Gene expression analysis of MDA-MB-231 cells 48 hours after mock transfected (NT) or transfected with 100 nM of indicated siRNA for 48 hrs. PORCN and WLS differentially regulate a distinct subset of genes. B. To illustrate genes differentially regulated by PORCN, the abundance of the indicated transcripts were assessed by qRT-PCR 48 hours after transfection of MDA-MB-231 cells with the indicated siRNAs C. Two independent PORCN siRNAs cause decrease in APEH protein. Immunoblot analysis of APEH and Actin from MDA-MB-231 cells treated with 100 nM Control (C), PORCN7 (P7) or PORCN8 (P8) siRNA for 72 hrs. D. PORCN knockdown triggers loss of APEH in multiple cell lines. Immunoblot analysis of APEH from MCF7, T47D, and DLD-1 cells transfected with 100 nM Control (C), or PORCN7 (P7) siRNAs for 72 hrs. Top panel is 10 s exposure, middle panel is 30 s exposure. Actin (bottom panel) serves as a load control. E. Loss of APEH is blocked by RNAi-immune wildtype and mutant PORCN. Immunoblot analysis of APEH and HA-tagged PORCN in MDA-MB-231 cells co-transfected with P7-resistant WT or H341A PORCN and 100 nM Control (C) or PORCN7 (P7) siRNA for 48 hrs. * = non-specific band recognized by anti-HA antibody, used as load control. Experiment done in duplicate with same results. F. Model of PORCN's function in transformed epithelial cells. PORCN is involved in the acylation and secretion of Wnt proteins. Independent of its role in Wnt secretion, PORCN regulates proliferation and gene expression. The role in gene expression may lead to changes in proliferation.