Inhibition of the Wnt palmitoyltransferase porcupine suppresses cell growth and downregulates the Wnt/β-catenin pathway in gastric cancer

Abstract

Similarly to the Wnt protein palmitoyltransferase, porcupine (PPN) is essential to the activation of the Wnt/β-catenin signaling pathway. However, little is known about the role of PPN activity in human gastric cancer, one of the most common causes of cancer-related mortality. Real-time quantitative PCR was used to detect the expression levels of PPN in paired gastric cancer tissues. Cell proliferation, migration and invasion assays were performed following treatment using a newly developed small molecule PPN inhibitor (inhibitors of Wnt production, IWP-2) in the gastric cancer MKN28 cell line. Expression of downstream target genes and transcriptional activity of the Wnt/β-catenin signaling pathway were examined following IWP-2 treatment in MKN28. We identified that PPN was overexpressed in human gastric cancer tissue samples and cell lines. Following treatment of the gastric cancer cell line MKN28 with IWP-2, we detected that IWP-2 decreased MKN28 cell proliferation, migration and invasion, and elevated caspase 3/7 activity. Further analysis demonstrated that IWP-2 downregulated the transcriptional activity of the Wnt/β-catenin signaling pathway and downregulated the expression levels of downstream Wnt/β-catenin target genes in MKN28 cells. As current Wnt pathway-targeting strategies used for anticancer therapy have mainly focused on Wnt-receiving cells, our data shed light on the potential use of Wnt palmitoyltransferase PPN inhibitors to abrogate Wnt production in Wnt-producing cells, thus providing a potential therapeutic option for gastric cancer.

Keywords: Wnt; gastric cancer; palmitoylation; palmitoyltransferase; porcupine.

Figure 1

Overexpression of PPN in gastric cancer tissue samples and MKN28 cell line. (A) Real-time PCR analysis of PPN expression levels in paired gastric cancer tissue samples. The 2^−ΔΔCt value demonstrates the fold change of the relative PPN expression (relative to internal control) in cancer tissue normalized to adjacent normal tissue. (B) Real-time PCR analysis of PPN expression levels in gastric cancer cell line MKN28 and three normal gastric tissue samples. The 2^−ΔCt value demonstrates the relative PPN expression (relative to internal control). ^**P<0.01, ^***P<0.001. PPN, porcupine.

Figure 2

PPN inhibitor suppressed MKN28 cell growth. (A) The effect of IWP-2 at different concentrations (0–50 μM) on MKN28 cell proliferation. Optical density, 490 nm. (B) Anchor-dependent colony formation images. (C) Anchor-dependent colony number counts. The cells were treated with IWP-2 for 3 weeks. (D) Anchor-independent colony number counts. The cells were treated with IWP-2 for 3 weeks. ^*P<0.05, ^**P<0.01 and ^***P<0.001. PPN, porcupine; IWP, inhibitors of Wnt production.

Figure 3

PPN inhibitor downregulated MKN28 cell migration and invasion, and induced cell apoptosis. (A) Transwell migration assay. (B) Invasion assay. (C) Caspase 3/7 activity assay. The cells were treated with IWP-2 for 3 days. Optical density, 570 nm. ^***P<0.001. PPN, porcupine.

Figure 4

PPN inhibitor downregulated the activity of Wnt/β-catenin signaling pathway. (A) Transcriptional activity of two transcription factors (TCF and LEF). FOP was used as the negative control. (B–E) Real-time PCR analysis of downstream Wnt/β-catenin signaling pathway target genes (AXIN2, C-MYC, CCND1 and BIRC5, respectively). (F) Western blot for key regulators of the Wnt/β-catenin signaling pathway. The cells were treated with IWP-2 for 3 days. ^*P<0.05, ^**P<0.01 and ^***P<0.001. PPN, porcupine; IWP, inhibitors of Wnt production.