Investigating the Role of Porcupine and WNTless: Components of WNT Signalling Pathway in Response to Endoplasmic Reticulum, Oxidative, Hypoxia and Environmental Toxins Stesses

Abstract

Porcupine (PORCN) and WNTless (WLS) are factors that control the production of WNT; a protein involved in the early stages of gastric cancer development. The WNT proteins play a role in stem cell development and cell differentiation. However, the response of PORCN and WLS to stressors remains to be studied. Previously, we investigated how modifications of PORCN and WLS result in changes in WNT expression and secretion from cells under stress conditions that are found in the tumor microenvironment (hypoxia, oxidative stress, endoplasmic reticulum (ER) stress). The mRNA and protein expression of both PORCN and WLS were notably altered with treatments in human colon cancer (HCT116) cells and Human Intestinal epithelium (HIEC-6) cells. Our results demonstrated significant changes in expression, which led us to investigate the role of different transcription factors during stresses including (GRP78, HIF1α, NFE2L1, NFEL2L2 and MT1). These transcription factors were altered both at levels of the translation and transcriptional, when overexpressed by PORCN, WLS and WNT3A. WNT and PORCN levels significantly at translation and transcriptional levels when overexpressed by PORCN, WLS and WNT3A. WNT and PORCN undergo palmitoyleation; a modification implicated in cancer cell signalling. The inhibition of palmitoyleation by 2-bromohexdecanoic acid (2-BHDA) resulted in a decrease in the modified forms of PORCN in HCT116 cells. Furthermore, the data showed that palmitoyleation plays an essential role in the expression of WNT proteins; specifically, inhibiting WNT3A and decreasing WLS protein expression in HCT116 cells. However, WLS and WNT3A are modified by N-linked glycosylation. Emerging evidence indicates the critical role of glycosylation in tumor aggressiveness, progression, and metastasis. Cell lysates from hypoxic HCT116 cells, containing induced WLS and WNT3A proteins, HCT116 cells were subjected for deglycosylation. The production of glycosylated WLS and WNT3A was inhibited. We also investigated the role of cadmium in activating WNT signaling and its components, our results showed a significant increase of the WNT proteins, and the transcription factors, when exposed to dose-specific concentration of cadmium at 5μM. These notable findings show the essential role of post-translation modification of PORCN and WLS and how it alters WNT signalling during stress conditions