Responses of Porcupine and Wntless proteins to oxidative, hypoxic and endoplasmic reticulum stresses.

The WNT (Wingless and Int-1) proteins play a role in stem cell development and cell differentiation. Mutations in the WNT proteins lead to the development of various tumours, including gastric tumours. Porcupine (PORCN) is a palmitoyltransferase and Wntless (WLS) is a dedicated WNT transport protein that modify and fold the WNT proteins respectively and are involved in their proper secretion and binding to the frizzled (FZD) receptor and the lipoprotein receptor-related protein 5 or 6 (LRP5/6). We investigated how modifications of PORCN and WLS result in changes in WNT expression and secretion from cells under stress conditions that occur in the tumour microenvironment (hypoxia, oxidative stress, endoplasmic reticulum (ER) stress). In the present study, we found the mRNA expression of both PORCN and WLS were significantly increased with treatments inducing oxidative stress (antimycin A) and proteasome inhibition (MG-132), in human colon cancer (HCT116) and human intestinal epithelial cell-6 (HIEC-6) cells. Treatment with ER stressors thapsigargin, tunicamycin, and dithiolthreitol significantly increased PORCN gene expression, while treatment with thapsigargin and dithiolthreitol increased WLS gene expression. The expression of PORCN and WLS proteins increased with hypoxia and ER stressor treatments in both HCT116 and HIEC-6 cells. All stressors used in this study increased beta-catenin (β-catenin) expression in HCT116 cells. Our results suggest that these stressors alter PORCN, WLS and β-catenin expression and function which may, in turn, alter WNT secretion. Silencing the expression of PORCN and WLS with siRNA expression reduced the expression of WLS and WNT3A in HCT116 cells. The possibility exists that PORCN specifically may be involved in a novel signaling pathway, independent of its palmitoleation of the WNT proteins and its role in their secretion, that is rate-limiting for cancer cell growth and tumorigenesis, within the tumour microenvironment.