Attenuation of Hedgehog Acyltransferase-Catalyzed Sonic Hedgehog Palmitoylation Causes Reduced Signaling, Proliferation and Invasiveness of Human Carcinoma Cells

Abstract

Overexpression of Hedgehog family proteins contributes to the aetiology of many cancers. To be highly active, Hedgehog proteins must be palmitoylated at their N-terminus by the MBOAT family multispanning membrane enzyme Hedgehog acyltransferase (Hhat). In a pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1 and transfected HEK293a cells Hhat localized to the endoplasmic reticulum. siRNA knockdown showed that Hhat is required for Sonic hedgehog (Shh) palmitoylation, for its assembly into high molecular weight extracellular complexes and for functional activity. Hhat knockdown inhibited Hh autocrine and juxtacrine signaling, and inhibited PDAC cell growth and invasiveness in vitro. In addition, Hhat knockdown in a HEK293a cell line constitutively expressing Shh and A549 human non-small cell lung cancer cells inhibited their ability to signal in a juxtacrine/paracrine fashion to the reporter cell lines C3H10T1/2 and Shh-Light2. Our data identify Hhat as a key player in Hh-dependent signaling and tumour cell transformed behaviour.

Sonic hedgehog processing and release are regulated by glypican heparan sulfate proteoglycans

“…The latter conclusion is supported by the detection of increased soluble product amounts from non-palmitoylated Shh C25A in comparison to palmitoylated protein products (compare relative amounts of soluble and cellular material produced in CHO-K1 tg cells; Fig. 2C,D, left lanes) and the findings of others (Chamoun et al, 2001;Konitsiotis et al, 2014).…”

“…2B, asterisk). These results demonstrate that both Hh lipidations contribute to Hh association with the cell membrane (Chamoun et al, 2001;Konitsiotis et al, 2014;Ohlig et al, 2011) to ensure that only fully processed Hh is released (Ohlig et al, 2012). This raises the question of how Hh shedding is controlled.…”

Synthetic protein lipidation

“…Due to the palmitate amide linkage on Hh ligands, a product of S to N acyl migration from a cysteine side chain to the free amino terminus, Hh proteins do not undergo cycles of acylation and deacylation, a behavior similar to O-palmitoylated Wnt proteins [10 ,25]. Recently, it was shown that palmitoylation is important for sonic Hedgehog signaling and its role in proliferation and invasiveness of human carcinoma cells [26].…”

The Deleterious Effects of Oxidative and Nitrosative Stress on Palmitoylation, Membrane Lipid Rafts and Lipid-Based Cellular Signalling: New Drug Targets in Neuroimmune Disorders

“…• EW12/PERL tumour suppressor function [300] • Influence proliferation and invasiveness of human carcinoma [301] • Breast cancer cell migration [302] • Migration and adhesion of cancer cells [303] Resistance to virus and bacterial infection…”