Protein acyltransferases (PATs), a group of enzymes catalysing fatty acylation of a variety of proteins, play an important role in protein sorting and signalling in the cell. In the Hedgehog (Hh) signalling pathway the PAT Hedgehog acyltransferase (Hhat) is responsible for addition of a palmitic acid to the N-terminal cysteine of Hh proteins. This modification is necessary for proper packaging and release of Hh ligands from the signalling cell and for transmitting a potent signal to the receiving cell. Hh signalling regulates cell growth and differentiation. Improper activation of the Hh signalling pathway in cancer arises either through an upregulation of ligand-dependent autocrine or paracrine signalling, or oncogenic mutations in the signal receiving cell. To date, drug therapies targeting the pathway in cancer have been directed at pathway regulators downstream of ligand binding. In this thesis, I investigate the potential of Hhat as a therapeutic target in lung cancer cell lines that express Sonic hedgehog (Shh). Studies of lung cancer cell lines A549, H209 and H82 treated with Shh pathway inhibitors showed no autocrine dependence on Shh signalling. Nonetheless, A549 cells were capable of inducing a Shh signal response in C3H10T½ cells in co-culture in a juxtacrine/paracrine fashion indicating that Shh in lung cancer cells may be involved in juxtacrine/paracrine signalling to the surrounding stroma. This effect was effectively attenuated by siRNA-mediated Hhat knock-down confirming the potential of Hhat as therapeutic target in cancer. I further provide a membrane topology model of Hhat which will aid in understanding the molecular mechanism of this enzyme. Membrane topology determination resulted in a topology model where Hhat consists of 11 transmembrane regions with the signal peptide preserved in the mature protein. Surprisingly, the suggested catalytic site, a conserved His, is localised in a transmembrane region near the cytosol away from the site of Hh palmitoylation which occurs in the lumen of the endoplasmic reticulum. Taken together, my results provide a firm basis for further investigation of the mechanism of action of Hhat and its role as a potential drug target in multiple human cancers.