Abstract A83: c-Met requires palmitoylation for proper stability and trafficking in cancer cells

The influence of growth factor receptors on cancer progression as both early promoters as well as drivers of late-stage invasion and metastasis has been thoroughly studied over the last several decades. Overexpression and activating mutations of several receptor tyrosine kinases (RTKs) are commonly detected in most cancer types. Despite this, there still remains a great deal not fully understood about RTK expression and regulation that may reveal novel strategies for therapeutic targeting. In particular, c-Met is an RTK whose downstream signal transduction can promote both mitogenic and motogenic phenotypes in cancer cells and whose expression is correlated with poor prognosis and resistance to therapy. c-Met is activated by either autocrine or paracrine ligand stimulation. Alternatively, receptor overexpression allows for ligand-independent dimerization and therefore constitutive activation. In addition, multiple reports have identified the enzyme fatty acid synthase (FASN) as being commonly overexpressed in prostate cancer, and that this aberrant expression is an early event that becomes more pronounced with aggressive androgen-independent and metastatic disease. FASN is the sole enzyme responsible for de novo synthesis of the 16-carbon saturated fatty acid palmitate. In cancer, de novo lipids are more selectively partitioned into lipid rafts as phospholipids as well as utilized for post-translational acyl-modifications of signaling proteins. Previous findings have led us to identify a novel mechanism by which FASN activity regulates c-Met expression. Our work has determined that inhibition or shRNA knockdown of FASN results in a post-translational downregulation of already synthesized c-Met protein. This downregulation is prevented by the addition of exogenous palmitate. Based on these findings we have subsequently acquired convincing data that the c-Met receptor tyrosine kinase is palmitoylated and that this palmitoylation regulates its stability. Inhibition of palmitoylation reduces the expression of c-Met in multiple cancer cell lines. This protein loss occurs post-transcriptionally and is associated with accumulation of c-Met in Golgi compartments. Using inhibitors to a number of internalization pathways, as well as surface biotinylation studies, confocal microscopy, and metabolic-ortholog labeling we determined that inhibition of palmitoylation reduces the stability of newly synthesized, c-Met as opposed to inducing internalization and degradation. Moreover, both an acyl-biotin exchange technique and a click-chemistry based palmitate-labeling protocol suggest c-Met itself is palmitoylated. Observing palmitoylation kinetics has provided evidence that c-Met is palmitoylated in the ER prior to cleavage of the 170kd c-Met precursor into its mature 140kd form. Taken together, these findings suggest inhibition of palmitoylation or FASN activity could be a novel target for preventing invasion and metastasis driven by c-Met overexpression. Citation Format: David T. Coleman, James A. Cardelli. c-Met requires palmitoylation for proper stability and trafficking in cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A83.