Anti-HNMT monoclonal antibody, clone FQS9422

Cancer-associated fibroblasts (CAF) influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4), lymph node metastasis (N+, n = 3) and bone marrow (BM, n = 4) obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01). Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF) and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF). Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs.

N-terminal myristoylation is a cotranslational lipid modification common to many signaling proteins that often serves an integral role in the targeting and/or function of these proteins. Myristoylation is catalyzed by an enzyme activity, N-myristoyltransferase (NMT), which transfers myristic acid from myristoyl coenzyme A to the amino group of a protein's N-terminal glycine residue. While a single human NMT cDNA has been isolated and characterized (hNMT-1), biochemical evidence has indicated the presence of several distinct NMTs in vivo, often varying in either apparent molecular weight and/or subcellular distribution. We now report the cloning and characterization of a second, genetically distinct human NMT (hNMT-2), as well as the isolation of the respective mouse NMT homologue for each human enzyme. The mouse and human versions of each NMT are highly homologous, displaying greater than 95% amino acid sequence identity. Comparisons between the NMT-1 and NMT-2 proteins revealed reduced levels of sequence identity (76-77%), indicating that NMT-1 and NMT-2 comprise two distinct families of N-myristoyltransferases. Transient transfection of either the hNMT-1 or hNMT-2 cDNA into COS-7 cells resulted in the expression of high levels of NMT enzyme activity. Both hNMT-1 and hNMT-2 were found to myristoylate several commonly studied peptide substrates with similar, hut distinguishable, relative selectivities. Western analysis revealed that while hNMT-2 appeared as a single 65-kDa protein in transfected COS-7 cells, hNMT-1 was processed to provide four distinct protein isoforms ranging from 49 to 68 kDa in size. Collectively, these studies demonstrate a heretofore unappreciated level of genetic complexity underlying the enzymology of N-terminal myristoylation and suggest that the specific inhibition or regulation of either NMT in vivo may in turn allow for the selective control of particular myristoylation-dependent cellular functions.