Proteomics reveals NNMT as a master metabolic regulator of cancer associated fibroblasts

High grade serous carcinoma (HGSC) has a poor prognosis primarily due to its early dissemination throughout the abdominal cavity. Genomic and proteomic approaches have provided snapshots of the proteogenomics of ovarian cancer (OvCa)(1,2), but a systematic examination of both the tumor and stromal compartments is critical to understanding OvCa metastasis. We developed a label-free proteomic workflow to analyze as few as 5,000 formalin-fixed, paraffin embedded cells microdissected from each compartment. The tumor proteome was stable during progression from in situ lesions to metastatic disease; however, the metastasis-associated stroma was characterized by a highly conserved proteomic signature, prominently including the methyltransferase nicotinamide N-methyltransferase (NNMT) and several proteins it regulates. Stromal NNMT expression was necessary and sufficient for functional aspects of the cancer associated fibroblast (CAF) phenotype, including the expression of CAF markers and the secretion of cytokines and oncogenic extracellular matrix. Stromal NNMT expression supported OvCa migration, proliferation, and in vivo growth and metastasis. Expression of NNMT in CAFs led to a depletion of S-adenosyl methionine (SAM) and a reduction in histone methylation associated with widespread gene expression changes in the tumor stroma. This work supports the use of ultra-low input proteomics to identify candidate drivers of disease phenotypes. NNMT is a central, metabolic regulator of CAF differentiation and cancer progression in the stroma that may be therapeutically targeted.