Compartment-Resolved Proteomics Reveal Nnmt As A Master Metabolic Regulator Of Cancer Associated Fibroblasts

The poor outcome of ovarian cancer patients is due to late detection and the ability of ovarian cancer cells to metastasize quickly throughout the abdominal cavity. Recent research, including histopathological studies and experiments with mouse models, suggest that ovarian cancer may actually arise in the fallopian tube as serous tubal intraepithelial carcinomas (STIC). Lesions of the ovary would, therefore, represent metastases from the fallopian tube. To better understand the molecular events that occur in both the tumor and stromal compartment during ovarian cancer progression, we performed shotgun proteomics on laser-microdissected tumor and stromal compartments from anatomic sites representing the hypothetical progression series for ovarian cancer (STIC → fallopian tube → ovary → omentum). With an optimized sample preparation technique, we successfully quantified 4-5000 proteins per anatomic site with high reproducibility and with as few as several thousand microdissected cells derived from formalin-fixed and paraffin-embedded (FFPE) biobank specimens. Proteomics of the tumor compartments revealed high inter-patient heterogeneity, with no conserved protein signatures associated with invasion or metastasis. In strong contrast, a highly conserved molecular signature of stromal proteins associated with metastasis to the omentum was identified. In particular, nicotinamide N-methyl transferase (NNMT) was highly upregulated in the stroma of omental and peritoneal metastases. Functionally, NNMT was necessary and sufficient for multiple aspects of the cancer associated fibroblast (CAF) phenotype, including expression of CAF markers and secretion of cytokines and oncogenic extracellular matrix. NNMT expression was necessary for the ability of CAFs to support ovarian cancer cell migration, proliferation, adhesion, and in vivo growth. Mechanistically, high expression of NNMT in CAFs led to a depletion of S-adenosyl methionine (SAM) and a reduction in histone methylation associated with gene expression changes in the tumor stroma. This work supports the use of ultra-low input proteomics for compartment-resolved identification of candidate drivers of disease phenotypes and identifies NNMT as a central, metabolic regulator of CAF differentiation. Citation Format: Mark A. Eckert, Fabian Coscia, Shawn Pan, Samantha M. Tienda, Agnieszka A. Chryplewicz, Chun-Yi Chiang, Anthony Montag, S. Diane Yamada, Matthias Mann, Ernst R. Lengyel. Compartment-resolved proteomics reveal NNMT as a master metabolic regulator of cancer associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5899. doi:10.1158/1538-7445.AM2017-5899