Netrin G1 Promotes Pancreatic Tumorigenesis through Cancer-Associated Fibroblast–Driven Nutritional Support and Immunosuppression

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identifi ed Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1 + cancer-associated fi broblasts (CAF) support PDAC survival, through a NetG1mediated effect on glutamate/glutamine metabolism. Also, NetG1 + CAFs are intrinsically immunosuppressive and inhibit natural killer cell-mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SIGNIFICANCE:This study demonstrates the feasibility of targeting a fi broblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.

Palladin isoforms 3 and 4 regulate cancer-associated fibroblast pro-tumor functions in pancreatic ductal adenocarcinoma

“…Patient derived CAFs 13,22 were treated with vehicle control or TGFβ1 signaling inhibitor (SB431542) during d-ECM production. Protein expression was analyzed in cell lysates while protein localization and d-ECM topographies were phenotypically characterized via indirect immunofluorescence.…”

“…CAFs participate in tumorigenesis is via secretion of inflammatory cytokines 4,13 . To assess if loss of palladin affects the ability of PDAC CAFs to secrete these and other known CAF-relevant cytokines, conditioned media collected from palladin KD CAFs were analyzed via enzyme-linked immunosorbent assay (ELISA) in which the concentrations of secreted TGFβ1, IL-6, and IL-8 were calculated.…”

“…Palladin isoforms are required to produce pro-tumor desmoplastic ECM. CAFs are known to produce d-ECMs that aid tumorigenesis by supporting PDAC cell growth and survival 4,13,29 . We questioned whether the altered ECM (i.e., sufficient ECM production with reduced ECM alignment), produced by CAFs in experiments described above, could impact the growth of K-Ras driven tumorigenic human pancreatic ductal epithelial cells like KRas-HPNE 30 and the PDAC cell line Panc-1.…”

“…We also tested the ability of the various CAF-generated ECMs to rescue KRas-HPNE and Panc-1 cells from nutritional stress-induced death. For this, tumorigenic cell lines were plated in the assorted ECMs for 3 days under serum and glutamine deprivation 13 . Results show that ECMs produced by SB431542 treated CAFs (inhibited TGFβ1 signaling) failed to show differences in supporting KRas-HPNE survival and presented with a modest reduction in Panc-1 cell survival compared to d-ECM controls (Fig.…”

Transporters at the Interface between Cytosolic and Mitochondrial Amino Acid Metabolism

“…In cells with high proliferative rates (e.g., cancer cells, activated T lymphocytes), glutamine demand outweighs supply and environmental access becomes "conditionally essential" [8][9][10][11][12]. When nutrients become locally limited, several cancers, including pancreatic, glioblastoma, and ovarian, hijack stromal glutamine synthesis as an alternative supply line to fulfill their increased demands [13][14][15]. Furthermore, glutamine deprivation suppresses expansion of activated T lymphocytes, and competition for nutrients within tissues may affect immune responses to pathological states that exhibit hallmark increases in nutrient consumption (e.g., viral infection, cancer) [10,[16][17][18].…”