Lipocalin-2 (LCN2) promotes malignant development in many cancer types. LCN2 is upregulated in patients with pancreatic ductal adenocarcinoma (PDAC) and in obese individuals, but whether it contributes to PDAC development is unclear. In this study, we investigated the effects of Lcn2 depletion on diet-induced obesity, inflammation and PDAC development. Mice with acinar cell-specific expression of KrasG12D were crossed with Lcn2-depleted animals and fed isocaloric diets with varying amounts of fat content. Pancreas were collected and analyzed for inflammation, pancreatic intraepithelial neoplasia (PanIN) and PDAC. We also used a syngeneic orthotopic PDAC mouse model to study tumor growth in the presence or absence of Lcn2 expression. In addition, to understand the mechanistic role of how LCN2 could be mediating PDAC, we studied LCN2 and its specific receptor solute carrier family 22 member 17 (SLC22A17) in human pancreatic cancer stellate cells (PSC), key mediators of the PDAC stroma. Depletion of Lcn2 diminished extracellular matrix deposition, immune cell infiltration, PanIN formation and tumor growth. Notably, it also increased survival in both obesity-driven and syngeneic orthotopic PDAC mouse models. LCN2 modulated the secretion of pro-inflammatory cytokines in PSC of the PDAC tumor microenvironment, while downregulation of LCN2-specific receptor SLC22A17 blocked these effects. Our results reveal how LCN2 acts in the tumor microenvironment links obesity, inflammation and PDAC development.

Chronic inflammation (CI) is a risk factor for pancreatic cancer (PC) including the most common type, ductal adenocarcinoma (PDAC), but its role and the mechanisms involved are unclear. To investigate the role of CI in PC, we generated genetic mouse models with pancreatic specific CI in the presence or absence of TP53. Mice were engineered to express either cyclooxygenase-2 (COX-2) or IκB kinase-2 (IKK2), and TP53+/+ or TP53f/f specifically in adult pancreatic acinar cells by using a full-length pancreatic elastase promoter-driven Cre. Animals were followed for >80 weeks and pancreatic lesions were evaluated histologically and immunohistochemically. The presence of K-ras mutations was assessed by direct sequencing, locked nuclei acid (LNA)-based PCR, and immunohistochemistry. We observed that sustained COX-2/IKK2 expression caused histological abnormalities of pancreas, including increased immune cell infiltration, proliferation rate and DNA damage. A minority of animals with CI developed pre-neoplastic lesions, but cancer was not observed in any TP53+/+ animals within 84 weeks. In contrast, all animals with CI-lacking TP53 developed various subtypes of PC, including acinar cell carcinoma, ductal adenocarcinoma, sarcomatoid carcinoma and neuroendocrine tumors, and all died within 65 weeks. No evidence of K-ras mutations was observed. Variations in the activity of the Hippo, pERK and c-Myc pathways were found in the diverse cancer subtypes. In summary, chronic inflammation is extremely inefficient at inducing PC in the presence of TP53. However, in the absence of TP53, CI leads to the development of several rare K-ras-independent forms of PC, with infrequent PDAC. This may help explain the rarity of PDAC in persons with chronic inflammatory conditions.

<p>Figure S1. LCN2 plasma levels do not correlated with BMI of patients with metastatic PDAC. Figure S2. Lcn2 and SLC22A17 expression in SVF from mice. Figure S3. Lcn2 expression is depleted in Lcn2-/-/KRasG12D/CRE animals and experience lower changes in body weight and effects of caerulein treatments in mice with and without Lcn2 expression. Figure S4. KPC-LUC cells expressed higher levels of Lcn2 compared to normal pancreas from wild type mice. Figure S5. KPC-LUC cells injected in the pancreas do not survive in all Lcn2-/- mice. Figure S6. Lcn2 depletion attenuates ECM remodeling, immune infiltration and tumor cell proliferation in Lcn2 null mice with tumors and invasion of peritoneal macrophages was not impaired in Lcn2 null mice. Figure S7. Lcn2 null mice expressed lower levels of the Lcn2 receptor SLC22A17. Figure S8. LCN2 induces a pro-inflammatory response in PSCs that is reduced after downregulation of the SLC22A17 receptor. Figure S9. SLC22A17 expression in adipose tissue from mice.</p>

<p>Supplementary Table 1. List of antibodies used for immunohistochemical staining of mouse pancreas.</p>

<p>Figure S1. LCN2 plasma levels do not correlated with BMI of patients with metastatic PDAC. Figure S2. Lcn2 and SLC22A17 expression in SVF from mice. Figure S3. Lcn2 expression is depleted in Lcn2-/-/KRasG12D/CRE animals and experience lower changes in body weight and effects of caerulein treatments in mice with and without Lcn2 expression. Figure S4. KPC-LUC cells expressed higher levels of Lcn2 compared to normal pancreas from wild type mice. Figure S5. KPC-LUC cells injected in the pancreas do not survive in all Lcn2-/- mice. Figure S6. Lcn2 depletion attenuates ECM remodeling, immune infiltration and tumor cell proliferation in Lcn2 null mice with tumors and invasion of peritoneal macrophages was not impaired in Lcn2 null mice. Figure S7. Lcn2 null mice expressed lower levels of the Lcn2 receptor SLC22A17. Figure S8. LCN2 induces a pro-inflammatory response in PSCs that is reduced after downregulation of the SLC22A17 receptor. Figure S9. SLC22A17 expression in adipose tissue from mice.</p>