Iron and Lipocalin-2 Modulate Cellular Responses in the Tumor Micro-environment of Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease with poor outcomes. Iron is known to signal cellular responses, and its levels are regulated by lipocalin-2 (LCN2) expression, a PDAC pro-tumorigenic molecule. However, how iron and LCN2 function in PDAC is unclear. Here we demonstrate that iron levels regulate PDAC cell proliferation, invasion, expression of epithelial to mesenchymal tumor markers, and pro-inflammatory cytokines. Iron chelation increased the expression of the LCN2 receptor SLC22A17 in pancreatic stellate cells and the anti-metastatic gene NDRG1 in PDAC cells. Deletion of Lcn2 in mouse tumor cells modulated the expression of genes involved in extracellular matrix deposition and cell migration. Moreover, cellular iron responses were dependent on the Kras mutation status of cells, and LCN2 expression levels. Deletion of Lcn2 expression in PDAC suggests a protective role against metastasis. Thus, iron modulation and LCN2 blockade could serve as potential therapeutic approaches against PDAC.


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Iron levels regulate proliferation of PDAC and pancreatic stellate cells 127 Iron is known to modulate cell proliferation (12,13). Therefore, we tested whether

Iron levels modulate the expression of pro-inflammatory and iron-transport genes in
147 PDAC and pancreatic stellate cells 148 TME-associated inflammation can mediate tumor growth (38,39). Therefore, we tested 149 whether modulating iron levels could influence the expression of pro-inflammatory genes (IL6 150 and IL1β), known to be regulated by the expression of LCN2, a PDAC-associated cytokine 151 involved in cellular iron uptake (5). We also measured the expression of two iron-transport 152 genes, ferritin heavy chain 1 (FTH1), and solute carrier family 22 member 17 (SLC22A17), 153 (LCN2 receptor) to verify cellular iron storage, and iron-bound LCN2 transport into the cells 154 respectively. BXPC3, PANC-1, and HPSC were treated with FAC at 150µM (a physiologically 155 relevant dose of iron) (40) for 24 hours and showed elevated expression of IL6, IL1β,and FTH1,156 in the presence of FAC relative to control (Figure 2). However, SLC22A17 expression was not 157 responsive to iron treatment in any of the cell lines tested (Figure 2). In addition, we treated cells 158 Page 7 of 30 with 20 µM of DFO, and showed that expression of IL6 and IL1β decreased in HPSC. 159 Expression of IL6 was not stimulated in PANC-1 and BXPC3 while IL1β increased in BXPC3 160 after iron chelation (Figure 2B, D). FTH1 expression was reduced in PANC-1, while it was 161 increased in HPSCs after DFO treatment ( Figure 2F). Moreover, SLC22A17 expression was 162 upregulated only in HPSCs after iron chelation ( Figure 2H), which could be the result of an 163 adaptation mechanism in response to low levels of iron in HPSCs, to preserve iron transport in 164 the TME. Thus, increased iron levels seem to promote inflammation and iron transport in PDAC 165 and HPSCs, but do not affect the expression of the LCN2 receptor. Iron chelation for the most 166 part blocked some of those effects and specifically increased iron transport molecules in HPSCs. 167 These results indicate that stromal cells of the TME respond differently to reduced iron levels 168 than cancer cells.

Iron treatment promotes EMT and cancer cell invasion of human PDAC cell lines in a 170
Kras-dependent matter. 171 Iron promotes changes in EMT which is known to precede invasion (10). Therefore, to 172 understand whether iron levels modulate the EMT phenotype of PDAC cells, we examined cell regulates the invasive potential of BXPC3 and PANC-1, we measured invasion via transwell 188 assay with a BME coated membrane. We showed that iron treatments significantly increased the

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Iron is known to downregulate the expression of the iron related metastasis suppressor, 196 N-myc downstream regulated gene 1 (NDRG1), and this downregulation is associated with 197 increased proliferation and invasion of PDAC (34,41,42). Therefore, we measured the 198 expression of NDRG1 after treating BXPC3 and PANC-1 cells with 150μM and 20mM FAC. 199 We showed that NDRG1 expression decreases 33-fold in BXPC3 and 22-fold in PANC-1 cells     mutation in PDAC patients, was found to regulate cellular iron transport and storage (51).

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Moreover, triapine, an iron chelator and an inhibitor of the M2 subunit of the ribonucleotide 297 reductase, has been used to improve radiation therapy outcomes in PDAC patients (52). In 298 addition, iron chelation has also been effective in reducing tumor growth alone or in combination 299 with other treatments in vitro and in PDAC xenograft models (53)(54)(55)(56).

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Considering the interaction of iron with the pro-inflammatory cytokine LCN2, we sought  (Figure 6 B, C, D). These results validate and build upon our prior work supporting the finding 362 that stromal cells treated with LCN2 increase inflammation in the TME of PDAC and that lack 363 of Lcn2 expression in mice delays PDAC tumor formation and increases survival (5). In particular, the processes of cell adhesion, proteinaceous extracellular matrix, and integrin 365 pathway were strongly over-represented in the tumor cells that lack Lcn2 expression (Figure 7). 366 Cell adhesion is associated with an epithelial phenotype and it is known to be reduced during   Sequencing reads were aligned to mouse reference genome GRCm38 with hisat2 (74).