Ance to gemcitabine in insensitive lines.LCN2-associated Global Transcriptional ChangesSeveral studies have identified LCN2 as an upregulated gene in cancer. However no studies have yet examined the effect of LCN2 on gene expression. To examine how LCN2 affects gene expression in PDAC cell lines, transcriptional profiling was performed on the BxPC3 cell lines and xenografts. LCN2 expression upregulated the expression of 623 genes (Table S1) and downregulated the expression of 538 genes (Table S2). The putative LCN2 target genes were annotated to GO biological processes and were significantly enriched for processes involved in apoptosis (28/623; p = 0.008), cell cycle (32/623; p = 0.02), and adhesion (14/623, p = 0.02). The downregulated genes annotated to GO biological processes were significantly enriched for genes involved in apoptosis (36/538; p = 0.004). The genes involved in apoptosis were analysed and revealed 57 of the upregulated genes were involved in survival, and 67 of the downregulated genes were pro-apoptotic (Fig. 4A). To validate this we performed Q-PCR in the BxPC3, HPAF-II, and PANC1 cell lines. The pro-apoptotic gene AIFM1 was identified to have higher expression in the BxPC3 and HPAF-II cell lines after LCN2 was knocked-down (Fig. 4C). Whereas, expressing LCN2 in PANC1 cells enhanced expression of anti-apoptotic genes BIRC2, FAIM, and MCL-1 compared to the control (p,0.05; Fig. 4D ). Additionally, the genes enriched for attachment were examined (Fig. 4B). 44 of the genes promoted cell to cell attachment, Epigenetics Whereas the remaining genes positively regulated cell to ECM adhesion. Q-PCR validation demonstrated that expressing LCN2 in the PANC1 cell lines promoted expression of LAMAC2, MMP7, CDH11, and ITGA2 (p,0.05; Fig. 4G ). Whereas depleting LCN2 expression in the BxPC3 and HPAF-II cell lines downregulated expression of MMP-7 and CDH11 (p,0.05; Fig. 4H, I). We identified that LCN2 enhances 15755315 expression of genes annotated to adhesion and survival in PDAC.DiscussionIn the present study, the use of multiple modalities has provided a cohesive study into the function of LCN2 in PDAC and its pattern of expression during pancreatic carcinogenesis. We haveLCN2 in Pancreatic Cancershown that LCN2 expression is associated with the progression of PanIN lesions and PDAC. Through expression profiling studies, we have also demonstrated that LCN2 upregulates genes involved in survival, adhesion, and cell cycle, and downregulates proapoptotic genes. We have provided strong evidence that LCN2 promotes attachment, invasion, tumor growth, and gemcitabine resistance in multiple 
PDAC cell lines. By modifying LCN2 expression we were able to demonstrate by gelatin zymography that it modulates MMP-9 enzymatic activity. Depleting LCN2 abrogates invasion through basement membrane substrata, Matrigel, and collagen IV by PDAC cells. Since MMP-9 is a collagenase, depleting LCN2 in the BxPC3 and HPAF-II cell lines attenuated invasion through collagen IV. However, invasion through Matrigel was hindered in the BxPC3 cell line only. Matrigel is composed of other extracellular matrix proteins besides collagen such as laminins and proteoglycans, and represents a more complex substratum. Therefore, altering LCN2 may elicit diverse invasive phenotypes in different PDAC cell lines. Our Epigenetics findings that LCN2 expression promotes MMP-9 activity are consistent with other cancer cell types. Depletion of LCN2 in colon [21], gastric [16], and breast cancer models [13,.Ance to gemcitabine in insensitive lines.LCN2-associated Global Transcriptional ChangesSeveral studies have identified LCN2 as an upregulated gene in cancer. However no studies have yet examined the effect of LCN2 on gene expression. To examine how LCN2 affects gene expression in PDAC cell lines, transcriptional profiling was performed on the BxPC3 cell lines and xenografts. LCN2 expression upregulated the expression of 623 genes (Table S1) and downregulated the expression of 538 genes (Table S2). The putative LCN2 target genes were annotated to GO biological processes and were significantly enriched for processes involved in apoptosis (28/623; p = 0.008), cell cycle (32/623; p = 0.02), and adhesion (14/623, p = 0.02). The downregulated genes annotated to GO biological processes were significantly enriched for genes involved in apoptosis (36/538; p = 0.004). The genes involved in apoptosis were analysed and revealed 57 of the upregulated genes were involved in survival, and 67 of the downregulated genes were pro-apoptotic (Fig. 4A). To validate this we performed Q-PCR in the BxPC3, HPAF-II, and PANC1 cell lines. The pro-apoptotic gene AIFM1 was identified to have higher expression in the BxPC3 and HPAF-II cell lines after LCN2 was knocked-down (Fig. 4C). Whereas, expressing LCN2 in PANC1 cells enhanced expression of anti-apoptotic genes BIRC2, FAIM, and MCL-1 compared to the control (p,0.05; Fig. 4D ). Additionally, the genes enriched for attachment were examined (Fig. 4B). 44 of the genes promoted cell to cell attachment, whereas the remaining genes positively regulated cell to ECM adhesion. Q-PCR validation demonstrated that expressing LCN2 in the PANC1 cell lines promoted expression of LAMAC2, MMP7, CDH11, and ITGA2 (p,0.05; Fig. 4G ). Whereas depleting LCN2 expression in the BxPC3 and HPAF-II cell lines downregulated expression of MMP-7 and CDH11 (p,0.05; Fig. 4H, I). We identified that LCN2 enhances 15755315 expression of genes annotated to adhesion and survival in PDAC.DiscussionIn the present study, the use of multiple modalities has provided a cohesive study into the function of LCN2 in PDAC and its pattern of expression during pancreatic carcinogenesis. We haveLCN2 in Pancreatic Cancershown that LCN2 expression is associated with the progression of PanIN lesions and PDAC. Through expression profiling studies, we have also demonstrated that LCN2 upregulates genes involved in survival, adhesion, and cell cycle, and downregulates proapoptotic genes. We have provided strong evidence that LCN2 promotes attachment, invasion, tumor growth, and gemcitabine resistance in multiple PDAC cell lines. By modifying LCN2 expression we were able to demonstrate by gelatin zymography that it modulates MMP-9 enzymatic activity. Depleting LCN2 abrogates invasion through basement membrane substrata, Matrigel, and collagen IV by PDAC cells. Since MMP-9 is a collagenase, depleting LCN2 in the BxPC3 and HPAF-II cell lines attenuated invasion through collagen IV. However, invasion through Matrigel was hindered in the BxPC3 cell line only. Matrigel is composed of other extracellular matrix proteins besides collagen such as laminins and proteoglycans, and represents a more complex substratum. Therefore, altering 
LCN2 may elicit diverse invasive phenotypes in different PDAC cell lines. Our findings that LCN2 expression promotes MMP-9 activity are consistent with other cancer cell types. Depletion of LCN2 in colon [21], gastric [16], and breast cancer models [13,.
