LOXL2 is upregulated in gemcitabine-resistant pancreatic cancer cell lines
Patients who underwent pancreatic cancer surgery and received gemcitabine adjuvant chemotherapy were classified into resistant and sensitive groups based on recurrence within 1 year (Table 1). Following immunohistochemical staining with LOXL2, the resistant group showed a higher ratio at strong intensity compared to the sensitive group (Fig. 1A, B). Total RNA was isolated from tissues of patients with pancreatic cancer and the difference in LOXL2 expression was compared. Therefore, it was possible to confirm the high LOXL2 expression in the resistant patient group (Fig. 1C). To elucidate the role of LOXL2 in vitro, we established gemcitabine-resistant Mia-Paca2 (Mia GR) by continuous exposure to gemcitabine in stepwise increments from each parental cell line (Mia Con) (Fig. 1D). When the final treatment concentration of gemcitabine-resistant cells was 10 μM, the most consistent resistance was confirmed. Gemcitabine-resistant cell lines demonstrated a clear increase in gemcitabine tolerance compared with parental cell lines, with half-maximal inhibitory concentration (IC50) of gemcitabine increasing from 12.03 μM to 774.5 μM (Fig. 1E). We identified known factors related to gemcitabine resistance in the prepared Mia GR through qPCR [29]. Several ATP-binding cassette (ABC) transporters and ALDH were confirmed to increase in Mia GR compared to Mia Con (Fig. S1A, B). In addition, it was found that among the factors related to gemcitabine metabolism, RRM1 increased and, conversely, hENT and DCK decreased in Mia GR (Fig. 1F) [30]. By western blotting, gemcitabine metabolism-related proteins were shown to increase (Fig. 1H). Therefore, it could be confirmed that Mia GR was generated accurately and consistently as a cell model to study the mechanism of gemcitabine resistance. LOXL2 mRNA and protein expression were confirmed to increase significantly compared to Mia Con (Fig. 1G, H). In addition, overexpression of LOXL2 was confirmed when a gemcitabine-resistant cell line was created using the PANC1 cell line (Fig. S1C, E). We could hypothesize that LOXL2 was overexpressed in tissues and cells of patients with gemcitabine-resistant pancreatic cancer based on confirmed results and that it would play an important role in the resistance-related mechanism.
Overview of mRNA expression and enrichment analyses of gemcitabine-resistant pancreatic cancer cell lines
Given the different phenotypes between Mia GR and Mia Con, we next analyzed the mRNA profiles between these cells using RNA sequencing. In addition, by using Mia GR transfected with siLOXL2, we attempted to check the mRNA profile change according to LOXL2. From the scatterplot data, we identified many genes with distribution differences between Mia Con and Mia GR cells (Fig. 2A). Using the DAVID analysis tool, the gene that showed high expression in the Mia GR group belonged to the gene ontology and the pathway through KEGG analysis was confirmed (Fig. 2B). Genes were included in biological processes expected to be associated with chemical resistance, such as negative regulation of apoptotic processes, positive regulation of cell proliferation, positive regulation of migration, and wound healing. Processes related to glucose metabolism were also included. The pathway was confirmed to include MAPK-JNK, TNF, and NF-κB signaling processes, and genes with high expression in Mia GR are included in stem-related signaling processes. In Mia GR transfected with siLOXL2, genes involved in apoptosis, proliferation, ECM, and migration-related biological processes were present. Through KEGG analysis, it was confirmed that these genes belong to the PI3K-Akt, Focal adhesive, MAPK, and TNF signaling pathways (Fig. 2B). From the gene set enrichment analysis (GSEA) results, the gene ontology to which the genes increased in Mia GR belonged was confirmed (Fig. 2C). Among them, glucose metabolism and NF-κB signaling were expected to be associated with increased LOXL2 expression.
Glucose metabolism is activated in gemcitabine-resistant pancreatic cancer
As seen from the mRNA seq results, genes more highly expressed in Mia GR compared to Mia Con belong to glucose metabolism. The Mia GR group exhibited a high expression of glut, which is involved in glucose uptake (Fig. 3A). Glucose uptake was compared using 2-NBDG, and a substantial increase in Mia GR was confirmed (Fig. 3B). Glucose uptake was reduced when Mia GR was treated with the glut inhibitor WZB117 and GLUT siRNA (Fig. 3B). Production of lactic acid, a product of glucose utilization, was found to increase in the MIA GR group (Fig. 3C, D). Previously, treatment with siGLUT3 and WZB117 revealed a decrease in glucose uptake and downregulated LOXL2 expression, and it was confirmed through qPCR and western blotting that LOXL2 expression was reduced even after treatment with 2DG, a glycolysis inhibitor (Fig. 3E, H). Therefore, it was concluded that increased glucose metabolism in gemcitabine-resistant cells induces and regulates LOXL2 expression.
NF-κB signaling is regulated by glucose metabolism in gemcitabine-resistant cell lines
One of the signaling pathways activated in chemoresistance, NF-κB, was highly regulated in mRNA seq analysis; therefore, we attempted to confirm the association with glucose metabolism. By western blot analysis, we showed that phospho-NF-κB expression was increased in Mia GR (Fig. 3I). In contrast, treatment with the GLUT inhibitors WZB117 and 2DG reduced NF-κB phosphorylation (Fig. 3J, K). Treatment with siGLUT3 was found to reduce phosphor-NF-κB expression of phosphor-NF-κB and decrease nuclear translocation (Fig. S2A, B). Therefore, it was confirmed that glucose metabolism regulates NF-κB activation.
Activated NF-κB binds to the LOXL2 promoter and transcriptionally regulates overexpression
NF-κB is phosphorylated and regulates the transcription of several genes. First, JSH-23, known as NF-κB inhibitor, significantly inhibits the nuclear translocation of phosphorylated NF-κB in gemcitabine-resistant cells (Fig. 4A). It was confirmed that the expression of LOXL2 and ZEB1 was regulated with time when Mia GR was administered to JSH-23, an inhibitor of NF-κB (Fig. 4B, C). To determine whether NF-κB transcriptionally regulates LOXL2 expression, we analyzed the NF-κB binding region within the LOXL2 promoter using JASPAR (Fig. 4D). We could predict the 5'-GGGGACCACCG-3' region and confirm that a high binding was formed in the vicinity, using chIP (Fig. 4E). Furthermore, treatment with 2DG, a glycolysis inhibitor, was shown to decrease binding to the expected NP3 region (Fig. 4F).
ZEB1, like LOXL2, is a representative factor that regulates EMT and is transcriptionally regulated by NF-κB [31]. We treated each siRNA to confirm the regulatory relationship between LOXL2 and ZEB1, and by western blotting and qPCR it was possible to confirm that LOXL2 was regulated by ZEB1 (Fig. 4G, H). Based on the JASPAR program, the LOXL2 promoter binding region of ZEB1 was specified and chIP was performed (Fig. 4I). High binding was confirmed in the ZP4 region (Fig. 4J). JSH-23 significantly inhibited the binding of ZEB1 to the LOXL2 promoter region (Fig. 4K). Therefore, it was concluded that LOXL2 overexpression caused by NF-κB activation in gemcitabine-resistant cells occurred either directly or through ZEB1.
LOXL2 and ZEB1 are together involved in the regulation of EMT in gemcitabine-resistant pancreatic cancer
Mia GR was morphologically closer to mesenchymal cells than Mia Con, and migration-related genes were highly regulated in mRNA seq. Therefore, we attempted to confirm the change in invasiveness and migration ability of LOXL2-ZEB1. Based on the invasion assay, the invasiveness of LOXL2-ZEB1 knockdown cells was significantly reduced and a decrease in migration activity was confirmed by wound analysis (Fig. 5A, B). In addition, it was verified through qPCR that the expression of EMT-related genes and protein markers was also reduced (Fig. 5C, D). Therefore, the high EMT process in gemcitabine-resistant pancreatic cancer was confirmed to be regulated by the ZEB1-LOXL2 axis that contributes to maintaining chemoresistant characteristics.
MAPK activation by LOXL2 regulates cancer stemness of EPCAM-dependent gemcitabine-resistant pancreatic cancer
Cancer stemness is a key chemoresistant characteristic and contributes to recurrence and drug resistance in pancreatic cancer with high heterogeneity. From mRNA seq results, it was confirmed that the stem cell-related pathway was related according to LOXL2. Therefore, the stemness of the LOXL2 knockdown Mia GR was confirmed through spheroid formation and 3D culture. Therefore, the size of the spheroid was well-formed in Mia GR compared to Mia Con, but did not increase when LOXL2 was knocked down (Fig. 6A). In the 3D culture system, LOXL2 knockdown was confirmed to inhibit the growth of 3D shaped cells (Fig. 6B). Expression of the CD326 gene and protein (EPCAM), a representative surface marker of cancer stemness, was reduced, and it was found through flow cytometry that CD326 positive cells were decreased by LOXL2 (Fig. 6C). In addition, expression change of “Yamanaka factors,” factors related to cancer stemness, was confirmed, and reduction of Oct4 and c-myc was confirmed through qPCR and western blot (Fig. 6D, E). Conversely, by sorting CD326+ Mia GR, LOXL2 and stemness factors (oct4, c-myc) were overexpressed, through qPCR and western blot (Supple. Fig. 3A, B). Based on the previous mRNA seq results, MAPK was assumed as a sub-factor for regulating LOXL2 stemness and its activity was confirmed by western blot analysis (Fig. S4A and C). In Mia GR, p38 phosphorylation was increased compared to Mia Con, and its activity was decreased by LOXL2 knockdown (Supplementary Fig. S4D). When cells were treated with siLOXL2 and p38 inhibitor SB202190 alone or in combination, the expression of stemness markers was reduced (Fig. 6F, G). Furthermore, the CD326+ cell population and spheroid formation were significantly decreased in the cotreatment environment (Fig. 6H, I). Furthermore, inhibitor and LOXL2 knockdown showed the lowest viability after 8 days of 3D culture (Fig. 6J). Gemcitabine-resistant pancreatic cancer exhibits cancer stemness, which is regulated by LOXL2 and is achieved through the regulation of EPCAM and oct4, c-myc by MAPK signaling.
LOXL2 promotes tumorigenesis and modulates gemcitabine resistance in xenograft models
Increased gemcitabine sensitivity by LOXL2 knockdown was observed through the proliferation assay (Fig. 7A). We constructed a xenograft mouse model in Mia GR expecting the EMT process-preferring trait and maintenance of high cancer stemness will affect cancer growth. First, we created a Mia GR cell line that stably knocked down LOXL2 (Fig. S5A, B). It was confirmed that Mia GR tumorigenicity was higher than Mia Con, and that LOXL2 knockdown had an inhibitory effect on tumor volume (Fig. 7C, Fig. S5C). After 35 days of cell administration, the difference was more clearly defined when weight and size were compared (Fig. 7D and F). After administrating cells in the same way as in the previous xenograft mouse model, the reaction was confirmed by treatment with gemcitabine (Fig. 7G). Tumor growth was slow in mice administered with Mia GR, in which LOXL2 was knocked down (Fig. 7H, Fig. S5D). Furthermore, sensitivity to gemcitabine was higher in the LOXL2 knockdown mouse group when volume and weight were sacrificed 6 weeks after drug injection (Fig. 7I and K).