OSBPL10-CNBP axis mediates hypoxia-induced pancreatic cancer development

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of malignancies with worst outcomes among digestive system tumors. Identification of novel biomarkers is of great significance for treatment researches and prognosis prediction of pancreatic cancer patients. Due to OSBPL10 known involvement in oncogenic activity in other tumors, we elucidated the mechanism underlying its contribution to pancreatic cancer progression. We employed data from the Gene Expression Omnibus database to detect the expression of OSBPL10 in normal and pancreatic cancer tissues. A series of assays were conducted to assess the impact of OSBPL10 on the proliferation and metastatic capacities of pancreatic cancer cells and the influence of OSBPL10 on macrophages were evaluated by Flow cytometry. In addition, Co-immunoprecipitation, mass spectrometry, and western blot assays were utilized to investigate the potential mechanisms of OSBPL10 activity. From our study, OSBPL10 is revealed to be upregulated in pancreatic cancer, with poor prognosis. The overexpression promotes malignant behaviors of pancreatic cancer cells and has an impact on tumor immune microenvironment by stimulating the transformation M1 macrophages into M2 macrophages. Mechanistically, hypoxia induces the expression of OSBPL10 through interaction between hypoxia-inducible factor 1-α and the promoter region of OSBPL10. Additionally, OSBPL10 directly bound to CNBP, mediating CNBP expression and ultimately regulating the proliferation and metastasis capacity of pancreatic cancer cells, as well as influencing macrophage polarization. The research emphasized the oncogenic role of OSBPL10 in pancreatic cancer, uncovering key mechanisms involving hypoxia, HIF-1α, and CNBP. The finding suggests that OSBPL10 is a novel biomarker in pancreatic cancer, making it a potential therapeutic target for intervention in this malignancy.

Keywords: CNBP; OSBPL10; hypoxia; oncogene; pancreatic ductal adenocarcinoma; tumor progression.

FIGURE 1

OSBPL10 is in a high level and corresponding to grave prognosis in pancreatic cancer. (A) Venn diagram identifies the high expression of OSBPL10 through GSE16515, GSE27890, GSE46234 and GSE60979 database. (B) Tumor and normal tissues show differential expressions of OSBPL10 in TCGA database. (C, D) Overall and DFS analysis from TCGA database. (E) The expression of OSBPL10 in tumor and matched normal tissues was measured by IHC. (F, G) Overall and Disease free survival analysis were applied through the data from our clinical center. (H) The expression of OSBPL10 in HPNE and other pancreatic cancer cell lines.

FIGURE 2

OSBPL10 stimulates proliferation and metastasis. (A–F) Transwell assays were carried out for measuring cell migration in PANC1 (A), PATU8988 (B) and MiaPaCa2 (C) cells. The images and analyses of colony formation assays in PANC1 (D), PATU8988 (E), and MiaPaCa2 (F). (G–I) Transwell assays were performed for assessing cell migration in PANC1 (G), PATU8988 (H) and MiaPaCa2 (I) cells. (J–L) CCK‐8 assays in PANC1 (J), PATU8988 (K), and MiaPaCa2 (L) cells were used for detecting proliferation capacity.

FIGURE 3

OSBPL10 regulates macrophage polarization and cell apoptosis in pancreatic cancer. (A, B) Flow cytometry was carried out for evaluating the percent of M1 (A) and M2 macrophagy (B) in pancreatic tumors. (C, D) The cell apoptosis rates are much higher in knockdown groups compared to NC group in PANC1 and PATU8988 cells. (E) The results of cell apoptosis rate in MiaPaCa2cells.

FIGURE 4

OSBPL10 knockdown makes an inhibition on proliferation and metastasis abilities of cancer cells in vivo. (A, B) PANC1 and PATU8988 cells knocking down OSBPL10 or empty vector were selected for subcutaneous xenograft tumors generation in nude mice. (C) MiaPaCa cells overexpressing OSBPL10 were used for tumors generation in nude mice as well. (D–F) Subcutaneous xenograft tumors weight of PANC1, PATU8988 and MiaPaCa2. (G–I) Subcutaneous xenograft tumors volume treated by PANC1, PATU8988 and MiaPaCa2. (J–M) IHC analysis using these tumor tissues staining with Ki‐67, TUNEL, E‐Cad, N‐Cad antibodies.

FIGURE 5

Hypoxia potentiates the expression of OSBPL10. (A–C) The expression of OSBPL10 and in pancreatic cancer cells treated by hypoxia for 24,48 h in PANC1, PATU8988, and MiaPaCa2 cells. (D) The data from TCGA was applied for analyzing the relationship between OSBPL10 and HIF‐1α. (E, F) The prediction on the sequence of HIF‐1α from JASPAR database. (G–I) ChIP assays were operated for verifying the binding domain of OSBPL10 promter and HRE1 and HRE2 under hypoxia in PANC1, PATU8988, and MiaPaCa2 cells.

FIGURE 6

CNBP directly binds to OSBPL10. (A, B) CoIP was used for confirming the combination of OSBPL10 and CNBP antibodies in PANC1 (A) and PATU8988 (B) cells. (C, D) The correlation between OSBPL10 and CNBP was measured by data from TCGA and our center.

FIGURE 7

CNBP stimulates the malignant biological behaviors of pancreatic cancer. (A, B) The differential concentrations of IL‐6 in different groups. (C, D) The CCK‐8 assays were operated in PANC1 (C) and PATU8988 (D) cells for detecting cell viability. (E, F) Transwell assays were carried out for measuring cell migration in PANC1 and PATU898 cells. (G, H) The corresponding results of colony Wound Healing assays in PANC1 and PATU8988 cells.

FIGURE 8

CNBP and immune microenvironment of M1 and M2 macrophagy.

FIGURE 9

Single‐cell analysis. (A) The chart illustrates the proportional representation of all cellular subtypes. (B) Bar graphs show the distribution of cellular subgroups for each pancreatic cancer patient. (C, D) UMAP visualization demonstrates the clustering of cellular subpopulations. (E) The interaction counts among cellular subgroups. (F) The Interactions among different cellular subgroups in pancreatic cancer. (G, H) Distribution patterns of genes OSBPL10, CNBP, HIF‐1α, TP53, Bax, CDK4, CDK6 between different cells are depicted.

FIGURE 10

Analysis of GO terms in pancreatic cancer patients. (A, B) Upregulated and downregulated genes in GOMF. (C, D) Upregulated and downregulated gene sets in GOBP. (E, F) Upregulated and downregulated gene sets in IMMUNOLOGIC.

FIGURE 11

Signal pathway regulation. (A–C) The expression of key Hypoxia, TP53, PI3K‐AKT pathways in various cell subpopulations of pancreatic tumor. (D) Transcription factor regulation of pancreatic cancer cells.