The novel driver gene ASAP2 is a potential druggable target in pancreatic cancer

Abstract

Targeting mutated oncogenes is an effective approach for treating cancer. The 4 main driver genes of pancreatic ductal adenocarcinoma (PDAC) are KRAS, TP53, CDKN2A, and SMAD4, collectively called the "big 4" of PDAC, however they remain challenging therapeutic targets. In this study, ArfGAP with SH3 domain, ankyrin repeat and PH domain 2 (ASAP2), one of the ArfGAP family, was identified as a novel driver gene in PDAC. Clinical analysis with PDAC datasets showed that ASAP2 was overexpressed in PDAC cells based on increased DNA copy numbers, and high ASAP2 expression contributed to a poor prognosis in PDAC. The biological roles of ASAP2 were investigated using ASAP2-knockout PDAC cells generated with CRISPR-Cas9 technology or transfected PDAC cells. In vitro and in vivo analyses showed that ASAP2 promoted tumor growth by facilitating cell cycle progression through phosphorylation of epidermal growth factor receptor (EGFR). A repositioned drug targeting the ASAP2 pathway was identified using a bioinformatics approach. The gene perturbation correlation method showed that niclosamide, an antiparasitic drug, suppressed PDAC growth by inhibition of ASAP2 expression. These data show that ASAP2 is a novel druggable driver gene that activates the EGFR signaling pathway. Furthermore, niclosamide was identified as a repositioned therapeutic agent for PDAC possibly targeting ASAP2.

Keywords: ASAP2; driver gene; drug repositioning; niclosamide; pancreatic cancer.

FIGURE 1

Clinical significance of ASAP2 expression in PDAC. A, DNA copy number variations according to chromosome arm in 184 PDAC tissues from TCGA dataset. B, ASAP2 mRNA expression in PDAC and normal pancreatic tissues in TCGA GSE15471 and GSE28735 datasets. T: tumor tissue, N: normal tissue. C, Immunohistochemical staining for ASAP2 in PDAC tissues and normal tissues that were surgically removed (n = 6). The cells surrounded by the dotted line are normal pancreatic duct epithelial cells. Bar graph represents the percentage of ASAP2‐positive cells in normal and tumor tissues. T: tumor tissue, N: normal tissue. D, Correlation between mRNA expression and DNA copy number of ASAP2 in TCGA and CCLE datasets. R is the Pearson correlation coefficient. E, An integrated view of mRNA expression of ASAP2 and DNA copy number of ASAP2 in 184 PDAC cases from TCGA. Samples are sorted according to ASAP2 mRNA expression level. F, Positions and frequencies of mutations in ASAP2 among PDAC cases in TCGA dataset. Mutation was only observed in 1 case, for a frequency of 0.57%. G, Kaplan‐Meier OS and RFS curves of patients with PDAC according to ASAP2 mRNA expression in TCGA dataset. H, GSEA using TCGA dataset

FIGURE 2

RNA sequencing of ASAP2 knockout cells and wild‐type cells. A, MA plot of differentially expressed genes (DEGs) comparing wild‐type and ASAP2 knockout Panc1 and MiaPaCa2 cells. DEGs are represented as red dots. B, GO analysis of significantly upregulated genes in wild‐type cells. C, GSEA plots for selected hallmark gene sets

FIGURE 3

ASAP2 promotes cell cycle progression. A, Direct sequencing analysis confirmed successful genome editing of ASAP2 exon 1. Approximately 200 nucleotides, including the initiator codon, were deleted (left). RT‐PCR of gene‐targeted ASAP2. WT: 1220 bp, KO: approximately 1000 bp (top right). ASAP2 western blot analysis in ASAP2 knockout cells and wild‐type cells (bottom right). B, Knockout of ASAP2 suspends cell cycle progression of PDAC cells. Cell cycle assay after refeeding of FBS using flow cytometry in wild‐type cells and ASAP2 knockout cells. n.s. not significant; (*) P < .05; (**) P < .01; (***) P < .001

FIGURE 4

ASAP2 is associated with growth of PDAC cells. A, MTT assays using ASAP2 knockout Panc1 and MiaPaCa2 cells. (***) P < .001. B, Colony formation assays using ASAP2 knockout Panc1 and MiaPaCa2 cells. (*) P < .05; (***) P < .001. C, ASAP2 protein expression was assessed by western blot analysis of MiaPaCa2 cells stably overexpressing ASAP2 and control cells. D, MTT assay using MiaPaCa2 cells stably overexpressing ASAP2. (***) P < .001. E, Colony formation assay using MiaPaCa2 cells stably overexpressing ASAP2. (*) P < .05. F, In vivo analysis using a tumor xenograft model. The growth curve of xenograft tumors from MiaPaCa2 cells stably overexpressing ASAP2 (n = 6) and control cells (n = 6) (left). Bar graphs represent the tumor volume, respectively (right). (**) P < .01. G, Immunohistochemical staining for ASAP2 and Ki67 in tumor tissues from control cells and MiaPaCa2 cells stably overexpressing ASAP2 (left). Bar graphs represent the percentage of Ki67‐positive cells in tumor tissues from MiaPaCa2 cells stably overexpressing ASAP2 (n = 6) and control tumor tissues (n = 6) (right). Original magnification, ×600. (***) P < .001. H, ASAP2 protein levels in the indicated cells. ASAP2 protein expression using western blot analysis in these rescued cells. I, MTT assay using indicated cells

FIGURE 5

ASAP2 promotes cell migration and positively regulates the EGFR/ERK signaling pathway in PDAC cells. A, Wound healing assays using ASAP2 knockout Panc1 and MiaPaCa2 cells. The migrated distance was quantified by measuring the difference at 0, 24, and 48 h and was normalized to 0 h. n.s. not significant; (*) P < .05; (**) P < .01; (***) P < .001. B, Wound healing assays using MiaPaCa2 cells stably overexpressing ASAP2. The migration distance was quantified by measuring the difference at 0, 24, and 48 h and was normalized to 0 h. n.s. not significant; (***) P < .001. C, ASAP2 overexpressing or control MiaPaCa2 cells and ASAP2 knockout or wild‐type Panc1 and MiaPaCa2 cells were analyzed for levels of phosphorylated or total EGFR and ERK using western blotting. β‐Actin was used as the loading control for relative protein quantification. The normalized intensities of each protein and ratios of pEGFR/EGFR and pERK/ERK are shown

FIGURE 6

Prediction of potential ASAP2 inhibitors. A, B, Niclosamide was identified as a compound for which the gene expression profiles induced by compound administration and ASAP2 knockout were correlated and the gene expression profiles induced by compound administration and ASAP2 overexpression were inversely correlated

FIGURE 7

Niclosamide inhibited the growth of PDAC cells in vitro and in vivo. A, B, Sensitivity of PDAC cells to niclosamide. Cell viability was measured using MTT assays in PDAC cells treated with the indicated concentrations of niclosamide for 48 h. A, MTT assay using Panc1 and MiaPaCa2 cells. B, MTT assay using MiaPaCa2 cells stably overexpressing ASAP2 and control cells. C, Panc1 and MiaPaCa2 tumor growth in control (n = 4) and niclosamide‐treated mice (n = 4). Bar graphs represent the tumor volume, respectively. (*) P < .05. D, Subcutaneous tumors from control and niclosamide‐treated mice. E, Body weights of control and niclosamide‐treated mice during the entire experimental period. F, Immunohistochemical staining for ASAP2 and Ki67 in tumor tissues from control and niclosamide‐treated mice. Bar graphs represent the percentage of Ki67‐positive cells in tumor tissues from control (n = 4) and niclosamide‐treated mice (n = 4). Original magnification, ×600. (***) P < .001. G, Panc1 and MiaPaCa2 cells treated with the indicated concentrations of niclosamide for 48 h were analyzed to determine the levels of phosphorylated or total ERK and the expression of ASAP2 using western blotting. β‐Actin was used as the loading control for relative protein quantification. The normalized intensities of each protein and ratios of pEGFR/EGFR and pERK/ERK are shown