pubmed.ncbi.nlm.nih.gov

PAICS, a De Novo Purine Biosynthetic Enzyme, Is Overexpressed in Pancreatic Cancer and Is Involved in Its Progression - PubMed

PAICS, a De Novo Purine Biosynthetic Enzyme, Is Overexpressed in Pancreatic Cancer and Is Involved in Its Progression

Sumit Agarwal et al. Transl Oncol. 2020 Jul.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with an extremely poor prognosis. There is an urgent need to identify new therapeutic targets and also understand the mechanism of PDAC progression that leads to aggressiveness of the disease. To find therapeutic targets, we analyzed data related to PDAC transcriptome sequencing and found overexpression of the de novo purine metabolic enzyme phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS). Immunohistochemical analysis of PDAC tissues showed high expression of the PAICS protein. To assess the biological roles of PAICS, we used RNA interference and knock down of its expression in PDAC cell lines that caused a reduction in PDAC cell proliferation and invasion. Furthermore, results of chorioallantoic membrane assays and pancreatic cancer xenografts demonstrated that PAICS regulated pancreatic tumor growth. Our data also showed that, in PDAC cells, microRNA-128 regulates and targets PAICS. PAICS depletion in PDAC cells caused upregulation in E-cadherin, a marker of the epithelial-mesenchymal transition. In PDAC cells, a BET inhibitor, JQ1, reduced PAICS expression. Thus, our investigations show that PAICS is a therapeutic target for PDAC and, as an enzyme, is amenable to targeting by small molecules.

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1
Figure 1

Overexpression of PAICS in PDAC. Oncomine gene expression profiling showing PAICS expression from PDAC tissues as reported by (A) Segara et al., 2005 [36] and (B) Pei et al., 2009 [37]. (C) PAICS expression, determined by use of UALCAN [39], for PDACs compared to normal pancreatic tissue. (D) Stage-wise PAICS expression in pancreatic tumors. (E) Kaplan-Meier analysis of survival time for PDACs determined by use of UALCAN. (F) Representative images showing PAICS expression, assessed by immunohistochemistry, in PDACs. Hematoxylin was used for nuclear staining.

Figure 2
Figure 2

PAICS knockdown reduced PDAC cell proliferation and colony formation. (A) Immunoblot analyses of PAICS protein levels in PANC-1 and S2VP10 cells treated with PAICS shRNA compared to levels in cells treated with NT shRNA. β-Actin was used as a loading control. (B) PAICS knockdown reduced cell proliferation at 2, 4, and 6 days. (C) Colony formation was suppressed at 10 days after seeding of 1000 shRNA-treated cells in six-well plates. Statistically significant differences (*P < .05) are indicated by an asterisk (*).

Figure 3
Figure 3

PAICS knockdown suppressed invasion and motility of PDAC cells. (A) Representative images of PDAC cells with PAICS knockdown showing less invasion through Transwell Matrigel membranes compared to cells treated with NT shRNA. (B) Representative images showing wound healing assays of PANC-1 and S2VP10 cells lacking PAICS. Images were taken at 0 and 24 hours. Scale bar- 1000 μm. Comparative analysis of wound healing after PAICS knockdown. Asterisk (*) represents statistically significant difference. (C) PDAC cells with reduced PAICS formed smaller spheroids compared with cells treated with NT shRNA as shown by phase-contrast microscopy and GFP (due to the presence of pGreenPuro shRNA expression lentivector for GFP) images. Scale bar- 1000 μm. Histographs showed the spheroid formation capacity of PAICS shRNA relative to NT shRNA (*P < .05).

Figure 4
Figure 4

miR-128 targets and regulates PAICS in PDAC. (A) Pictorial diagram of predicted binding sites of miR-128 with the 3′-UTR of PAICS as shown by Targetscan. (B) In S2VP10 PDAC cells, NT-pre-miR or miR-128 was co-transfected with luciferase constructs of either PAICS-3′UTR wild type or mutant. (C) NT-pre-miR, pre-miR-128, 141, or 146 was transfected into S2VP10 cells, and immunoblot analyses were performed for PAICS protein expression. β-Actin was used as an internal control. Ectopic expression of pre-miR-128 compared to NT-pre-miR largely canceled S2VP10 expansion in cell proliferation (D), colony formation (E) and invasion (F) assays.

Figure 5
Figure 5

PAICS regulates apoptosis, the EMT, and pancreatic tumor growth. Immunoblot analyses of stable PAICS knockdown S2VP10 cells showing (A) PARP-1 cleavage, (B) E-cadherin levels, and (C) immunofluorescence staining of E-cadherin (red) in these cells. Nuclear staining was accomplished with DAPI (blue). Merged images are shown. (D) Immunoblot analysis of JQ1-treated PDAC cells for PAICS and c-MYC. NSG mice were implanted in right dorsal flank with 1 million S2VP10 pancreatic cancer cells. (E) Representative histogram to show tumors weight of control NT shRNA (n = 7) and PAICS shRNA1 (n = 7) xenograft groups, mean ± SD; *P < .01. (F) Immunoblot analysis of S2VP10 xenografts treated with NT shRNA or PAICS shRNA.

Similar articles

Cited by

References

    1. Siegel R.L., Miller K.D., Jemal A. Cancer statistics, 2019. CA Cancer J. Clin. 2019;69:7–34. doi: 10.3322/caac.21551. - DOI - PubMed
    1. Kosmidis C., Sapalidis K., Kotidis E., Mixalopoulos N., Zarogoulidis P., Tsavlis D., Baka S., Man Y.G., Kanellos J. Pancreatic cancer from bench to bedside: molecular pathways and treatment options. Ann Transl Med. 2016;4:165. doi: 10.21037/atm.2016.05.11. - DOI - PMC - PubMed
    1. Quaranta V., Rainer C., Nielsen S.R., Raymant M.L., Ahmed M.S., Engle D.D., Taylor A., Murray T., Campbell F., Palmer D.H., Tuveson D.A., Mielgo A., Schmid M.C. Macrophage-derived granulin drives resistance to immune checkpoint inhibition in metastatic pancreatic cancer. Cancer Res. 2018;78:4253–4269. doi: 10.1158/0008-5472.CAN-17-3876. - DOI - PMC - PubMed
    1. Teague A., Lim K.H., Wang-Gillam A. Advanced pancreatic adenocarcinoma: a review of current treatment strategies and developing therapies. Ther Adv Med Oncol. 2015;7:68–84. doi: 10.1177/1758834014564775. - DOI - PMC - PubMed
    1. Wang J.P., Wu C.Y., Yeh Y.C., Shyr Y.M., Wu Y.Y., Kuo C.Y., Hung Y.P., Chen M.H., Lee W.P., Luo J.C., Chao Y., Li C.P. Erlotinib is effective in pancreatic cancer with epidermal growth factor receptor mutations: a randomized, open-label, prospective trial. Oncotarget. 2015;6:18162–18173. doi: 10.18632/oncotarget.4216. - DOI - PMC - PubMed