Discovery of the inhibitor of DNA binding 1 as a novel marker for radioresistance in pancreatic cancer using genome-wide RNA-seq

Abstract

Purpose/Objective(s): Discovery of genetic drivers of radioresistance is critical for developing novel therapeutic strategies to combine with radiotherapy of radioresistant PDAC. In this study, we used genome-wide RNA-seq to identify genes upregulated in generated radioresistant PDAC cell lines and discovered the Inhibitor of DNA Binding 1 (ID1) gene as a potential regulator of radioresistance in PDAC.

Materials/Methods: Radioresistant clones of the PDAC cell lines MIA PaCa-2 and PANC-1 were generated by delivering daily ionizing irradiation (IR) (2 Gy/day) in vitro over two weeks (total 20 Gy) followed by standard clonogenic assays following one week from the end of IR. The generated RR and parental cell lines were submitted for RNA-seq analysis to identify differentially expressed genes. The Limma R package was used to calculate differential expression among genes. Log2 fold change values were calculated for each sample compared to the control. Genes with an absolute fold change > 1 were considered significant. RNA sequencing expression data from the Cancer Genome Atlas (TCGA) database was analyzed through the online databases GEPIA, cBioPortal, and the Human Protein Atlas.

Results: Following exposure to two weeks of 2 Gy daily IR in vitro, the two PDAC cell lines showed significantly greater clonogenic cell survival than their parental cell lines, indicating enhanced RR in these cells. RNA-seq analysis comparing parental and RR cell lines found upregulated seven genes (TNS4, ZDHHC8P1, APLNR, AQP3, SPP1, ID1, ID2) and seven genes downregulated (PTX3, ITGB2, EPS8L1, ALDH1L2, KCNT2, ARHGAP9, IFI16) in both RR cell lines. Western blotting confirmed increased expression of the ID1 protein in the RR cell lines compared to their parental cell lines. We found that ID1 mRNA was significantly higher in PDAC tumors compared to matched normal and high ID1 expression correlated with significantly worse disease-free survival (DFS) in PDAC patients (HR = 2.2, log rank P = 0.009). ID1 mRNA expression was also strongly correlated in tumors with TP53 mutation, a known driver of radioresistance.

Conclusion: Our analysis indicates a novel role of ID1 in PDAC radioresistance. ID1 expression is higher in tumor tissue compared to normal, and high expression correlates with both worse DFS and association with the TP53 mutation, suggesting that targeting ID1 prior to IR is an attractive strategy for overcoming radioresistance in PDAC.

Keywords

Pancreatic cancer / radiation resistance / ID1 / RNA-seq

References

[1] Siegel RL,Jemal A.Cancer statistics, 2016.CA Cancer J Clin2016;66:7-30

[2] Rahib L,Matrisian LM.Estimated projection of US cancer incidence and death to 2040.JAMA Netw Open2021;4:e214708 PMCID:PMC8027914

[3] Khorana AA,Berlin J.Potentially curable pancreatic cancer: American society of clinical oncology clinical practice guideline.J Clin Oncol2016;34:2541-56

[4] Hong SB,Kim JH.Pancreatic cancer CT: prediction of resectability according to NCCN criteria.Radiology2018;289:710-8

[5] Hall WA,Nickleach D.The influence of radiation therapy dose escalation on overall survival in unresectable pancreatic adenocarcinoma.J Gastrointest Oncol2014;5:77-85 PMCID:PMC3999628

[6] Wolfe AR.Altering the response to radiation: radiosensitizers and targeted therapies in pancreatic ductal adenocarcinoma: preclinical and emerging clinical evidence.Ann Pancreat Cancer2018;1:26-26 PMCID:PMC7351115

[7] Treviño JG,Kunigal S.Nicotine induces inhibitor of differentiation-1 in a Src-dependent pathway promoting metastasis and chemoresistance in pancreatic adenocarcinoma.Neoplasia2012;14:1102-14 PMCID:PMC3540937

[8] Huang YH,Chen F.ID1 mediates escape from TGFβ tumor suppression in pancreatic cancer.Cancer Discov2020;10:142-57 PMCID:PMC6954299

[9] Liao Y,Shi W.featureCounts: an efficient general purpose program for assigning sequence reads to genomic features.Bioinformatics2014;30:923-30

[10] Robinson MD.A scaling normalization method for differential expression analysis of RNA-seq data.Genome Biol2010;11:R25 PMCID:PMC2864565

[11] Ritchie ME,Wu D.limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res2015;43:e47 PMCID:PMC4402510

[12] Cerami E,Dogrusoz U.The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.Cancer Discov2012;2:401-4 PMCID:PMC3956037

[13] Tang Z,Kang B,Li C.GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses.Nucleic Acids Res2017;45:W98-W102 PMCID:PMC5570223

[14] Uhlén M,Hallström BM.Proteomics. Tissue-based map of the human proteome.Science2015;347:1260419

[15] Bae IH,Oh S.Mannosylerythritol lipids ameliorate ultraviolet A-induced aquaporin-3 downregulation by suppressing c-Jun N-terminal kinase phosphorylation in cultured human keratinocytes.Korean J Physiol Pharmacol2019;23:113-20 PMCID:PMC6384198

[16] Cook PJ,Kingsley PJ.Cox-2-derived PGE2 induces Id1-dependent radiation resistance and self-renewal in experimental glioblastoma.Neuro Oncol2016;18:1379-89 PMCID:PMC5035519

[17] Gujar AD,Mao DD.An NAD+-dependent transcriptional program governs self-renewal and radiation resistance in glioblastoma.Proc Natl Acad Sci U S A2016;113:E8247-56 PMCID:PMC5187672

[18] Castañon E,López I.Id1 and Id3 co-expression correlates with clinical outcome in stage III-N2 non-small cell lung cancer patients treated with definitive chemoradiotherapy.J Transl Med2013;11:13 PMCID:PMC3567999

[19] Guo Q,Mao Q.ID1 affects the efficacy of radiotherapy in glioblastoma through inhibition of DNA repair pathways.Med Oncol2013;30:325

[20] Baghdoyan S,Castel D.Id2 reverses cell cycle arrest induced by {gamma}-irradiation in human HaCaT keratinocytes.J Biol Chem2005;280:15836-41

[21] Sun G,Liu W.SPP1 regulates radiotherapy sensitivity of gastric adenocarcinoma via the wnt/beta-catenin pathway.J Oncol2021;2021:1642852 PMCID:PMC8337119

[22] Lee JM.p53 mutations increase resistance to ionizing radiation.Proc Natl Acad Sci U S A1993;90:5742-6 PMCID:PMC46798

[23] Kong X,Wang Z.Relationship between p53 status and the bioeffect of ionizing radiation.Oncol Lett2021;22:661 PMCID:PMC8299044

[24] Crane CH.Hypofractionated ablative radiotherapy for locally advanced pancreatic cancer.J Radiat Res2016;57 Suppl 1:i53-7 PMCID:PMC4990109

[25] Hassanzadeh C,Bommireddy A.Ablative five-fraction stereotactic body radiation therapy for inoperable pancreatic cancer using online MR-guided adaptation.Adv Radiat Oncol2021;6:100506 PMCID:PMC7897757

[26] Krishnan S,Suh Y.Focal radiation therapy dose escalation improves overall survival in locally advanced pancreatic cancer patients receiving induction chemotherapy and consolidative chemoradiation.Int J Radiat Oncol Biol Phys2016;94:755-65 PMCID:PMC4792191

[27] Henke L,Robinson C.Phase I trial of stereotactic MR-guided online adaptive radiation therapy (SMART) for the treatment of oligometastatic or unresectable primary malignancies of the abdomen.Radiother Oncol2018;126:519-26

[28] Souchek JJ,Lin C.Unbiased analysis of pancreatic cancer radiation resistance reveals cholesterol biosynthesis as a novel target for radiosensitisation.Br J Cancer2014;111:1139-49 PMCID:PMC4453840

[29] Ogawa K,Mimori K.Differential gene expression profiles of radioresistant pancreatic cancer cell lines established by fractionated irradiation.Int J Oncol2006;

[30] Wiechmann S,Schilling D.Radiosensitization by kinase inhibition revealed by phosphoproteomic analysis of pancreatic cancer cells.Mol Cell Proteomics2020;19:1649-63 PMCID:PMC8014995

[31] Schröter P,Osen W.Radiation-induced alterations in immunogenicity of a murine pancreatic ductal adenocarcinoma cell line.Sci Rep2020;10:686 PMCID:PMC6971029

[32] Benezra R,Lockshon D,Weintraub H.The protein Id: a negative regulator of helix-loop-helix DNA binding proteins.Cell1990;61:49-59

[33] Maruyama H,Wildi S.Id-1 and Id-2 are overexpressed in pancreatic cancer and in dysplastic lesions in chronic pancreatitis.The American Journal of Pathology1999;155:815-22 PMCID:PMC1866883

[34] Qian Y.ID1, inhibitor of differentiation/DNA binding, is an effector of the p53-dependent DNA damage response pathway.J Biol Chem2008;283:22410-6 PMCID:PMC2504896