Cancers (Basel). 2020;12(2):cancers12020334.

TDP1 and TOP1 Modulation in Olaparib-Resistant Cancer Determines the Efficacy of Subsequent Chemotherapy.

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제1저자 김진원, 민아름 · 교신저자 임석아

PMID 32028591 PMC7072281 DOI 10.3390/cancers12020334 IF 4.8 혈액종양내과

소속 (7)

Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, Korea.
Division of Hematology and Medical Oncology, Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam 13620, Korea.
Translational Medicine, Seoul National University College of Medicine, Seoul 03080, Korea.
Cancer Research Institute, Seoul National University, Seoul 03080, Korea.
Biomedical Research Institute, Seoul National University Hospital, Seoul 03080, Korea.
Division of Hematology and Medical Oncology, Department of Internal Medicine, Seoul National University Hospital, Seoul 03080, Korea.
Department of Internal Medicine, Chung-Ang University College of Medicine, Seoul 06973, Korea.

Abstract

The aim of this study was to elucidate the carryover effect of olaparib to subsequent chemotherapy and its underlying mechanisms. We generated olaparib-resistant SNU-484, SNU-601, SNU-668, and KATO-III gastric cancer cell lines and confirmed their resistance by cell viability and colony forming assays. Notably, olaparib-resistant cell lines displayed cross-resistance to cisplatin except for KATO-III. Inversely, olaparib-resistant SNU-484, SNU-668, and KATO-III were more sensitive to irinotecan than their parental cells. However, sensitivity to paclitaxel remained unaltered. There were compensatory changes in the ATM/ATR axis and p-Chk1/2 protein expression. ERCC1 was also induced in olaparib-resistant SNU-484, SNU-601, and SNU-668, which showed cross-resistance to cisplatin. Olaparib-resistant cells showed tyrosyl-DNA phosphodiesterase 1 (TDP1) downregulation with higher topoisomerase 1 (TOP1) activity, which is a target of irinotecan. These changes of TOP1 and TDP1 in olaparib-resistant cells was confirmed as the underlying mechanism for increased irinotecan sensitivity through manipulated gene expression of TOP1 and TDP1 by specific plasmid transfection and siRNA. The patient-derived xenograft model established from the patient who acquired resistance to olaparib with BRCA2 mutation showed increased sensitivity in irinotecan. In conclusion, the carryover effects of olaparib to improve antitumor effect of subsequent irinotecan were demonstrated. These effects should be considered when determining the subsequent therapy with olaparib.

Keywords

TDP1TOP1 activitycarryover effectirinotecanolaparib

Figures

**Figure 1** The sensitivity of chemotherapeutic agents was altered in olaparib-resistant cells. Cell viability was calculated through counting cell colonies by using GELCOUNT in SNU-484 (A), SNU-601 (B), SNU-668 (C), and KATO-III (D) cells. * indicates p < 0.001.

**Figure 2** The expressions of DNA-damage response proteins were altered after acquisition of olaparib resistance. Western blot analysis for p-ATR, ATR, p-ATM, ATM, p-Chk1, Chk1, p-Chk2, Chk2, and ERCC1 was conducted to evaluate the altered expression after acquisition of resistance to olaparib. There were compensatory changes in the DNA-damage response proteins.

**Figure 3** The nuclei were larger in olaparib-resistant cells than in parental cells. The degree of DNA condensation was examined by 4′,6-diamidino-2-phenylindole (DAPI) staining and confocal laser microscopy. Representative images are presented (80× original magnification). Scale bars represent 50 μm. In olaparib-resistant cells, increased nucleus indicates a relaxation of DNA condensation (A). The proportion of cells with a DNA content of more than 4n was calculated by flow cytometry with PI staining. The increased nuclear size of olaparib-resistant cells was not related to aneuploidy (B).

**Figure 4** Topoisomerase 1 (TOP1) activity was increased and tyrosyl-DNA phosphodiesterase 1 (TDP1) expression was downregulated in olaparib-resistant cells. TOP1 activity was increased in all olaparib-resistant cells compared with parental cells (A), although the protein expression of TOP1 was not altered (B). TDP1 protein expression was downregulated in olaparib-resistant SNU-484, SNU-668, and KATO-III cells compared with their parental cells (B). * indicates p < 0.001.

**Figure 5** The sensitivities to irinotecan with modulating TOP1 and TDP1 expressions were evaluated by MTT assay. TOP1-siRNA mix, TDP1-siRNA mix, and non-targeting siRNA (160nM each) were transfected into olaparib-resistant cells or parental cells, and then treated with irinotecan for 5 d. Cell viability percentages were then measured by MTT assay, and TOP1 or TDP1 silencing was determined by western blotting (A,B). pCMV-tag2A-TOP1 or pCMV-tag2A-TDP1 and/or siTOP1 or siTDP1 were transfected into parental or olaparib-resistant cells. The sensitivity to irinotecan was calculated by MTT assay and the efficacy of transfection was confirmed by western blotting (C,D).

**Figure 6** Parental cells were transfected with siRNA targeting TDP1 and/or pCMV-tag2A-TOP1 for 5 d. The size of nuclei was measured by DAPI staining and confocal laser microscopy. Representative images are presented (80× original magnification). Scale bars represent 50 μm.

**Figure 7** Irinotecan impeded tumor growth in a BRCA2 mutant PDX model with acquisition of olaparib resistance. (A) PDX model were treated with 50 mg/kg olaparib or 10 mg/kg irinotecan for 21 days (n = 4/group). The mean of tumor volumes was presented in a graph with the standard deviation (SD). * indicates p < 0.001 versus vehicle and olaparib group. (B) Changes in body weight were measured every three days for 21 days.

그림 출처: PubMed Central 공개 논문 (PMC7072281)

Conflict of interest statement

Seock-Ah Im is a recipient of research funds from AstraZeneca Inc. and Pfizer and has consultant and advisory role for AstraZeneca, Hanmi Corp, Novartis, Pfizer, Roche, and Spectrum. The other authors declared that they have no conflict of interest.

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