Jensen Niels Frank, Agama Keli, Roy Amit, Smith David Hersi, Pfister Thomas D, Rømer Maria Unni, Zhang Hong-Liang, Doroshow James H, Knudsen Birgitta R, Stenvang Jan, Brünner Nils, Pommier Yves
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, Section for Molecular Disease Biology, University of Copenhagen, Strandboulevarden 49, DK-2100, Copenhagen, Denmark.
National Institutes of Health, National Cancer Institute, Center for Cancer Research, Laboratory of Molecular Pharmacology, 37 Convent Drive, Building 37, Room 5068, Bethesda, MD, 20892-4255, USA.
J Exp Clin Cancer Res. 2016 Mar 31;35:56. doi: 10.1186/s13046-016-0335-x.
DNA topoisomerase I (Top1) is a DNA unwinding protein and the specific target of the camptothecin class of chemotherapeutic drugs. One of these, irinotecan, acting through its active metabolite SN-38, is used in the treatment of metastatic colorectal cancer. However, resistance to irinotecan represents a major clinical problem. Since molecular alterations in Top1 may result in resistance to irinotecan, we characterized Top1 in three human colon cancer cell lines with acquired resistance to SN-38.
Three SN-38 resistant (20-67 fold increased resistance) cell lines were generated and compared to wild-type parental cells with regards to: TOP1 gene copy number and gene sequence, Top1 expression (mRNA and protein), Top1 enzymatic activity in the absence and presence of drug, and Top1-DNA cleavage complexes in drug treated cells. TOP1 mutations were validated by PCR using mutant specific primers. Furthermore, cross-resistance to two indenoisoquinoline Top1-targeting drugs (NSC 725776 and NSC 743400) and two Top2-targeting drugs (epirubicin and etoposide) was investigated.
Two of three SN-38 resistant cell lines carried TOP1 gene copy number aberrations: A TOP1 gene copy gain and a loss of chromosome 20, respectively. One resistant cell line harbored a pair of yet unreported TOP1 mutations (R364K and G717R) in close proximity to the drug binding site. Mutant TOP1 was expressed at a markedly higher level than wild-type TOP1. None or very small reductions were observed in Top1 expression or Top1 activity in the absence of drug. In all three SN-38 resistant cell lines Top1 activity was maintained in the presence of high concentrations of SN-38. None or only partial cross-resistance were observed for etoposide and epirubicin, respectively. SN-38 resistant cells with wild-type TOP1 remained sensitive to NSC 743400, while cells with mutant TOP1 was fully cross-resistant to both indenoisoquinolines. Top1-DNA cleavage complex formation following drug treatment supported the other findings.
This study adds to the growing knowledge about resistance mechanisms for Top1-targeting chemotherapeutic drugs. Importantly, two yet unreported TOP1 mutations were identified, and it was underlined that cross-resistance to the new indenoisoquinoline drugs depends on the specific underlying molecular mechanism of resistance to SN-38.
DNA拓扑异构酶I（Top1）是一种DNA解旋蛋白，是喜树碱类化疗药物的特异性靶点。其中一种药物伊立替康通过其活性代谢产物SN-38发挥作用，用于治疗转移性结直肠癌。然而，对伊立替康的耐药性是一个主要的临床问题。由于Top1的分子改变可能导致对伊立替康的耐药性，我们对三株对SN-38获得性耐药的人结肠癌细胞系中的Top1进行了特征分析。
构建了三株对SN-38耐药（耐药性增加20-67倍）的细胞系，并与野生型亲本细胞在以下方面进行比较：TOP1基因拷贝数和基因序列、Top1表达（mRNA和蛋白质）、有无药物时的Top1酶活性以及药物处理细胞中的Top1-DNA裂解复合物。使用突变特异性引物通过PCR验证TOP1突变。此外，研究了对两种茚并异喹啉类Top1靶向药物（NSC 725776和NSC 743400）和两种Top2靶向药物（表柔比星和依托泊苷）的交叉耐药性。
三株对SN-38耐药的细胞系中有两株存在TOP1基因拷贝数异常：分别是TOP1基因拷贝数增加和20号染色体缺失。一株耐药细胞系在药物结合位点附近存在一对尚未报道的TOP1突变（R364K和G717R）。突变型TOP1的表达水平明显高于野生型TOP1。在无药物时，未观察到Top1表达或Top1活性有任何降低或仅有非常小的降低。在所有三株对SN-38耐药的细胞系中，在高浓度SN-38存在时Top1活性得以维持。分别观察到对依托泊苷和表柔比星无交叉耐药或仅有部分交叉耐药。具有野生型TOP1的SN-38耐药细胞对NSC 743400仍敏感，而具有突变型TOP1的细胞对两种茚并异喹啉类药物完全交叉耐药。药物处理后Top1-DNA裂解复合物的形成支持了其他研究结果。
本研究增加了对Top1靶向化疗药物耐药机制的认识。重要的是，鉴定出了两个尚未报道的TOP1突变，并强调对新型茚并异喹啉类药物的交叉耐药性取决于对SN-38耐药的具体潜在分子机制。
