Characterization of DNA topoisomerase I in three SN-38 resistant human colon cancer cell lines reveals a new pair of resistance-associated mutations.

Abstract

BACKGROUND:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSIONS:

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.

• Authors: Niels Frank Jensen, Keli Agama, Amit Roy, David Hersi Smith, Thomas D Pfister, Maria Unni Rømer, Hong-Liang Zhang, James H Doroshow, Birgitta R Knudsen, Jan Stenvang, Nils Brünner, Yves Pommier
• Journal: Journal of experimental & clinical cancer research : CR (J Exp Clin Cancer Res) Vol. 35 Pg. 56 (Mar 31 2016) ISSN: 1756-9966 [Electronic] England
• PMID: 27029323 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• 7-nitro-1H-indole-2-carboxylic acid (4-(1-(guanidinohydrazone)ethyl)phenyl)amide
• Benzodioxoles
• Guanidines
• Hydrazones
• Isoquinolines
• NSC 725776
• Epirubicin
• Etoposide
• Irinotecan
• DNA Topoisomerases, Type I
• TOP1 protein, human
• Camptothecin

Topics

• Benzodioxoles (pharmacology)
• Binding Sites
• Camptothecin (analogs & derivatives, pharmacology)
• Cell Line, Tumor
• Chromosome Deletion
• Colonic Neoplasms (genetics, metabolism)
• DNA Topoisomerases, Type I (genetics, metabolism)
• Drug Resistance, Neoplasm
• Epirubicin (pharmacology)
• Etoposide (pharmacology)
• Gene Dosage
• Guanidines (pharmacology)
• HCT116 Cells
• HT29 Cells
• Humans
• Hydrazones (pharmacology)
• Irinotecan
• Isoquinolines (pharmacology)
• Mutation