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

Abstract

BackgroundDNA 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.MethodsThree 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.ResultsTwo 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.ConclusionsThis 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.

Assessing chemotherapeutic effectiveness using a paper-based tumor model

“…While previous studies have already characterized the IC 50 of SN-38 for HCT116 cells in 2D cultures, these values range three orders of magnitude (50 - 0.5 nM) making comparisons with literature values difficult. 34,51 The IC 50 value for the HCT116 cells in the paper scaffolds (0.014 ± 0.006 μM) was much higher than for monolayer cultures (0.005 ± 0.001 μM). These data are summarized in Figure S5.…”

“…9,11,19,34 Engineering the HCT116 cells to constitutively express mCherry allowed us to relate fluorescence intensity to viability, as mCherry fluorescence intensity and cell number are linearly related for HCT116 mCHR cells imaged on a Typhoon scanner (Fig. S2).…”

<p>Irinotecan Induces Autophagy-Dependent Apoptosis and Positively Regulates ROS-Related JNK- and P38-MAPK Pathways in Gastric Cancer Cells</p>

“…9,10 Nonetheless, the therapeutic effect of IRI is still hampered by acquired chemotherapy resistance. 11 Therefore, it is necessary to devise a promising and safe treatment regimen to overcome drug resistance and reduce drug side effects in gastric cancer.…”

Predictive biomarkers for sacituzumab govitecan efficacy in Trop-2-expressing triple-negative breast cancer

“…Acquired resistance can be multifaceted because a patient's tumor may have developed multidrug resistance through ABCG2 or through mutation of TOP1 [46,47]. Loss of SN-38 activity through the tumor cell's lack of dependence on TOP1 would make further treatment with SG futile [46,47]. Further, we had previously demonstrated that SG is unable to overcome SN-38 resistance mediated through the ABCG2 multidrug resistance pump [48].…”