Tetraploidy-Associated Genetic Heterogeneity Confers Chemo-Radiotherapy Resistance to Colorectal Cancer Cells

Abstract

Tetraploidy, or whole-genome duplication, is a common phenomenon in cancer and preludes chromosome instability, which strongly correlates with disease progression, metastasis, and treatment failure. Therefore, it is reasonable to hypothesize that tetraploidization confers multidrug resistance. Nevertheless, the contribution of whole-genome duplication to chemo-radiotherapy resistance remains unclear. Here, using isogenic diploid and near-tetraploid clones from three colorectal cancer cell lines and one non-transformed human epithelial cell line, we show a consistent growth impairment but a divergent tumorigenic potential of near-tetraploid cells. Next, we assessed the effects of first-line chemotherapeutic drugs, other commonly used agents and ionizing radiation, and found that whole-genome duplication promoted increased chemotherapy resistance and also conferred protection against irradiation. When testing the activation of apoptosis, we observed that tetraploid cells were less prone to caspase 3 activation after treatment with first-line chemotherapeutic agents. Furthermore, we found that pre-treatment with ataxia telangiectasia and Rad3 related (ATR) inhibitors, which targets response to replication stress, significantly enhanced the sensitivity of tetraploid cells to first-line chemotherapeutic agents as well as to ionizing radiation. Our findings provide further insight into how tetraploidy results in greater levels of tolerance to chemo-radiotherapeutic agents and, moreover, we show that ATR inhibitors can sensitize near-tetraploid cells to commonly used chemo-radiotherapy regimens.

Keywords: chemotherapy; colorectal cancer; drug resistance; radiotherapy; tetraploidy; whole-genome duplication.

Figure 1

Proliferative and tumorigenic capacities of 2N and 4N cells. Quantification of colony formation assay by (A) number of colonies and (B) area of occupancy in one 2N and two 4N clones derived from the different cell lines. Please note that analysis of the number of colonies for the RKO cell line was not possible due to its dispersed phenotype in the colony-forming assay. Mean of at least two replicates from three independent experiments with SD is shown. Measurements of tumor volumes after injecting (C–E) CRC and (F) non-transformed RPE1 2N and 4N cells subcutaneously in athymic nude mice. A.u., arbitrary units. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2

Cellular viability response upon treatment with first-line chemotherapeutic agents. Dose-response curves for increasing concentrations of 5-fluorouracil (A–C), oxaliplatin (D–F) and the combination of both compounds (G–I) in one 2N and two 4N clones of DLD-1, RKO and SW837 CRC cell lines. Each cellular viability was normalized based on its corresponding non-treated counterpart. Fitted curves for two replicates from three independent experiments are plotted. ANOVA test with post-hoc Tukey was performed to test significance. Data are reported as means ± SD. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 3

Clonogenic capacity of 2N and 4N cells after treatment with first-line chemotherapeutic agents. Graphs showing the clonogenic capacity in one 2N and two 4N clones of DLD-1 (A), RKO (B), SW837 (C) and RPE1 (D) cells treated with 5 µM 5-fluorouracil, 5 µM oxaliplatin or the combination of both compounds. Clonogenic capacity was assessed by area of occupancy over the untreated control at 14 to 21 days after treatment. Data are reported as means ± SD (n = 6). n.s., not significant; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.

Figure 4

Caspase 3 activation assay in 2N and 4N cells upon treatment with FOLFOX. Quantification of caspase3 activity as a surrogate marker of apoptosis in 2N and 4N clones of (A) DLD-1, (B) RKO, (C) SW837 and (D) RPE1 cell lines in both non-treated and after treatment (25 µM of 5-fluorouracil and 20 µM of oxaliplatin) conditions. For all these analyses, caspase3 activity was calculated as pmol of pNA/min·mg of protein at 24 h after treatment. Three independent experiments were performed for each cell line. Data are represented as means ± SD. n.s., not significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 5

Cellular viability response upon increasing doses of ionizing radiation. Graphs depicting cellular viability curves of one 2N clone and two 4N clones of CRC cells (A) DLD-1, (B) RKO, (C) SW837 and (D) the non-transformed cell line RPE1 in response to irradiation at the indicated doses. Each cellular viability was normalized based on its corresponding non-irradiated counterpart. Fitted curves for two replicates from three independent experiments are plotted. ANOVA test with post-hoc Tukey was performed to test significance. Data are reported as means ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 6

Effect of FOLFOX and ionizing radiation upon treatment of 2N and 4N cells with the ATR inhibitor VE-821. Histograms showing cell viability in response to VE-821 treatment prior to FOLFOX treatment (A–C) or ionizing radiation (D,E). One 2N and two 4N clones of (A) DLD-1, (B) RKO and (C) SW837 were treated with FOLFOX (25 µM 5-fluorouracil and 20 µM oxaliplatin) for 72 h, when indicated 10 µM VE-821 was added for 24 h prior to FOLFOX treatment. Same clones of (D) DLD-1 and (E) RKO were irradiated with a dose of 10 Gy, 10 µM VE-821 was added for 24 h before irradiation when indicated. Each cellular viability was normalized based on its corresponding non-treated or non-irradiated counterpart. Mean of at least three independent experiments with SD is shown. n.s., not significant; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.