Molecular characterisation of isogenic taxane resistant cell lines identify novel drivers of drug resistance

Abstract

Background: Taxanes such as paclitaxel and docetaxel are used successfully to treat breast cancer, usually in combination with other agents. They interfere with microtubules causing cell cycle arrest; however, the mechanisms underlying the clinical effects of taxanes are yet to be fully elucidated.

Methods: Isogenic paclitaxel resistant (PACR) MDA‒MB‒231, paclitaxel resistant ZR75‒1 and docetaxel resistant (DOCR) ZR75‒1 cell lines were generated by incrementally increasing taxane dose in native cell lines in vitro. We used aCGH analysis to identify mechanisms driving taxane resistance.

Results: Taxane resistant cell lines exhibited an 18-170 fold increased resistance to taxanes, with the ZR75-1 resistant cell lines also demonstrating cross resistance to anthracyclines. Paclitaxel treatment of native cells resulted in a G2/M block and a decrease in the G1 phase of the cell cycle. However, in the resistant cell lines, minimal changes were present. Functional network analysis revealed that the mitotic prometaphase was lost in the resistant cell lines.

Conclusion: This study established a model system for examining taxane resistance and demonstrated that both MDR and mitosis represent common mechanism of taxane resistance.

Figure 1

MDA-MB-231 25PACR cells maintained resistance to paclitaxel after prolonged culture without exposure to taxane. MDA-MB-231 25PACR cells were separated into two groups: one maintained and passaged, as normal in the presence of paclitaxel (white bar), the other was maintained and passaged in the absence of drug (grey bar)for a period of six months. The native cells are represented by the black bar. Cells were incubated with varying concentrations of paclitaxel and cell viability determined by CCK-8 assay. The X axis shows the increasing paclitaxel concentration measured in nM. The Y axis represents the percentage of cells with untreated cells being used as a baseline of 100%.

Figure 2

Cell cycle analysis of native and resistant MDA-MB-231 and ZR75-1 by flow cytometry after synchronisation. The native and respective resistant cell lines were treated with 25nM or 50nM of paclitaxel. The DNA content was measured by flow cytometry to determine the distribution of cell in each phase. The histograms demonstrate the cell cycle distribution within the cell population. A MDA-MB-231 native and MDA-MB231 25PACR cells with or without 25nM paclitaxel. B MDA-MB-231 native and MDA-MB231 25PACR cells with or without 50nM paclitaxel. C ZR75-1 native and ZR75-1 25PACR cells with or without 25nM paclitaxel. D ZR75-1 native and ZR75-1 25PACR cells with or without 50nM paclitaxel. E ZR75-1 native and ZR75-1 25DOCR cells with or without 25nM docetaxel. F ZR75-1 native and ZR75-1 50DOCR cells with or without 50nM docetaxel. Standard deviation of three experiments are shown in brackets.

Figure 3

aCGH of taxane resistant cell lines. The plots show Log2Ratios of test to reference signal intensity from BAC clines in an aCGH experiment using DNA from native cells as a reference samples and DNA from resistant cells as a test sample. Navy dots represent BAC clones which remain unchanged, the green dots represent the BAC clones in which there is an area of gain on the genome, and the red dots represent the BAC clones in which there is an area of loss of the genome. The Log2ratio is measured on the Y axis and on the X axis the genome runs in chromosome order from 1 to the sex chromosomes. The p or short arm on each chromosome is followed by the q or long arm. The dotted lines represent the position of the centromere. The cbs algorithm recursively split chromosomes into segments based on the maximum t statistic estimated by each permutation (re Mathworks.com). A. MDA-MB-231 Natives vs MDA-MB-231 25PACR. B. MDA-MB-231 Natives vs MDA-MB-231 50PACR. C. ZR75-1 native cells vs ZR75-1 25PACR. D. ZR75-1 native cells vs ZR75-1 50PACR. E. ZR75-1 native cells vs ZR75-1 25DOCR. F. ZR75-1 native cells vs ZR75-1 50DOCR.

Figure 4

Network-based analysis of MDA-MB-231 25PACR, ZR75-1 25PACR and ZR75-1 25DOCR taxane resistant cell lines. A. Venn diagram of genes within significant areas of gain. B. Venn diagram of genes within significant areas of loss in 3 cell lines. C. Mitotic prometaphase module identified from functional interaction network analysis. D. Bar graph shows relative expression levels of AHCTH1, CENPF, PPP2R5A, NUP133, MLP1P and NSL1 in the MDA-MB-231 native, 25PACR, 50 PACR ZR75-1 25PACR, 50PACR and ZR75-1 25DOCR and 50DOCR taxane resistant cell lines. Error bars show standard deviation of three experiments. Asterisks indicate statistical difference * = p < 0.05, ** = p < 0.001.

Figure 5

Effects of MDR-1 knockdown in ZR75-1 paclitaxel resistant cells A Western blot analysis of proteins extracted from the cell lines and probed with MDR1. Actin was used as a loading control. B. Western blot analysis of proteins extracted from ZR75-1 25PACR cells after transfections with MDR1 siRNA. Control cells were untreated, or with transfections reagents only or with siRNA targeting GAPDH; actin was used as a loading control. C. Graph shows the average IC₅₀ of ZR75-1 25PACR cells after transfections with MDR1 siRNA. D. Graph shows the average IC₅₀ of ZR75-1 50PACR cells after transfections with MDR1 siRNA. Asterisks indicate statistical difference * = p < 0.05, ** = p < 0.001.