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. 2012;7(9):e45268.
doi: 10.1371/journal.pone.0045268. Epub 2012 Sep 18.

Differential gene and microRNA expression between etoposide resistant and etoposide sensitive MCF7 breast cancer cell lines

Karobi Moitra  1 , Kate Im, Katy Limpert, Alexander Borsa, Julie Sawitzke, Rob Robey, Naoya Yuhki, Ram Savan, Da Wei Huang, Richard A Lempicki, Susan Bates, Michael Dean
Affiliations

Differential gene and microRNA expression between etoposide resistant and etoposide sensitive MCF7 breast cancer cell lines

Karobi Moitra et al. PLoS One. 2012.

Abstract

In order to develop targeted strategies for combating drug resistance it is essential to understand it's basic molecular mechanisms. In an exploratory study we have found several possible indicators of etoposide resistance operating in MCF7VP cells, including up-regulation of ABC transporter genes, modulation of miRNA, and alteration in copy numbers of genes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cellular cytotoxicity assays determined that MCF7VP cells are more resistant to etoposide than the parental MCF7 cells.
Curve fitting of cellular cytotoxicity data with GraphPad prism. (MCF7VP- red squares and MCF7- blue circles). The dotted line represents the actual curve and the solid line depicts the fitted curve. The assay was carried out using 10,000 cells per well of a 96 well plate and 100 µl of various concentrations (0–100 µM) of etoposide (the graph depicts log concentrations of etoposide).
Figure 2
Figure 2. Heat map depicting the upregulation of ABCC6 and ABCC1 genes in MCF7VP cells.
It can be seen that both ABCC6 (red) and ABCC1 (red) genes are upregulated in the MCF7VP cell lines. cDNA from the MCF7 and MCF7VP cells was used to run the Taqman Low Density ABC transporter arrays. The data was analyzed using Microsoft excel and R-Bioconductor to generate heat maps of the ABC array data.
Figure 3
Figure 3. Expression of ABCC1 and ABCC6 in MCF7VP cells, Fig. 3A.
Up-regulation of ABCC1 in MCF7VP cells validated by qRT-PCR. The gene expression level of ABCC1 was found to be up-regulated in the MCF7VP cell line using microfluidic arrays, this was validated using quantitative RT PCR with taqman probes against ABCC1. The fold change was calculated using the delta delta CT method. Fig. 3B. Up-regulation of ABCC6 in MCF7VP cells validated by qRT-PCR. ABCC6 up-regulation was validated with quantitative qRT PCR and taqman probes against ABCC6. The fold change in gene expression between the MCF7 and MCF7VP was calculated using the dd CT method.
Figure 4
Figure 4. Gene expression microarray data and GO analysis revealed differential gene expression between MCF7 & MCF7VP cell lines.
Figure 4A. Heatmap depicting differential gene expression in etoposide resistant and etoposide sensitive MCF7 cells using Affymetrix U133 arrays (>2fold change in expression, p<0.05). Up-regulated genes depicted in red, down-regulated genes in blue (see color bar). Fig. 4B. DAVID analysis of microarray data depicting the number of genes expressed in different cellular mechanisms and pathways (Top 219 genes, p<0.05).
Figure 5
Figure 5. GSEA analysis of microarray data showing enrichment of the JAK-STAT and MAP Kinase pathways and upregulation of ECM structural component genes along with qRT-PCR validation of the down-regulation of topoisomerase 2 gene expression in MCF7VP cells.
Fig. 5A. Gene set enrichment analysis of microrray data depicting the enrichment of genes in the JAK-STAT signaling pathway. The GSEA software was used to calculate the enrichment levels. Fig. 5B. Gene set enrichment analysis of microrray data depicting the enrichment of genes in the MAP kinase signaling pathway. The GSEA software was used to calculate the enrichment levels. Figure 5C. Gene set enrichment analysis depicting enrichment of ECM (extra-cellular matrix) genes in MCF7VP cells. The GSEA algorithm was used to calculate the enrichment levels. Figure 5D. Down-regulation of TOPO2A (the drug target of etoposide) in MCF7VP cells. The microarray data was validated by qRTPCR as depicted in the bar chart which shows differences in fold change.
Figure 6
Figure 6. Circos plot incorporating differential gene expression and LOH.
Chromosome numbers and bands are identified in the outer-most ring. Other tracks from outer to inner represent: amplifications (red) and deletions (blue) in MCF7VP compare to MCF7 using Affy 6.0 SNP data (Partek); differential expression (p<0.05) between MCF7 and MCF7VP (AffyU133), ln(MCF7/MCFVP) values plotted; LOH in MCF7 derived from Affy 6.0 SNP data (green); LOH in MCF7VP derived from Affy 6.0 SNP data (black).
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