Gene expression and pathway analysis of ovarian cancer cells selected for resistance to cisplatin, paclitaxel, or doxorubicin

Abstract

Background: Resistance to current chemotherapeutic agents is a major cause of therapy failure in ovarian cancer patients, but the exact mechanisms leading to the development of drug resistance remain unclear.

Methods: To better understand mechanisms of drug resistance, and possibly identify novel targets for therapy, we generated a series of drug resistant ovarian cancer cell lines through repeated exposure to three chemotherapeutic drugs (cisplatin, doxorubicin, or paclitaxel), and identified changes in gene expression patterns using Illumina whole-genome expression microarrays. Validation of selected genes was performed by RT-PCR and immunoblotting. Pathway enrichment analysis using the KEGG, GO, and Reactome databases was performed to identify pathways that may be important in each drug resistance phenotype.

Results: A total of 845 genes (p < 0.01) were found altered in at least one drug resistance phenotype when compared to the parental, drug sensitive cell line. Focusing on each resistance phenotype individually, we identified 460, 366, and 337 genes significantly altered in cells resistant to cisplatin, doxorubicin, and paclitaxel, respectively. Of the 845 genes found altered, only 62 genes were simultaneously altered in all three resistance phenotypes. Using pathway analysis, we found many pathways enriched for each resistance phenotype, but some dominant pathways emerged. The dominant pathways included signaling from the cell surface and cell movement for cisplatin resistance, proteasome regulation and steroid biosynthesis for doxorubicin resistance, and control of translation and oxidative stress for paclitaxel resistance.

Conclusions: Ovarian cancer cells develop drug resistance through different pathways depending on the drug used in the generation of chemoresistance. A better understanding of these mechanisms may lead to the development of novel strategies to circumvent the problem of drug resistance.

Figure 1

Establishment of drug resistant cell lines and gene expression profiling. A. IC₅₀ values for the various cell lines used in this study. Thick outlined squares show resistance levels for the drug against which the corresponding cell lines were derived. White squares denote lack of resistance, and light gray squares, moderate resistance. Dark gray indicates drug resistance over 10-fold compared to the parental OV90 line. B. Multi-dimensional scaling plot indicating the cell lines used for the gene expression profiling analysis. Each of the two different resistant clones obtained from the 3 different drugs were cultured and analyzed in duplicate. Two cultures were analyzed for the parental OV90 (OV90-1 and OV90-2). C. Venn diagram representing the number of genes significantly altered in each type of drug resistance. A total of 68 genes were found altered in all three types of resistance generated following exposure to cisplatin, doxorubicin, and paclitaxel.

Figure 2

Genes differentially expressed following the development of drug resistance. A. Heat map showing the expression of all the significant genes analyzed using the Illumina bead array (845 genes). Changes in gene expression for the 3 pairwise comparisons are included in this analysis (OV90C vs OV90, OV90D vs OV90, and OV90P vs OV90). B. Heat map representing the clustering of genes significantly altered in cisplatin-derived drug resistance. C. Heat map representing the clustering of genes significantly altered in doxorubicin-derived drug resistance. D. Heat map representing the clustering of genes significantly altered in paclitaxel-derived drug resistance.

Figure 3

Validation of selected differentially expressed genes. A. RT-PCR analysis of genes elevated in drug resistant cells. The y-axis represents fold up-regulation in the different drug resistant cell lines over the parental OV90 cell line. B. RT-PCR analysis of genes decreased in drug resistant cells. The y-axis represents the fold down-regulation of the different resistant cell lines compared to the parental OV90 cell line. C. Immunoblot analysis of selected gene products identified by microarray and RT-PCR as altered in drug resistant cells.

Figure 4

Network of genes identified using Ingenuity Pathway Analysis. A. Network including ECM and other genes altered in cisplatin derived resistant cells. B. Network including proteasome genes and other genes altered in doxorubicin resistant cells. C. Network containing translation genes as well as other genes differentially expressed in paclitaxel-derived drug-resistant cells

Figure 5

Model for the development of various resistance phenotypes in ovarian cancer. Following selection for drug resistance with the indicated drugs, a number of molecular pathways are altered. The molecular pathways identified as altered in the different conditions may be functionally related to the development of drug resistance.