New markers for human ovarian cancer that link platinum resistance to the cancer stem cell phenotype and define new therapeutic combinations and diagnostic tools

Abstract

Background: Ovarian cancer is the leading cause of gynecologic cancer-related death, due in part to a late diagnosis and a high rate of recurrence. Primary and acquired platinum resistance is related to a low response probability to subsequent lines of treatment and to a poor survival. Therefore, a comprehensive understanding of the mechanisms that drive platinum resistance is urgently needed.

Methods: We used bioinformatics analysis of public databases and RT-qPCR to quantitate the relative gene expression profiles of ovarian tumors. Many of the dysregulated genes were cancer stem cell (CSC) factors, and we analyzed its relation to therapeutic resistance in human primary tumors. We also performed clustering and in vitro analyses of therapy cytotoxicity in tumorspheres.

Results: Using bioinformatics analysis, we identified transcriptional targets that are common endpoints of genetic alterations linked to platinum resistance in ovarian tumors. Most of these genes are grouped into 4 main clusters related to the CSC phenotype, including the DNA damage, Notch and C-KIT/MAPK/MEK pathways. The relative expression of these genes, either alone or in combination, is related to prognosis and provide a connection between platinum resistance and the CSC phenotype. However, the expression of the CSC-related markers was heterogeneous in the resistant tumors, most likely because there were different CSC pools. Furthermore, our in vitro results showed that the inhibition of the CSC-related targets lying at the intersection of the DNA damage, Notch and C-KIT/MAPK/MEK pathways sensitize CSC-enriched tumorspheres to platinum therapies, suggesting a new option for the treatment of patients with platinum-resistant ovarian cancer.

Conclusions: The current study presents a new approach to target the physiology of resistant ovarian tumor cells through the identification of core biomarkers. We hypothesize that the identified mutations confer platinum resistance by converging to activate a few pathways and to induce the expression of a few common, measurable and targetable essential genes. These pathways include the DNA damage, Notch and C-KIT/MAPK/MEK pathways. Finally, the combined inhibition of one of these pathways with platinum treatment increases the sensitivity of CSC-enriched tumorspheres to low doses of platinum, suggesting a new treatment for ovarian cancer.

Keywords: Biomarkers; Cancer stem cells; Ovarian cancer; Therapy.

Fig. 1

Analysis of the survival probability of ovarian cancer patients in different datasets by the expression of the identified genes. Cox analysis of survival probability of patients according to mRNA levels of different genes. Kaplan-Meier curves of the genes up-regulated (a) or down-regulated (b) in ovarian cancer

Fig. 2

Gene expression analyses in tumorspheres from ovarian cancer cell lines. a Gene expression analyses by RT-qPCR of up- and down-regulated genes in tumorspheres and total adherent cell culture (total culture) samples from SKOV3 and OVCAR8 ovarian cancer cell lines. b Gene expression analyses by RT-qPCR of C-KIT, CD133, NANOG, CXCR4 and ABCG2 in tumorspheres and total culture samples from SKOV3 and OVCAR8 ovarian cancer cell lines. For (a) and (b), normalized expression values of tumorspheres compared to total culture samples (line at value 1) are shown. The average and SD of three independent experiments are shown. Statistical significance was assessed using the one-sample t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 3

Gene expression analyses and platinum resistance in tumoral samples from ovarian cancer patients. a Gene expression analyses by RT-qPCR of the candidate up-regulated genes in non-tumoral and tumoral samples from ovarian cancer patients, resistant or sensitive to platinum treatment. b Same analysis as in (a) with the down-regulated genes in ovarian cancer. For (a) and (b), the average and SD of three independent experiments are shown. c Proportion of platinum-resistant patients with expression levels for each gene above (Up) or below (Down) the median value of the sensitive ones. Statistical significance was assessed using the Fisher’s exact test by comparison with the proportions of sensitive patients. *, P < 0.05, ***, P < 0.001

Fig. 4

Gene expression analyses and platinum resistance in tumoral samples from ovarian cancer patients. Gene expression analyses by RT-qPCR of C-KIT, CD133, NANOG, CXCR4 and ABCG2 in non-tumoral and tumoral samples from ovarian cancer patients, resistant or sensitive to platinum treatment. The average and SD of three independent experiments are shown. Statistical significance was assessed using the Student’s t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 5

Notch, C-KIT, MAPK, PI3K and PARP inhibition reduces tumorsphere formation in ovary tumor cells. a Tumorsphere formation in OVCAR8 and SKOV3 cells treated with cis-platin (IC30; 0.3 μM), with or without gamma-secretase inhibitor DAPT (5 μM), tyrosine kinase inhibitor Imatinib (8 μM), PARP inhibitor olaparib (10 μM), MAPK inhibitor PD98059 (15 μM) or PI3K inhibitor BEZ235 (10 μM). b Tumorsphere formation in OVCAR8 and SKOV3 cells treated with carboplatin (IC30; 0.3 μM), with or without the inhibitors described in (A). The average and SD of at least three independent experiments are shown. Statistical significance was assessed using the Student’s t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001