Identifying novel hypoxia-associated markers of chemoresistance in ovarian cancer

Abstract

Background: Ovarian cancer is associated with poor long-term survival due to late diagnosis and development of chemoresistance. Tumour hypoxia is associated with many features of tumour aggressiveness including increased cellular proliferation, inhibition of apoptosis, increased invasion and metastasis, and chemoresistance, mostly mediated through hypoxia-inducible factor (HIF)-1α. While HIF-1α has been associated with platinum resistance in a variety of cancers, including ovarian, relatively little is known about the importance of the duration of hypoxia. Similarly, the gene pathways activated in ovarian cancer which cause chemoresistance as a result of hypoxia are poorly understood. This study aimed to firstly investigate the effect of hypoxia duration on resistance to cisplatin in an ovarian cancer chemoresistance cell line model and to identify genes whose expression was associated with hypoxia-induced chemoresistance.

Methods: Cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) ovarian cancer cell lines were exposed to various combinations of hypoxia and/or chemotherapeutic drugs as part of a 'hypoxia matrix' designed to cover clinically relevant scenarios in terms of tumour hypoxia. Response to cisplatin was measured by the MTT assay. RNA was extracted from cells treated as part of the hypoxia matrix and interrogated on Affymetrix Human Gene ST 1.0 arrays. Differential gene expression analysis was performed for cells exposed to hypoxia and/or cisplatin. From this, four potential markers of chemoresistance were selected for evaluation in a cohort of ovarian tumour samples by RT-PCR.

Results: Hypoxia increased resistance to cisplatin in A2780 and A2780cis cells. A plethora of genes were differentially expressed in cells exposed to hypoxia and cisplatin which could be associated with chemoresistance. In ovarian tumour samples, we found trends for upregulation of ANGPTL4 in partial responders and down-regulation in non-responders compared with responders to chemotherapy; down-regulation of HER3 in partial and non-responders compared to responders; and down-regulation of HIF-1α in non-responders compared with responders.

Conclusion: This study has further characterized the relationship between hypoxia and chemoresistance in an ovarian cancer model. We have also identified many potential biomarkers of hypoxia and platinum resistance and provided an initial validation of a subset of these markers in ovarian cancer tissues.

Fig. 1

Hypoxia matrix treatment pathway. In the pre-treatment phase, cells received no, acute or chronic hypoxia. During treatment, cells that had prior hypoxia exposure were either treated in normoxia or hypoxia. Cells that had no prior hypoxia exposure were treated in normoxia for the full 3-day treatment period or in hypoxia for 1, 2 or 3 days, with any remaining treatment time in normoxia

Fig. 2

Response of A2780 and A2780cis to Cisplatin following Acute Hypoxia. a. A2780cis were 9-fold more resistant to cisplatin in normal oxygen. b. Following acute hypoxia, A2780 cells were 8-fold more resistant to cisplatin if the treatment was also carried out in hypoxia. This was attenuated (2.4-fold) if the treatment was carried out in normoxia. c. A2780cis were approximately 2-fold more resistant to cisplatin following acute hypoxia if the treatment was carried out in hypoxia. When acutely hypoxic A2780cis were placed in normal oxygen for the treatment period, the resistance returned to the same level as cells which were never exposed to hypoxia. n = 3 *p < 0.05 ***p < 0.001

Fig. 3

Response of A2780 and A2780cis to chronic hypoxia and hypoxia during treatment. a. A2780 cells exposed to chronic hypoxia before treatment with cisplatin resulted in a 10-fold increase in resistance when the treatment was also carried out in hypoxia. The resistance returned to that of normoxia when the chronically hypoxic cells were returned to normal oxygen for the treatment period. b. A2780cis displayed more modest changes in resistance (<2-fold) following hypoxia although this was still significant. c. Hypoxia naïve cells (cells not exposed to hypoxia before treatment) were exposed to hypoxia during cisplatin treatment for all or part of the 72 h treatment period for A2780. d. Hypoxia naïve cells (cells not exposed to hypoxia before treatment) were exposed to hypoxia during cisplatin treatment for all or part of the 72 h treatment period for and A2780cis. Both cell lines developed resistance when cells were challenged with cisplatin and hypoxia at the same time without any previous exposure, although the fold changes were more modest in A2780cis. e. Timecourse of HIF-1α protein expression (120 kDa). Loading control β-actin also shown (42 kDa). HIF-1α protein was absent in normoxia in A2780 and A2780cis. The levels fluctuated slightly over time, with an increase in HIF-1α expression at 3 days in A2780 and a decrease in HIF-1α expression in A2780cis at 3 days. A2780 cells were exposed to 50 μM CoCl₂ for 24 h for a positive control. n = 3 *p < 0.05 **p < 0.01 ***p < 0.001

Fig. 4

Expression of potential and known hypoxia biomarkers in ovarian cancer samples. The expression of ANGPTL4 (a), HER3 (b) and HIF1α (c) was examined in 35 serous ovarian adenocarcinoma samples. The samples were divided into responders (n = 16), partial responders (n = 11) and non-responders (n = 8). Expression of ANGPTL4 trended towards up-regulation in partial and down-regulation in non-responders compared to responders to chemotherapy. Expression of HER3 trended towards down-regulation in partial and non-responders to chemotherapy compared to responders. Expression of HIF-1α trended towards down-regulation in non-responders compared to responders to chemotherapy. There were missing data in one patient for HER3 expression and in three patients for ANGPTL4 expression in the responder group; and in one patient for ANGPTL4 expression in the non-responder group