Abstract B9: PTEN expression level is important in determining sensitivity to cytotoxic agents in vitro, but is not representative of PTEN function observed in primary ovarian cancer cultures

Abstract

Abstract Introduction: The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumour suppressor protein is a central negative regulator of the PI3K/AKT signalling cascade and induces apoptosis and suppresses cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. The role of PTEN in cancer however is still not fully understood. PTEN loss has been suggested to impair double strand break repair by homologous recombination and has therefore been suggested to improve sensitivity to PARP inhibitors. However, conflicting results suggest that PTEN loss is associated with cisplatin resistance development. In this study we assess the role of PTEN in ovarian cancer using experimental knockdown models and primary ovarian cancer cultures. Methods: PTEN knockdown models were created in a normal epithelial and clear cell carcinoma cell lines using MISSION®shRNA lentiviral transduction particles and grown in purimycin selection media. Sensitivity of the knockdown models to common therapies were assessed using SRB assay (these included cisplatin, paplitaxol, camptothecin, doxorubicin and irradiation). We screened 28 unselected primary epithelial ovarian cancer cultures derived from ascitic fluid collected at the time of surgery and matched genomic DNA for PTEN mutations in exons 2-5, 6-7 and 9-10 using PCR amplification and Sanger sequencing. We also assessed mRNA expression of PTEN in this panel of ovarian cancer cultures using qRT-PCR. Sensitivity to cisplatin was assessed in the primary ovarian cultures using SRB assay and HR status was determined using γH2AX/RAD51 immunofluorescence assay. Results: In normal epithelial cells, PTEN knock-down had no effect on cytotoxic agent sensitivity whilst in clear cell carcinoma cells, PTEN knockdown enhanced sensitivity to cisplatin, doxorubicin and irradiation. PTEN knockdown produced G1 arrest in both normal epithelial and clear cell carcinoma cells. The primary ovarian cancer cultures included 20 high grade serous, 6 mixed endometroid / clear cell, 1 clear cell and 1 mucinous carcinoma. Whilst two point mutations were detected in primary cultures (1105T>TG, 25L>L in 6 cultures and 1508G>GA, 159R>R in 4 cultures), no somatic mutations were observed in any of the PTEN coding sequences. The mutations found did not correlate with histological type or the level of PTEN mRNA expression. PTEN mRNA expression varied over 40 fold between the cultures. PTEN expression did not correlate with HR status or cisplatin sensitivity in primary ovarian cancer cultures. Discussion: These data indicate that PTEN knockdown results in therapeutic sensitisation. However, we have also shown that in primary ovarian cancers mutations in PTEN gene are rare and that PTEN mRNA expression does correlate with cisplatin sensitivity or HR status. Therefore the results of experimental PTEN knockdown models need to be interpreted with caution and PTEN inhibition needs to be further assessed in primary cultures to assess its role ovarian cancer. Citation Format: Aiste McCormick, Eleanor Earp, Charlotte Leeson, Michelle Dixon, Rachel ODonnell, Richard J. Edmondson. PTEN expression level is important in determining sensitivity to cytotoxic agents in vitro, but is not representative of PTEN function observed in primary ovarian cancer cultures. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B9.