Anti-tumor activity of olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, in cultured endometrial carcinoma cells

Homologous recombination (HR) is a critical step for DNA repair, and certain types of cancers are HR defective, including BRCA1/2 deficiency [1, 2]. Poly (ADP-ribose) polymerase (PARP) plays a key role in the repair of DNA single-strand breaks (SSBs) [1], and PARP inhibition leads to the accumulation of SSBs, which results in the development of DNA double strand breaks (DSBs) via the collapse of replication forks [3‐5]. Tumor cells lacking functional BRCA1 and BRCA2 are deficient in the repair of DSBs by RAD51-mediated HR, which leads to cell cycle arrest and/or cell death [3]. Thus, targeting the HR defect, which is specific to cancer cells, and causing synthetic lethality by a PARP inhibitor is expected to be a promising therapeutic strategy in selected tumors [2]. Indeed, a PARP inhibitor, olaparib (AZD2281/ KU0059436), showed antitumor activity in cancer patients, especially with the BRCA 1/2 mutations in breast and ovarian cancers [6, 7]. However, BRCA status alone is not necessarily the only predictive biomarker for effective olaparib treatment because various types of genes are known to be involved in the HR process, including PTEN, ATM, RAD51[8‐10]. Therefore, PARP inhibition might be useful for various types of tumors with HR defects, independent of the BRCA status (BRCAness).

Endometrial cancer is the fourth most common malignancy among women in the United States [11]. In endometrial cancer, the constitutive activation of the phosphatidylinositol 3-kinase (PI3K) pathway is induced by various types of alternations, including frequent mutations of K-Ras (10–20%), PIK3CA (25–36%), AKT (2%), and PTEN (34–56%) [12‐15]. Additionally, the loss of heterozygosity (30–40%) of the PTEN locus at chromosome 10q23.31 is also associated with the inactivation of PTEN [16‐18].

In addition to a negative regulator of the PI3K/AKT signaling pathway, PTEN contributes to maintaining genomic stability and DNA repair processes by regulating the expression of RAD51, a key protein in HR DNA repair [19]. The lack of PTEN also impairs CHK1 function, which results in the accumulation of DNA DSBs [20, 21].

Dedes and coworkers showed that PTEN-deficient endometrial cell lines, which fail to elicit RAD51 to DNA damage sites, are sensitive to PARP inhibitors [3]. However, the correlation between PTEN status and RAD51 expression remains a debatable matter. For example, a recent study showed that PTEN deletion is not associated with the loss of RAD51 in prostate cancer cells [22].

The purpose of this study is to clarify the anti-tumor effect of olaparib on a panel of endometrial cancer cell lines and to assess the association among PTEN status, HR repair, and sensitivity to olaparib in endometrial cancer cells.

Inhibiting PARP is a promising strategy in cancer cells, especially in cells with a deficiency in HR repair. Although BRCA1/2 play key roles in HR repair, the association of other tumor suppressor genes with HR repair is still debatable. In phase II clinical trials conducted in high-grade serous ovarian carcinomas, olaparib has been reported to be effective in certain patients without BRCA1/2 mutations [27, 28]. The activity of olaparib (AZD2281, KU0059436) was demonstrated by Menear et al. [28]. The IC50 of olaparib (Compound 47 in the paper) was shown to be 6 nM and exposure to 100–300 nM olaparib inhibited PARP activity (quantified by a PAR formation) by 90–95% [6]. In addition, poly (ADP-ribose) expression was sufficiently suppressed by 1 μM or higher doses of olaparib, regardless of the SF50 values (ranging from 200 nM to 4,500 nM) [29]. Preclinical studies have suggested that PTEN deficiency causes HR defects and possibly induces sensitivity to PARP inhibitors [27]. Therefore, the inactivation of PTEN in cancer cells might be one of the possible mechanisms of HR defects independent from BRCA. In this study, using a panel of endometrial cell lines, we focused on (i) the anti-tumor effect of olaparib and its relationship with PTEN status, (ii) the association between PTEN and HR-related proteins (RAD51 and γ-H2AX), and (iii) the relationship between PTEN status and the response to IR exposure (another DNA damaging therapy).

Clonogenic assays revealed that sensitivity to olaparib is greatly distinct among endometrial cell lines, with SF50 values ranging from 8 nM to 2,500 nM. The high ratio (25%) of sensitive cells with SF50 values <100 nM suggests that endometrial cancers are good candidates for PARP inhibitors such as olaparib. Recently, the possibility of a relationship between PTEN status and sensitivity to PARP inhibitors has received much attention [3, 22, 30]. Dedes et al. reported that PTEN-deficient cells were more sensitive to a PARP inhibitor than PTEN-wild type endometrial cancer cells [31]. However, our results were not in agreement with their conclusion. In our study, the existence of PTEN mutations did not result in high sensitivity to olaparib. One of the 4 PTEN wild-type cells and three of the 12 PTEN mutant cells were classified as sensitive (SF50 ≦ 100 nM). Moreover, all the 4 resistant cell lines (SF50 > 1,000 nM) were PTEN mutant. The report by Dedes et al. included only 2 PTEN wild-type cell lines (HEC-1B and EFE-184), whereas 4 PTEN-wild type cell lines were included in our study. All the SF50 values in these 4 cell lines were 340 nM or lower. In addition, they did not include 3 of the 4 “resistant” (and PTEN mutant) cell lines in this study (HEC-6, HEC-116 and HEC-108) [31]. The contribution of PTEN inactivation was also negatively reported in prostate cancer cell lines (22RV1, DU145, and PC3) [22].

Therefore, we further examined the relationship between PTEN and HR-related proteins in endometrial cancer cells. RAD51 mediates the formation of DNA joints that link homologous DNA molecules [32]. Several recent reports have suggested that the loss of PTEN might be associated with the downregulation of RAD51 [19, 33]. Our data suggest that RAD51 expression levels were not associated with the PTEN status in a panel of endometrial cells and that the introduction of PTEN does not upregulate RAD51 expression in HEC-6 cells. Dedes et al. reported that expression of RAD51 was not associated with the status of PTEN, and that RAD51 expression was predominantly observed in cytoplasm, not in nucleus [31]. These data are compatible with previous reports in astrocytes and prostate cancer cell lines [22, 30] but not in agreement with reports in colorectal cancer cells [27]. Thus, the association of RAD51 and PTEN might be distinct among various types of tumors.

One of the earliest events in the signal transduction cascade initiating DSBs is the phosphorylation of serine 139 of histone H2AX (γH2AX) and RAD51 filament formation on DNA [34, 35]. Our data showed that foci formations of γ-H2AX and RAD51 after olaparib exposure did not differ between parental HEC-6 and HEC-6-PTEN + cell lines. The data was also in agreement with the previous report that phospho-γ-H2AX foci formation by exposure to PARP inhibitor was not associated with PTEN status [31]. In previous reports, a PARP inhibitor induced G2/M arrest, which led to cell death [36, 37]. In our study, olaparib induced both G2/M arrest and cleaved PARP expression in HEC-6 cell lines after 24 and 48 h of olaparib and IR exposure. It is crucial to note that these events are independent from PTEN status. Therefore, the response to olaparib-induced DNA damage is suggested to be independent from PTEN in endometrial cancer cells. Although PTEN deficiency was proposed as a predictive biomarker to PARP inhibition, our data with clear phenotypic change by olaparib but no impact of PTEN status suggests that PTEN is unlikely to be a predictive biomarker to PARP inhibitors in endometrial cancer.

We also examined whether the response to IR-induced DNA damage is affected by PTEN status. IR directly produces DNA DSBs [38]. After IR exposure, γH2AX and RAD51 foci formation was not significantly different between the parental HEC-6 cells and the HEC-6-PTEN + cells. Additionally, the cell cycle profile and cell proliferation were also not affected by exogenous PTEN when examined using flow cytometry and clonogenic assays. The response of RAD51 to IR exposure was similar in the endometrial cells without PTEN mutations.

Our study has some limitations. Predictive biomarkers for olaparib are still unknown. Further studies are warranted to elucidate whether PTEN inactivation is a biomarker for resistance to olaparib. The role of PTEN might be distinct when a PARP inhibitor was administered in combination with another drug (or irradiation), as the combination of a PARP inhibitor with cisplatin or irradiation was reported to be effective in PTEN deficient cells [39, 40]. Additionally, the mechanism of RAD51 expression should be elucidated further.

xIn conclusion, our findings have noteworthy implications: PTEN inactivation is not a good biomarker of olaparib treatment and PARP inhibitors might still provide a promising therapeutic strategy in certain endometrial carcinomas.