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Anti-tumor activity of olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, in cultured endometrial carcinoma cells
- Aki Miyasaka1,
- Katsutoshi Oda1Email author,
- Yuji Ikeda1,
- Osamu Wada-Hiraike1,
- Tomoko Kashiyama1,
- Atsushi Enomoto2,
- Noriko Hosoya2,
- Takahiro Koso1,
- Tomohiko Fukuda1,
- Kanako Inaba1,
- Kenbun Sone1,
- Yuriko Uehara1,
- Reiko Kurikawa1,
- Kazunori Nagasaka1,
- Yoko Matsumoto1,
- Takahide Arimoto1,
- Shunsuke Nakagawa3,
- Hiroyuki Kuramoto4,
- Kiyoshi Miyagawa2,
- Tetsu Yano5,
- Kei Kawana1,
- Yutaka Osuga1 and
- Tomoyuki Fujii1
© Miyasaka et al.; licensee BioMed Central Ltd. 2014
Received: 14 October 2013
Accepted: 27 February 2014
Published: 13 March 2014
PTEN inactivation is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in nucleus. Poly (ADP-ribose) polymerase (PARP) plays key roles in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in endometrial cancer cell lines.
The response to olaparib was evaluated using a clonogenic assay with SF50 values (concentration to inhibit cell survival to 50%) in 16 endometrial cancer cell lines. The effects of PTEN on the sensitivity to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN + (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and γH2AX, and induction of cleaved PARP. The effects of siRNA to PTEN were analyzed in cells with wild-type PTEN.
The SF50 values were 100 nM or less in four (25%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in four (25%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 746 ± 838 nM; Wild-type [n = 4]: 215 ± 85 nM, p = 0.26 by Student’s t test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and γH2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN + cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN + cells. In addition, knocking down PTEN by siRNA did not alter the sensitivity to olaparib in 2 cell lines with wild-type PTEN.
Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. Inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer.
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) , 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 . 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 . 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 . 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 . 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 . 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 .
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.
Cell lines and reagents
PTEN status in endometrial cancer cell lines
INTRON 4 (+2)
T TO C
1bp (A) del
TAC to TAC
Tyr (Y) to His (H)
CGA to TGA
CCG to CTG
Pro (P) to Leu (L)
1bp (A) del
CGA to GGA
Arg (R) to Gly (G)
CGC to TGC
Arg (R) to Cys (C)
GAT to TAT
Asp (D) to Tyr (Y)
TTT to TGT
Phe (F) to Cys (C)
2bp (AA) del
1bp (A) del
Intron 2 (-1)
G to A
CGC to TGC
Arg (R) to Cys (C)
CGA to TGA
3bp (ATT) del
In frame deletion
2bp (AT) del
1bp (A) del
1bp (A) del
1bp (G) del
1bp (A) del
4bp (ACTT) del
1bp (A) del and 1bp (A) ins
AGC to AAC
Ser (S) to Asn (N)
1bp (A) ins
Histologically, only the HEC-180 cell line was classified as a serous adenocarcinoma; the other cell lines were classified as endometrioid adenocarcinomas. The culture conditions of the 13 endometrial cancer cell lines were described previously . HEC-180, HEC-251, and HEC-265 cells were maintained in Eagle’s MEM with 10% FBS. HEC-6 cells stably expressing wild-type PTEN were generated by a retroviral infection, as described previously . Phoenix cells were transfected with retroviral vectors (pFB-neo) that contained tandem affinity purification (TAP)-tagged wild-type PTEN using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and the resulting supernatants were used to infect HEC-6 cells. Drug selection was used to purify cell populations after infections by neomycin (500 μg/mL, 7 days).
Olaparib (AZD2281/KU0059436) was provided by AstraZeneca (London, UK). Olaparib was solved in DMSO, and the concentration of DMSO in each assay was 0.1%.
Gene silencing and transient transfection
Cells were plated at approximately 30% confluence in 100-mm plates and incubated for 24 h before transfection with small interfering RNA (siRNA) duplexes at the concentrations indicated, using Lipofectamine 2000 RNAiMAX (Invitrogen, Carlsbad, CA) and Opti-MEM medium (Life Technologies, Grand Island, NY). The target sequence of siRNA specific for PTEN was described previously . A negative control kit was used as a control (Invitrogen, Carlsbad, CA). HA-tagged wild-type PTEN expression plasmid was generated and transfected into PTEN mutant cell lines using Effectene transfection reagent (Qiagen, Valencia, CA, USA). HA-tagged pcDNA plasmid was used as a control.
PCR and direct sequencing
The mutational status of PTEN (exons 1–9) was analyzed by PCR and direct sequencing as described previously . The mutational status in 13 of the 16 endometrial cell lines and the PCR primers have been described previously [12, 24]. The mutational status in the remaining three cell lines (HEC-180, HEC-251, and HEC-265) is presented in Table 1.
Cells were lysed as described previously [12, 25]. Antibodies specific for PTEN (138G6), phospho-PTEN (Ser380), AKT (Cell Signaling Technology), phospho-AKT (Ser473), PARP, cleaved PARP (Cell Signaling Technology, Beverly, MA), RAD51 (Millipore, MA, USA and Santa Cruz Biotechnology, CA, USA), and β-actin (Sigma-Aldrich, MO, USA) were used for western blotting, as recommended by the manufacturer. Proteins were visualized using an ECL western blot detection kit (GE Healthcare, Little Chalfont, UK).
Immunocytochemistry was performed as described previously . Primary antibodies to RAD51 (Millipore, MA, USA) (1:500 dilution) and γH2AX (Millipore, MA, USA) (1:500 dilution) and secondary antibodies to Alexa Fluor 488-conjugated chicken anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) (1:100 dilution) were used for analysis. Nuclei were visualized by staining with DAPI. The slides were briefly counterstained and analyzed by confocal fluorescence microscopy (Carl-Zeiss MicroImaging Inc., Oberkochen, Germany). The number of RAD51- and γH2AX-foci was evaluated in a mean of 100 cells.
Cell cycle analysis
Cell cycle analysis was performed by flow cytometry, as previously described . The cells were exposed to olaparib (10 μM) for the indicated time or were irradiated with 10 Gy after 24 h of irradiation. The cell cycle distribution was analyzed using CELL Quest pro ver. 3.1. (Beckman Coulter Epics XL Brea, CA). All experiments were repeated three times.
Cells were seeded in six-well plates at a concentration of 2,000 cells per well with olaparib (10 nM to 100 μM) or IR (2 Gy to 6 Gy). Cells were continuously exposed to olaparib with media during the incubation. After 14–21 days of incubation, the cells were fixed with methanol and stained with Giemsa (Wako). All experiments were repeated three times and the SF50 (surviving fractions at 50%) values, which indicate the concentration required to inhibit cell survival to 50%, were calculated by proliferation curves.
Cells were irradiated using a Shimadzu PANTAK HF-350 X-ray generator (1.0 mm Al +0.5 mm Cu filter; 200 kVp; 20 mA; Shimadzu, Kyoto, Japan).
Data are expressed as the means ± standard deviations of three independent determinations. The significance of differences between the two samples was analyzed using Student’s t-test, and a p-value of <0.05 was considered to denote a statistically significant difference.
RAD51 protein is expressed regardless of PTENmutation status in endometrial cancer cell lines
We generated a stable endometrial cancer cell line expressing wild-type PTEN (HEC-6 PTEN+) by introducing wild-type PTEN into a parental HEC-6 (PTEN null) cell line. The expression of exogenous PTEN was confirmed (Figure 1B) and the phosphorylation level of AKT (Ser-473) was decreased by PTEN introduction in HEC-6 PTEN + cells (Figure 1B), which suggests that the PI3K signaling is affected by wild-type PTEN. However, the expression of RAD51 was not increased by the introduction of PTEN (Figure 1B). Thus, RAD51 expression is indicated to be regulated in a PTEN-independent manner in endometrial cancers.
Anti-proliferative effect of olaparib in endometrial cancer cell lines
SF50 values to olaparib and the PTEN status in endometrial cancer cell lines
P value (Student t test)
We also examined the effect of PTEN in other cell lines. Knocking down PTEN by siRNA in two cell lines with wild-type PTEN (HEC-1B and HEC-50B) did not affect the sensitivity in clonogenic assay (Additional file 1: Figure S1A). In addition, introduction of wild-type PTEN plasmid (pcDNA3-HA-PTEN) into a PTEN mutant cell line (AN3CA) did not alter the sensitivity, compared with the control (Additional file 1: Figure S1B).
These results suggest that accumulated DSB, following PARP inhibition by olaparib, successfully induces anti-proliferative effects in specific endometrial cancer cell lines and that PTEN status is not a useful biomarker to predict the effectiveness.
γH2AX and RAD51 foci formation following olaparib exposure in PTEN + and PTEN-null endometrial cells
Sensitivity to IR was not affected by PTEN status in HEC-6 cells
Effects of olaparib and IR on the cell cycle of HEC-6 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. . 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% . 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) . Preclinical studies have suggested that PTEN deficiency causes HR defects and possibly induces sensitivity to PARP inhibitors . 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 . 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) . The contribution of PTEN inactivation was also negatively reported in prostate cancer cell lines (22RV1, DU145, and PC3) .
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 . 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 . 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 . 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 . 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 . 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.
We thank Keiko Shoji, Michihiro Tanikawa, Yuichiro Miyamoto, Kensuke Tomio, and Satoko Kojima for their support and assistance. We also thank Masato Nishida for generously providing the Ishikawa cells. This work was financially supported by The Grant-in-aid for Scientific Research (C), Grant Number 23592437 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K Oda). This study was also performed as a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), Ministry of Education, Culture, Sports, Science and Technology of Japan (to T Yano).
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