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

Aki Miyasaka, Katsutoshi Oda, Yuji Ikeda, Osamu Wada-Hiraike, Tomoko Kashiyama, Atsushi Enomoto, Noriko Hosoya, Takahiro Koso, Tomohiko Fukuda, Kanako Inaba, Kenbun Sone, Yuriko Uehara, Reiko Kurikawa, Kazunori Nagasaka, Yoko Matsumoto, Takahide Arimoto, Shunsuke Nakagawa, Hiroyuki Kuramoto, Kiyoshi Miyagawa, Tetsu Yano, Kei Kawana, Yutaka Osuga, Tomoyuki Fujii, Aki Miyasaka, Katsutoshi Oda, Yuji Ikeda, Osamu Wada-Hiraike, Tomoko Kashiyama, Atsushi Enomoto, Noriko Hosoya, Takahiro Koso, Tomohiko Fukuda, Kanako Inaba, Kenbun Sone, Yuriko Uehara, Reiko Kurikawa, Kazunori Nagasaka, Yoko Matsumoto, Takahide Arimoto, Shunsuke Nakagawa, Hiroyuki Kuramoto, Kiyoshi Miyagawa, Tetsu Yano, Kei Kawana, Yutaka Osuga, Tomoyuki Fujii

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figures

Figure 1
Figure 1
Correlation between PTEN status and RAD51 expression in endometrial cancer cell lines. (A) PTEN and RAD51 expression (western blot) in a panel of 16 endometrial cancer cell lines. Cell lines with a PTEN mutation are denoted as (●). (B) Establishment of the HEC-6-PTEN + cell line. Levels of PTEN, total/phosphorylated AKT, and RAD51 were evaluated by western blot analysis.
Figure 2
Figure 2
Status of PTEN in endometrial cancer cells is irrelevant to the response to olaparib. (A) (B) Each cell line was treated with 5 concentrations of olaparib, and the cell proliferation was evaluated by a clonogenic assay. Cells were cultured for 14–21 d. Cells were continuously exposed to olaparib with media during the incubation. All experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. PTEN mutant cells (n = 12) are shown in (A: 6 cells in upper left and the other 6 in lower left), and wild-type cells (n = 4) are shown in (B). (C) Clonogenic assay comparing HEC-6-PTEN + cells with parental HEC-6 cells. SF50 values were 1,800 nM in both cell lines.
Figure 3
Figure 3
γH2AX and RAD51 foci formation in HEC-6 cells after olaparib treatment. (A) Immunofluorescence images of PTEN-/+ HEC6 cell lines: Hoechst-stained nuclei (blue), γ-H2AX (red), and RAD51 (green) after olaparib exposure (10 μM) for 24 h. (B) The number of γ-H2AX and RAD51 foci following exposure to olaparib (10 μM) was counted in the HEC-6 cell lines. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. (C) Time course expression of cleaved PARP and PTEN in PTEN-/+ HEC-6 cell lines. Proteins were extracted after 24 h of olaparib (10 μmol/L) exposure.
Figure 4
Figure 4
Response to IR in HEC-6 PTEN + and parental HEC-6 cell lines. (A) Total/cleaved PARP and total/phospho-PTEN expression in HEC-6 PTEN + and parental HEC-6 cell lines were examined by western blot analysis. Proteins were extracted after 10 Gy of IR at the indicated times. (B) Immunofluorescence images of PTEN-/+ HEC-6 cell lines: Hoechst-stained nuclei (blue), γ-H2AX (red), and RAD51 foci (green) after IR exposure (2 Gy). (C) The number of γ-H2AX and RAD51 foci following exposure to IR (2 Gy) was counted in the PTEN-/+ HEC-6 cell lines. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. (D) Clonogenic assay in the PTEN-/+ HEC-6 cell lines after exposure to IR at the indicated doses (2–6 Gy).
Figure 5
Figure 5
Cell cycle population was not affected by PTEN status in the HEC-6 endometrial cancer cells. Cell cycle populations following exposure to olaparib (10 μM, 72 h) or IR (10 Gy, 48 h) were determined by flow cytometry in PTEN-/+ HEC-6 cell lines.

References

    1. Satoh MS, Lindahl T. Role of poly (ADP-ribose) formation in DNA repair. Nature. 1992;356:356–358. doi: 10.1038/356356a0.
    1. Tutt AN, Lord CJ, McCabe N, Farmer H, Turner N, Martin NM, Jackson SP, Smith GC, Ashworth A. Exploiting the DNA repair defect in BRCA mutant cells in the design of new therapeutic strategies for cancer. Cold Spring Harb Symp Quant Biol. 2005;70:139–148. doi: 10.1101/sqb.2005.70.012.
    1. Dedes KJ, Wilkerson PM, Wetterskog D, Weigelt B, Ashworth A, Reis-Filho JS. Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations. Cell Cycle. 2011;10:1192–1199. doi: 10.4161/cc.10.8.15273.
    1. Bryant HE, Petermann E, Schultz N, Jemth AS, Loseva O, Issaeva N, Johansson F, Fernandez S, McGlynn P, Helleday T. PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination. EMBO J. 2009;28:2601–2615. doi: 10.1038/emboj.2009.206.
    1. Yang YG, Cortes U, Patnaik S, Jasin M, Wang ZQ. Ablation of PARP-1 does not interfere with the repair of DNA double-strand breaks, but compromises the reactivation of stalled replication forks. Oncogene. 2004;23:3872–3882. doi: 10.1038/sj.onc.1207491.
    1. Menear KA, Adcock C, Boulter R, Cockcroft XL, Copsey L, Cranston A, Dillon KJ, Drzewiecki J, Garman S, Gomez S, Javaid H, Kerrigan F, Knights C, Lau A, Loh VM Jr, Matthews IT, Moore S, O'Connor MJ, Smith GC, Martin NM. 4-[3-(4-cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin- 1-one: a novel bioavailable inhibitor of poly (ADP-ribose) polymerase-1. J Med Chem. 2008;51:6581–6591. doi: 10.1021/jm8001263.
    1. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, Mortimer P, Swaisland H, Lau A, O'Connor MJ, Ashworth A, Carmichael J, Kaye SB, Schellens JH, de Bono JS. Inhibition of poly (ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med. 2009;361:123–134. doi: 10.1056/NEJMoa0900212.
    1. Forster MD, Dedes KJ, Sandhu S, Frentzas S, Kristeleit R, Ashworth A, Poole CJ, Weigelt B, Kaye SB, Molife LR. Treatment with olaparib in a patient with PTEN-deficient endometrioid endometrial cancer. Nat Rev Clin Oncol. 2011;8:302–306. doi: 10.1038/nrclinonc.2011.42.
    1. Williamson CT, Muzik H, Turhan AG, Zamo A, O'Connor MJ, Bebb DG, Lees-Miller SP. ATM deficiency sensitizes mantle cell lymphoma cells to poly (ADP-ribose) polymerase-1 inhibitors. Mol Cancer Ther. 2010;9:347–357. doi: 10.1158/1535-7163.MCT-09-0872.
    1. Min A, Im SA, Yoon YK, Song SH, Nam HJ, Hur HS, Kim HP, Lee KH, Han SW, Oh DY, Kim TY, O'Connor MJ, Kim WH, Bang YJ. RAD51C-deficient cancer cells are highly sensitive to the PARP inhibitor olaparib. Mol Cancer Ther. 2013;12:865–877. doi: 10.1158/1535-7163.MCT-12-0950.
    1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62:10–29. doi: 10.3322/caac.20138.
    1. Oda K, Stokoe D, Taketani Y, McCormick F. High frequency of coexistent mutations of PIK3CA and PTEN genes in endometrial carcinoma. Cancer Res. 2005;65:10669–10673. doi: 10.1158/0008-5472.CAN-05-2620.
    1. Oda K, Okada J, Timmerman L, Rodriguez-Viciana P, Stokoe D, Shoji K, Taketani Y, Kuramoto H, Knight ZA, Shokat KM, McCormick F. PIK3CA cooperates with other phosphatidylinositol 3’-kinase pathway mutations to effect oncogenic transformation. Cancer Res. 2008;68:8127–8136. doi: 10.1158/0008-5472.CAN-08-0755.
    1. Enomoto T, Inoue M, Perantoni AO, Buzard GS, Miki H, Tanizawa O, Rice JM. K-ras activation in premalignant and malignant epithelial lesions of the human uterus. Cancer Res. 1991;51:5308–5314.
    1. Shoji K, Oda K, Nakagawa S, Hosokawa S, Nagae G, Uehara Y, Sone K, Miyamoto Y, Hiraike H, Hiraike-Wada O, Nei T, Kawana K, Kuramoto H, Aburatani H, Yano T, Taketani Y. The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas. Br J Cancer. 2009;101:145–148. doi: 10.1038/sj.bjc.6605109.
    1. Salvesen HB, MacDonald N, Ryan A, Jacobs IJ, Lynch ED, Akslen LA, Das S. PTEN methylation is associated with advanced stage and microsatellite instability in endometrial carcinoma. Int J Cancer. 2001;91:22–26. doi: 10.1002/1097-0215(20010101)91:1<22::AID-IJC1002>;2-S.
    1. Toda T, Oku H, Khaskhely NM, Moromizato H, Ono I, Murata T. Analysis of microsatellite instability and loss of heterozygosity in uterine endometrial adenocarcinoma. Cancer Genet Cytogenet. 2001;126:120–127. doi: 10.1016/S0165-4608(00)00400-3.
    1. Peiffer SL, Herzog TJ, Tribune DJ, Mutch DG, Gersell DJ, Goodfellow PJ. Allelic loss of sequences from the long arm of chromosome 10 and replication errors in endometrial cancers. Cancer Res. 1995;55:1922–1926.
    1. Shen WH, Balajee AS, Wang J, Wu H, Eng C, Pandolfi PP, Yin Y. Essential role for nuclear PTEN in maintaining chromosomal integrity. Cell. 2007;128:157–170. doi: 10.1016/j.cell.2006.11.042.
    1. Puc J, Keniry M, Li HS, Pandita TK, Choudhury AD, Memeo L, Mansukhani M, Murty VV, Gaciong Z, Meek SE, Piwnica-Worms H, Hibshoosh H, Parsons R. Lack of PTEN sequesters CHK1 and initiates genetic instability. Cancer Cell. 2005;7:193–204. doi: 10.1016/j.ccr.2005.01.009.
    1. Puc J, Parsons R. PTEN loss inhibits CHK1 to cause double stranded-DNA breaks in cells. Cell Cycle. 2005;4:927–929. doi: 10.4161/cc.4.7.1795.
    1. Fraser M, Zhao H, Luoto KR, Lundin C, Coackley C, Chan N, Joshua AM, Bismar TA, Evans A, Helleday T, Bristow RG. PTEN deletion in prostate cancer cells does not associate with loss of RAD51 function: implications for radiotherapy and chemotherapy. Clin Cancer Res. 2012;18:1015–1027. doi: 10.1158/1078-0432.CCR-11-2189.
    1. Kuramoto H, Nishida M, Morisawa T, Hamano M, Hata H, Kato Y, Ohno E, Iida T. Establishment and characterization of human endometrial cancer cell lines. Ann N Y Acad Sci. 1991;622:402–421. doi: 10.1111/j.1749-6632.1991.tb37884.x.
    1. Ikeda Y, Oda K, Nakagawa S, Murayama-Hosokawa S, Yamamoto S, Ishikawa S, Wang L, Takazawa Y, Maeda D, Wada-Hiraike O, Kawana K, Fukayama M, Aburatani H, Yano T, Kozuma S, Taketani Y. Genome-wide single nucleotide polymorphism arrays as a diagnostic tool in patients with synchronous endometrial and ovarian cancer. Int J Gynecol Cancer. 2012;22:725–731. doi: 10.1097/IGC.0b013e31824c6ea6.
    1. Shoji K, Oda K, Kashiyama T, Ikeda Y, Nakagawa S, Sone K, Miyamoto Y, Hiraike H, Tanikawa M, Miyasaka A, Koso T, Matsumoto Y, Wada-Hiraike O, Kawana K, Kuramoto H, McCormick F, Aburatani H, Yano T, Kozuma S, Taketani Y. Genotype-dependent efficacy of a dual PI3K/mTOR inhibitor, NVP-BEZ235, and an mTOR inhibitor, RAD001, in endometrial carcinomas. PLoS One. 2012;7:e37431. doi: 10.1371/journal.pone.0037431.
    1. Tanikawa M, Wada-Hiraike O, Nakagawa S, Shirane A, Hiraike H, Koyama S, Miyamoto Y, Sone K, Tsuruga T, Nagasaka K, Matsumoto Y, Ikeda Y, Shoji K, Oda K, Fukuhara H, Nakagawa K, Kato S, Yano T, Taketani Y. Multifunctional transcription factor TFII-I is an activator of BRCA1 function. Br J Cancer. 2011;104:1349–1355. doi: 10.1038/bjc.2011.75.
    1. Banerjee S, Kaye S. PARP inhibitors in BRCA gene-mutated ovarian cancer and beyond. Curr Oncol Rep. 2011;13:442–449. doi: 10.1007/s11912-011-0193-9.
    1. Chen Y, Zhang L, Hao Q. Olaparib: a promising PARP inhibitor in ovarian cancer therapy. Arch Gynecol Obstet. 2013;288:367–374. doi: 10.1007/s00404-013-2856-2.
    1. Chuang HC, Kapuriya N, Kulp SK, Chen CS, Shapiro CL. Differential anti-proliferative activities of poly (ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer cells. Breast Cancer Res Treat. 2012;134:649–659. doi: 10.1007/s10549-012-2106-5.
    1. McEllin B, Camacho CV, Mukherjee B, Hahm B, Tomimatsu N, Bachoo RM, Burma S. PTEN loss compromises homologous recombination repair in astrocytes: implications for glioblastoma therapy with temozolomide or poly (ADP-ribose) polymerase inhibitors. Cancer Res. 2010;70:5457–5464. doi: 10.1158/0008-5472.CAN-09-4295.
    1. Dedes KJ, Wetterskog D, Mendes-Pereira AM, Natrajan R, Lambros MB, Geyer FC, Vatcheva R, Savage K, Mackay A, Lord CJ, Ashworth A, Reis-Filho J. PTEN deficiency in endometrioid endometrial adenocarcinomas predicts sensitivity to PARP inhibitors. Sci Transl Med. 2010;2:53ra75.
    1. San Filippo J, Sung P, Klein H. Mechanism of eukaryotic homologous recombination. Annu Rev Biochem. 2008;77:229–257. doi: 10.1146/annurev.biochem.77.061306.125255.
    1. Mendes-Pereira AM, Martin SA, Brough R, McCarthy A, Taylor JR, Kim JS, Waldman T, Lord CJ, Ashworth A. Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors. EMBO Mol Med. 2009;1:315–322. doi: 10.1002/emmm.200900041.
    1. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273:5858–5868. doi: 10.1074/jbc.273.10.5858.
    1. Sung P, Krejci L, Van Komen S, Sehorn MG. Rad51 recombinase and recombination mediators. J Biol Chem. 2003;278:42729–42732. doi: 10.1074/jbc.R300027200.
    1. McCabe N, Turner NC, Lord CJ, Kluzek K, Bialkowska A, Swift S, Giavara S, O'Connor MJ, Tutt AN, Zdzienicka MZ, Smith GC, Ashworth A. Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly (ADP-ribose) polymerase inhibition. Cancer Res. 2006;66:8109–8115. doi: 10.1158/0008-5472.CAN-06-0140.
    1. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature. 2005;434:917–921. doi: 10.1038/nature03445.
    1. Ward JF. The yield of DNA double-strand breaks produced intracellularly by ionizing radiation: a review. Int J Radiat Biol. 1990;57:1141–1150. doi: 10.1080/09553009014551251.
    1. Minami D, Takigawa N, Takeda H, Takata M, Ochi N, Ichihara E, Hisamoto A, Hotta K, Tanimoto M, Kiura K. Synergistic effect of olaparib with combination of cisplatin on PTEN-deficient lung cancer cells. Mol Cancer Res. 2013;11:140–148. doi: 10.1158/1541-7786.MCR-12-0401.
    1. Chatterjee P, Choudhary GS, Sharma A, Singh K, Heston WD, Ciezki J, Klein EA, Almasan A. PARP inhibition sensitizes to low dose-rate radiation TMPRSS2-ERG fusion gene-expressing and PTEN-deficient prostate cancer cells. PLoS One. 2013;8:e60408. doi: 10.1371/journal.pone.0060408.

Source: PubMed

Sponsorer och medarbetare

Medicinska tillstånd

Läkemedelsinterventioner

3
Prenumerera