Oncotarget, Vol. 7, No. 7
www.impactjournals.com/oncotarget/
Loss of CtIP disturbs homologous recombination repair and sensitizes breast cancer cells to PARP inhibitors Junhui Wang1,2,*, Qianshan Ding3,*, Hiroaki Fujimori1,5, Akira Motegi4, Yoshio Miki2, Mitsuko Masutani1,5 1
Division of Chemotherapy and Clinical Cancer Research, National Cancer Center Research Institute, Tokyo 104-0045, Japan
2
epartment of Molecular Genetics, Division of Medical Genomics, Medical Research Institute, Tokyo Medical and Dental D University, Tokyo 113-8510, Japan
3
Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, China
4
Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan
5
epartment of Frontier Life Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, D Japan
*
These authors have contributed equally to this work
Correspondence to: Mitsuko Masutani, e-mail: [email protected] Keywords: CtIP, breast cancer, PARP inhibitors, 53BP1 Received: September 18, 2015 Accepted: November 27, 2015 Published: December 22, 2015
ABSTRACT Breast cancer is one of the leading causes of death worldwide, and therefore, new and improved approaches for the treatment of breast cancer are desperately needed. CtIP (RBBP8) is a multifunctional protein that is involved in various cellular functions, including transcription, DNA replication, DNA repair and the G1 and G2 cell cycle checkpoints. CtIP plays an important role in homologous recombination repair by interacting with tumor suppressor protein BRCA1. Here, we analyzed the expression profile of CtIP by data mining using published microarray data sets. We found that CtIP expression is frequently decreased in breast cancer patients, and the patient group with low-expressing CtIP mRNA is associated with a significantly lower survival rate. The knockdown of CtIP in breast cancer MCF7 cells reduced Rad51 foci numbers and enhanced g H2AX foci formation after g-irradiation, suggesting that deficiency of CtIP decreases homologous recombination repair and delays DNA double strand break repair. To explore the effect of CtIP on PARP inhibitor therapy for breast cancer, CtIPdepleted MCF7 cells were treated with PARP inhibitor olaparib (AZD2281) or veliparib (ABT-888). As in BRCA mutated cells, PARP inhibitors showed cytotoxicity to CtIPdepleted cells by preventing cells from repairing DNA damage, leading to decreased cell viability. Further, a xenograft tumor model in mice with MCF7 cells demonstrated significantly increased sensitivity towards PARP inhibition under CtIP deficiency. In summary, this study shows that low level of CtIP expression is associated with poor prognosis in breast cancer, and provides a rationale for establishing CtIP expression as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients.
20 years in industrialized countries [1]. Therefore, the development of mechanism-based, targeted combination therapy that helps to improve disease-free survival and overall survival of breast cancer patients is still a major challenge.
INTRODUCTION Breast cancer is the most frequently diagnosed cancer and one of the leading causes of cancer-associated deaths in women. Incidence rates have risen over the past
www.impactjournals.com/oncotarget
7701
Oncotarget
Cells are continuously exposed to exogenous agents and biological processes that create DNA damage, which, if not repaired effectively and efficiently, can lead to genomic instability or cell death [2]. Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme that synthesizes the poly(ADP-ribose) polymer when activated by DNA single strand breaks (SSBs) [3]. Once single strand break (SSB) and double strand break (DSB) damage of DNA are produced, PARP-1 binds to the DNA and rapidly recruits x-ray repair complementation group 1(XRCC1) and tyrosyl DNA phosphodiesterase 1 (TDP1) to the site of damage to catalyze subsequent repair [4]. Repair of DSBs can be undertaken by two main pathways: homologous recombination (HR) repair (HRR) and non-homologous end-joining (NHEJ) repair [5]. BRCA1 and BRCA2 proteins normally function as important components of the HR pathway for the repair of DSBs. It has been reported that PARP inhibition leads to accumulation of DSBs by the failure of SSB repair and by replication fork collapse, which in turn requires HR-mediated repair [6-8]. In addition to blocking PARP catalytic activity at SSBs, PARP inhibitors can trap PARP enzyme at damaged DNA to form PARP-DNA complexes, which are more cytoxic [9]. PARP inhibition also leads to attenuation of alternative end joining repair through suppression of DNA polymerase θ recruitment [10, 11]. Cancers with BRCA1/2 mutations are defective in HRR and are therefore hypersensitive to PARP inhibitors [7]. Following this approach, recent clinical trials for the treatment of BRCA1/BRCA2-deficient breast and ovarian tumors using specific inhibitors targeting PARP have been performed with the concept of ‘synthetic lethality’ [4, 6, 12, 13]. However, whether synthetic lethality is applicable to human cancers that have acquired other mutations/deletions in DNA repair genes has not been widely investigated. Because CtIP (CtBP-interacting protein), which functions in the initial step of HRR with NBS1 and BRCA1 by acting as an end-resection enzyme to produce 3’-single stranded DNA, is known to be frequently downregulated in breast cancers as well as in other types of cancers, we focused on CtIP in this study and showed that breast cancer cells with defects in CtIP function are hypersensitive to the PARP inhibitors olaparib and veliparib. Thus, PARP inhibitors have therapeutic potential in the treatment of CtIP-deficient breast cancers, and our results might extend the concept of synthetic lethality to tumors bearing alterations in CtIP.
capped meiotic DSBs [15-20]. Therefore, we investigated the relationship of CtIP expression in breast cancers and their clinical outcome by using two publicly available microarray datasets in the Gene Expression Omnibus (GEO) database (GSE10780 [21] and GSE3744 [21]) that contain both normal and breast cancer samples. CtIP expression levels were measured as log2 (probe intensities) using Affymetrix microarrays. In both these two datasets, the levels of CtIP mRNA in breast cancers were statistically lower than those in normal breast tissues (Fig.1A and Supplemental Fig.1A). Because patients diagnosed with triple negative breast cancer (TNBC) have a higher risk of disease relapse within 5 years than patients treated for other breast cancer subtypes [22], we compared the CtIP mRNA level in TNBC to that in non-TNBC by using one publicly available microarray dataset (GSE47561 [23]). We found that CtIP expression was significantly lower in triple negative breast cancer (Fig.1B), which is consistent with the previous study [24]. These results indicate that the expression level of CtIP is decreased in breast cancers. Furthermore, CtIP gene alterations were observed in several cancer types with either mutation, deletion or copy number variation (Supplemental Fig. 1C). However, the incidence of deletion or mutation of CtIP was rather low in cancers. We thus speculated that CtIP down-regulation might be due to epigenetic or posttranscriptional regulation. To determine the clinical impact of reduced CtIP expression in human breast cancer, we assessed the association between CtIP mRNA levels and clinical outcome in three independent breast cancer cohorts [25-27] with clinical information (GEO database). To investigate the prognostic impact of CtIP expression in breast cancer, breast cancer patients were categorized into two groups based on CtIP mRNA expression. We found that patients with cancers displaying low CtIP expression levels had significantly shorter overall survival compared to those with high CtIP (Fig. 1C-1D, Supplemental Figure 1B). Additionally, we confirmed the correlation between CtIP expression and clinicopathological variables. Clinical data from GEO dataset GSE3494 was used for this analysis [25]. The samples pooled in the dataset were divided into two groups according to the expression level of CtIP in tumor tissue and χ 2 test was performed. As shown in Table 1, low level of CtIP expression was associated with p53 mutation (P=0.0025), PR status (P=0.0312), larger tumor (P=0.0082) and lymph nodes metastasis (P 20th percentile) CtIP expression (P = 0.018, log-rank test) (GSE3494). D. KaplanMeier survival curves comparing disease-free survival between cases with the lowest (≤ 20th percentile) vs. highest (> 20th percentile) CtIP expression (P = 0.0084, log-rank test) (GSE21653). www.impactjournals.com/oncotarget
7703
Oncotarget
Table 1: The correlation between CtIP expression and clinicopathological variables Characteristics
No. of patients
Expression High
Low
Chi-square value
P value
0.45
0.5029
0.04
0.8355
9.15
0.0025
2.27
0.1317
4.64
0.0312
4.94
0.0847
6.98
0.0082
19.06
www.impactjournals.com/oncotarget/
Loss of CtIP disturbs homologous recombination repair and sensitizes breast cancer cells to PARP inhibitors Junhui Wang1,2,*, Qianshan Ding3,*, Hiroaki Fujimori1,5, Akira Motegi4, Yoshio Miki2, Mitsuko Masutani1,5 1
Division of Chemotherapy and Clinical Cancer Research, National Cancer Center Research Institute, Tokyo 104-0045, Japan
2
epartment of Molecular Genetics, Division of Medical Genomics, Medical Research Institute, Tokyo Medical and Dental D University, Tokyo 113-8510, Japan
3
Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, China
4
Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan
5
epartment of Frontier Life Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, D Japan
*
These authors have contributed equally to this work
Correspondence to: Mitsuko Masutani, e-mail: [email protected] Keywords: CtIP, breast cancer, PARP inhibitors, 53BP1 Received: September 18, 2015 Accepted: November 27, 2015 Published: December 22, 2015
ABSTRACT Breast cancer is one of the leading causes of death worldwide, and therefore, new and improved approaches for the treatment of breast cancer are desperately needed. CtIP (RBBP8) is a multifunctional protein that is involved in various cellular functions, including transcription, DNA replication, DNA repair and the G1 and G2 cell cycle checkpoints. CtIP plays an important role in homologous recombination repair by interacting with tumor suppressor protein BRCA1. Here, we analyzed the expression profile of CtIP by data mining using published microarray data sets. We found that CtIP expression is frequently decreased in breast cancer patients, and the patient group with low-expressing CtIP mRNA is associated with a significantly lower survival rate. The knockdown of CtIP in breast cancer MCF7 cells reduced Rad51 foci numbers and enhanced g H2AX foci formation after g-irradiation, suggesting that deficiency of CtIP decreases homologous recombination repair and delays DNA double strand break repair. To explore the effect of CtIP on PARP inhibitor therapy for breast cancer, CtIPdepleted MCF7 cells were treated with PARP inhibitor olaparib (AZD2281) or veliparib (ABT-888). As in BRCA mutated cells, PARP inhibitors showed cytotoxicity to CtIPdepleted cells by preventing cells from repairing DNA damage, leading to decreased cell viability. Further, a xenograft tumor model in mice with MCF7 cells demonstrated significantly increased sensitivity towards PARP inhibition under CtIP deficiency. In summary, this study shows that low level of CtIP expression is associated with poor prognosis in breast cancer, and provides a rationale for establishing CtIP expression as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients.
20 years in industrialized countries [1]. Therefore, the development of mechanism-based, targeted combination therapy that helps to improve disease-free survival and overall survival of breast cancer patients is still a major challenge.
INTRODUCTION Breast cancer is the most frequently diagnosed cancer and one of the leading causes of cancer-associated deaths in women. Incidence rates have risen over the past
www.impactjournals.com/oncotarget
7701
Oncotarget
Cells are continuously exposed to exogenous agents and biological processes that create DNA damage, which, if not repaired effectively and efficiently, can lead to genomic instability or cell death [2]. Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme that synthesizes the poly(ADP-ribose) polymer when activated by DNA single strand breaks (SSBs) [3]. Once single strand break (SSB) and double strand break (DSB) damage of DNA are produced, PARP-1 binds to the DNA and rapidly recruits x-ray repair complementation group 1(XRCC1) and tyrosyl DNA phosphodiesterase 1 (TDP1) to the site of damage to catalyze subsequent repair [4]. Repair of DSBs can be undertaken by two main pathways: homologous recombination (HR) repair (HRR) and non-homologous end-joining (NHEJ) repair [5]. BRCA1 and BRCA2 proteins normally function as important components of the HR pathway for the repair of DSBs. It has been reported that PARP inhibition leads to accumulation of DSBs by the failure of SSB repair and by replication fork collapse, which in turn requires HR-mediated repair [6-8]. In addition to blocking PARP catalytic activity at SSBs, PARP inhibitors can trap PARP enzyme at damaged DNA to form PARP-DNA complexes, which are more cytoxic [9]. PARP inhibition also leads to attenuation of alternative end joining repair through suppression of DNA polymerase θ recruitment [10, 11]. Cancers with BRCA1/2 mutations are defective in HRR and are therefore hypersensitive to PARP inhibitors [7]. Following this approach, recent clinical trials for the treatment of BRCA1/BRCA2-deficient breast and ovarian tumors using specific inhibitors targeting PARP have been performed with the concept of ‘synthetic lethality’ [4, 6, 12, 13]. However, whether synthetic lethality is applicable to human cancers that have acquired other mutations/deletions in DNA repair genes has not been widely investigated. Because CtIP (CtBP-interacting protein), which functions in the initial step of HRR with NBS1 and BRCA1 by acting as an end-resection enzyme to produce 3’-single stranded DNA, is known to be frequently downregulated in breast cancers as well as in other types of cancers, we focused on CtIP in this study and showed that breast cancer cells with defects in CtIP function are hypersensitive to the PARP inhibitors olaparib and veliparib. Thus, PARP inhibitors have therapeutic potential in the treatment of CtIP-deficient breast cancers, and our results might extend the concept of synthetic lethality to tumors bearing alterations in CtIP.
capped meiotic DSBs [15-20]. Therefore, we investigated the relationship of CtIP expression in breast cancers and their clinical outcome by using two publicly available microarray datasets in the Gene Expression Omnibus (GEO) database (GSE10780 [21] and GSE3744 [21]) that contain both normal and breast cancer samples. CtIP expression levels were measured as log2 (probe intensities) using Affymetrix microarrays. In both these two datasets, the levels of CtIP mRNA in breast cancers were statistically lower than those in normal breast tissues (Fig.1A and Supplemental Fig.1A). Because patients diagnosed with triple negative breast cancer (TNBC) have a higher risk of disease relapse within 5 years than patients treated for other breast cancer subtypes [22], we compared the CtIP mRNA level in TNBC to that in non-TNBC by using one publicly available microarray dataset (GSE47561 [23]). We found that CtIP expression was significantly lower in triple negative breast cancer (Fig.1B), which is consistent with the previous study [24]. These results indicate that the expression level of CtIP is decreased in breast cancers. Furthermore, CtIP gene alterations were observed in several cancer types with either mutation, deletion or copy number variation (Supplemental Fig. 1C). However, the incidence of deletion or mutation of CtIP was rather low in cancers. We thus speculated that CtIP down-regulation might be due to epigenetic or posttranscriptional regulation. To determine the clinical impact of reduced CtIP expression in human breast cancer, we assessed the association between CtIP mRNA levels and clinical outcome in three independent breast cancer cohorts [25-27] with clinical information (GEO database). To investigate the prognostic impact of CtIP expression in breast cancer, breast cancer patients were categorized into two groups based on CtIP mRNA expression. We found that patients with cancers displaying low CtIP expression levels had significantly shorter overall survival compared to those with high CtIP (Fig. 1C-1D, Supplemental Figure 1B). Additionally, we confirmed the correlation between CtIP expression and clinicopathological variables. Clinical data from GEO dataset GSE3494 was used for this analysis [25]. The samples pooled in the dataset were divided into two groups according to the expression level of CtIP in tumor tissue and χ 2 test was performed. As shown in Table 1, low level of CtIP expression was associated with p53 mutation (P=0.0025), PR status (P=0.0312), larger tumor (P=0.0082) and lymph nodes metastasis (P 20th percentile) CtIP expression (P = 0.018, log-rank test) (GSE3494). D. KaplanMeier survival curves comparing disease-free survival between cases with the lowest (≤ 20th percentile) vs. highest (> 20th percentile) CtIP expression (P = 0.0084, log-rank test) (GSE21653). www.impactjournals.com/oncotarget
7703
Oncotarget
Table 1: The correlation between CtIP expression and clinicopathological variables Characteristics
No. of patients
Expression High
Low
Chi-square value
P value
0.45
0.5029
0.04
0.8355
9.15
0.0025
2.27
0.1317
4.64
0.0312
4.94
0.0847
6.98
0.0082
19.06
