PARP9 is overexpressed in human breast cancer and promotes cancer cell migration

Introduction

Breast cancer is the most common type of cancer in women (1). Although the prognosis of human breast cancer can be significantly improved by surgical therapy, radiotherapy, chemotherapy, endocrine therapy and molecular targeted therapy, breast cancer incidence has increased in recent decades (2–5). Breast cancer comprises a group of heterogeneous diseases, whose prognoses differ due to variation in molecular and pathological characteristics (6). Treatment of breast cancer is based on various clinicopathological parameters, including the status of lymph node metastasis and hormone receptor expression (7,8). Invasion of the lymph nodes implicates that tumor cells may spread, and combined therapies are required (9). Furthermore, the expression of estrogen receptor (ER) provides a basis for endocrine therapy (10).

Numerous oncogenes are involved in the occurrence and progression of breast cancer. PARP is a family of proteins involved in cellular processes, including DNA repair and apoptosis (11). The most well-established PARP protein is PARP1, which can be cleaved and inactivated by caspase during apoptosis (12). Notably, PARP9 lacks PARP activity, despite having similar carboxyl-terminal amino acid sequences to other members of the PARP family (13). Previous studies suggest that PARP9 may promote metastasis in a variety of tumors. PARP9 is overexpressed in lymphoma and prostate cancer and has been demonstrated to positively correlate with metastasis, recurrence and chemotherapy resistance (14,15). However, the expression of PARP9 and its implication in breast cancer remain elusive. In the present study, the expression of PARP9 in 57 pairs of breast cancer and normal breast tissues was investigated, as well as the association between PARP9 and clinicopathological parameters. Furthermore, the effects of PARP9 on breast cancer-cell migration were detected.

Materials and methods

Tissues samples

A total of 57 pairs of breast cancer and normal breast tissues were obtained from the tissue bank at West China Hospital (Chengdu, China). All patients were female, who underwent surgery between February 2010 and May 2015. The diagnosis of breast cancer was confirmed by two patologists at West China Hospital, following biopsy. The expression of human epithelial growth factor receptor-2 (Her-2) was defined as positive by immunohistochemistry (3+) or fluoresence in situ hybridisation examination (+). Ki-67 levels were considered high if the percentage was ≥14% detected by immunohistochemistry.

No radiotherapy or chemotherapy had been received prior to operation. The age of patients ranged from 25–81 years, with a median age of 52 years. There were 54 cases of invasive ductal carcinoma, 2 cases of invasive lobular carcinoma and only 1 case of mucinous carcinoma.

Reagents

The polyclonal rabbit anti-human PARP9 antibody (cat. no. 17535-1-AP) was purchased from Proteintech, Inc. (Wuhan, China). The monoclonal mouse GAPDH (cat. no. 200306) and actin (cat. no. 250132) antibodies, and horseradish peroxidase-conjugated goat anti-mouse IgG (cat. no. 511103) and goat anti-rabbit IgG (cat. no. 511203) secondary antibodies were purchased from Zen Bioscience Co., Ltd. (Chengdu, China).

Western blot analysis

Tissues were lysed with ice-cold lysis buffer containing 1% Triton X-100, 40 mM Hepes pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.5 mM orthovanadate, PMSF and aprotinin (Roche Diagnostics, Basel, Switzerland). Tissue lysates were ground for 30 sec/time (4 times) until the tissues were totally triturated, then the mixture were incubated on ice for 30 min and centrifuged for 15 min at 14,000 × g to remove cell debris. The supernatants were harvested and protein concentration was using a BCA assay (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The proteins were heated at 100°C in 4× loading buffer [containing 200 nM Tri-HCl, 40% glycerol, 10% SDS (w/v), 400 nM DTT, 0.04% Bromphenil blue (w/v)] for 10 min, samples containing 20 µg protein were resolved by 10% SDS-PAGE, then transferred to a polyvinylidene difluoride membrane (Millipore; Merck KGaA, Darmstadt, Germany). Each membrane was blocked with 5% non-fat dry milk in Tris buffered saline with Tween-20 (TBST) for 1 h at room temperature. The membranes were then incubated with PARP9 (1:1,500), actin (1:1,000) and GAPDH (1:1,000) primary antibodies overnight at 4°C and the appropriate secondary antibodies (1:1,500) for 1 h at room temperature. Detection was performed using a chemiluminescence kit (BeyoECL plus; cat. no. P0018; Beyotime Biotechnology, Shanghai, China). Images were collected by Alpha Innotech's FluorChem imaging system (Alpha Innotech, San Leandro, CA, USA). GAPDH was used as an internal control. Western blots were subjected to densitometric analysis using Quantity One software (version 4.6.2; Bio-RAD, Hercules, CA, USA). The PARP9/GAPDH ratio in all breast cancer samples was calculated. The level of PARP9 expression was considered high if the PARP9/GAPDH ratio exceeded the median value in these samples.

Cell culture

The breast cancer cell line, HCC1806, was a gift from Professor Ceshi Chen (Kunming Institute of Zoology, Chinese Academy of Sciences, Shanghai, China). The cells were maintained in Dulbecco's minimal essential medium (Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C in a humidified atmosphere with 5% CO2.

RNA interference

All small interfering RNAs (siRNAs) were synthesized by GenePharma, Co, Ltd. (Shanghai, China). The target sequence used for knockdown of PARP9 was 5′-GGACAGAGTTAGAGATTGAAAC-3′, and the sequence of si-Control was 5′-ACGUGACACGUUCGGAGAATT-3′. siRNA (10 µM) were transfected into HCC1806 cells (~7,000 cells per well) by Lipofectamine RNAimax reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Cells were cultured for 48 h prior to subsequent western blotting.

Wound-healing assay

Cells were seeded in 6-well plates at 60,000 cells/well. The cells were cultured in DMEM medium containing 5% fetal bovine serum for 24 h. To inhibit cell proliferation, the cells were treated with 2 µg/ml mitomycin C at 37°C for 96 h. 24 h after the transfection of si-PARP9 or si-Control, a 1-mm scratch was made in the confluent cultures with a pipette tip, and washed twice with PBS to remove debris. The area of the scratch was measured by taking images under a phase-contrast microscope. To ensure the consistency of observation, specific observing points along the wound were labeled, and the same fields were observed at multiple time points.

Statistical analysis

Statistical analysis was conducted using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA) and Graphpad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) was used to produce graphs. Fisher's exact test was applied to determine statistical significance on the relationship between PARP9 and clinicopathological parameters. Student's t-test was used to determine the statistical difference in migration rate between siControl- and siPARP9-transfected cells. P<0.05 was considered to indicate a statistically significant difference.

Results

PARP9 is frequently overexpressed in human breast cancer

PARP9 expression was not detected in 43 normal breast tissues (75.4%), whereas it was detected in all 57 breast cancer tissues. Low levels of PARP9 (PARP9/GAPDH ratio <10% of the median value in cancer tissues) were detected in 13 cases (22.8%), and modest levels of PARP9 (PARP9/GAPDH ratio ~1:1) were detected in only 1 case (1.7%). In all 57 cases, the levels of PARP9 in breast cancer tissues were higher than that in paired normal breast tissues (Fig. 1). In 57 cases of breast cancer tissues, high levels of PARP9 were detected in 25 cases (43.8%), while low levels of PARP9 were detected in 32 cases (56.1%) (Fig. 2).

Figure 1. The expression of PARP9 in paired breast cancer and normal breast tissues. Cell lysates from breast cancer and normal breast samples were subjected to western blot analysis of PARP9 expression. PARP9, Poly(ADP-Ribose) polymerase family member 9; N, normal breast tissue; T, breast cancer tissue.

Figure 2. The expression of PARP9 in multiple breast cancer samples. Cell lysates from breast cancer samples were subjected to western blot analysis of PARP9 expression. PARP9, Poly(ADP-Ribose) polymerase family member 9; T, breast cancer tissue.

PARP9 expression is associated with lymph node metastasis

In 57 breast cancer tissues, 35 cases were ERα-positive, while 22 cases were ERα-negative. Overexpression of PARP9 was negatively associated with ERα expression (Table I). Axillary lymph nodes metastasis was detected in 31 cases (54.4%), and high levels of PARP9 were detected in 25 cases (43.8%) of breast cancer. Overexpression of PARP9 was positively associated with axillary lymph nodes metastasis (P<0.01; Table I). PARP9 expression was not associated with age, Her-2 expression, Ki-67 or tumor size (P>0.05; Table I).

Table I. The association between PARP9 expression and clinicopathological parameters.

Table I.

The association between PARP9 expression and clinicopathological parameters.

Clinicopathological parameters	PARP9high (n=25)	PARP9low (n=32)	P-value
Age (years)			0.108
≤45	8	18
>45	17	14
ER status			0.017
Positive	11	24
Negative	14	8
PR status			0.117
Positive	14	11
Negative	11	21
Her-2 status			0.592
Positive	12	14
Negative	11	18
Axillary lymph node metastasis			0.007
Positive	19	12
Negative	6	20
Ki-67 index (%)			0.135
≤14	4	11
>14	21	20
Tumor size (cm)			0.513
≤2	4	6
>2	21	25
Stage			0.575
I/II	16	23
III/IV	9	9
Molecular type			0.087
Luminal A	2	8
Luminal B	9	13
Her-2-enriched	7	2
Basal-like	7	7

[i] PARP9, Poly(ADP-Ribose) polymerase family member 9; ER, estrogen receptor; PR, progesterone receptor; Her-2, Human epidermal growth factor receptor 2.

PARP9-knockdown inhibits breast cancer-cell migration

Since overexpression of PARP9 was positively associated with lymph node metastasis, the effects of PARP9 on breast cancer-cell migration were investigated. PARP9 siRNA was transfected into HCC1806 cells, followed by a wound-healing assay. The efficiency of PARP-knockdown was confirmed by western blotting, and it was demonstrated that PARP9-knockdown significantly inhibited HCC1806 cell migration (Fig. 3).

Figure 3. The effect of PARP9-knockdown on breast cancer cell migration. HCC1806 cells were transfected with control siRNA or PARP9 siRNA, followed by wound-healing assays. (A) The effects of PARP9 on cell migration. (B) The relative migration rate at different time points. The migration rate for siControl-transfected cells at 96 h was set as 1. *P<0.05; **P<0.001. (C) The efficiency of PARP9-knockdown was confirmed by western blotting. PARP9, Poly(ADP-Ribose) polymerase family member 9; si, small interfering RNA.

Discussion

The progression of breast cancer involves aberrant expression of hormone receptors, growth factor receptors and other oncogenes, including mechanistic target of rapamycin kinase (16,17). In the present study, it was demonstrated that PARP9 was overexpressed in 43.8% of human breast cancer cases. Overexpression of PARP9 was negatively associated with ER expression, but positively associated with axillary lymph node metastasis. PARP9-knockdown was indicated to inhibit breast cancer cell migration. These data suggest that PARP9 may promote breast cancer progression.

A previous study demonstrated that PARP9 is involved in the interferon (IFN)-γ-signal transducer and activator of transcription 1 (STAT1) pathway. PARP9 interacts with STAT1α and STAT1β, upregulates the expression of interferon regulatory factor (IRF)2 and downregulates that of IRF1, thereby promoting cell proliferation, survival and migration (18). Also, PARP9 has been demonstrated to promote recurrence, metastasis and chemotherapy-resistance in prostate cancer and lymphoma (14,15). The expression of PARP9 has been demonstrated to be more abundant in the androgen-resistant prostate cell lines, PC3 and DU145, compared with the AR-dependent cell line, LNCap, and normal prostate epithelial cell line, SUDHL7 (15). PARP9 has been indicated to enhance tumor cell survival and doxorubicin-resistance via the E3 ubiquitin ligase, DTX3L, which selectively ubiquitinates histone H4 and protects cells against DNA-damaging agents following binding to PARP in chemotherapy-resistant diffuse large B cell lymphoma (19).

Although STAT1 has been established to have anti-tumor effects, increasing the expression of STAT1 and IFN-γ-induced genes contributed to metastatic potential by enhancing the ability of malignant cells to invade the stroma and migrate to distant sites in node-positive triple negative breast tumors (20). PARP9 has also been demonstrated to form complex with STAT1 and DTX3L, which may affect the nuclear activities of STAT1 by antagonistically regulating the tyrosine phosphorylation of STAT1 on Y701 (21). In consistence with previous studies on PARP9 in prostate cancer, the present study demonstrates that PARP9 promotes breast cancer cell migration. Thus, overexpression of PARP9 may promote the progression of human breast cancer.

Acknowledgements

Not applicable.

Funding

No finding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

XT, JJ and YJ conceived the study and wrote the manuscript. XT and HZ performed the experiments. YL and HH assisted in collecting and analyzing the patient data regarding the clinicopathological parameters of breast cancer. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The study was approved by the ethics committee of West China Medical Center, Sichuan University and informed consent to participate was signed by the patients and/or guardians.

Patient consent for publication

Informed consent was signed by the patients and/or guardians.

Competing interests

The authors declare that they have no competing interests.

References