RNF126 as a biomarker of a poor prognosis in invasive breast cancer and CHK1 inhibitor efficacy in breast cancer cells

Abstract

Purpose

1) To investigate expression of the E3 ligase, RNF126, in human invasive breast cancer (BC) and its links with BC outcomes. 2) To test the hypothesis that RNF126 determines the efficacy of inhibitors targeting the cell cycle checkpoint kinase, CHK1.

Experimental Design

A retrospective analysis by immunohistochemistry (IHC) compared RNF126 staining in 110 invasive BC and 78 paired adjacent normal tissues with clinicopathologic data. Whether RNF126 controls CHK1 expression was determined by chromatin immunoprecipitation and a CHK1 promoter driven luciferase reporter. Staining for these two proteins by IHC using tissue microarrays was also conducted. Cell killing/replication stress induced by CHK1 inhibition was evaluated in cells, with or without RNF126 knockdown, by MTT/colony formation, replication stress biomarker immunostaining and DNA fiber assays.

Results

RNF126 protein expression was elevated in BC tissue samples. RNF126 was associated with a poor clinical outcome after multivariate analysis and was an independent predictor. RNF126 promotes CHK1 transcript expression. Critically, a strong correlation between RNF126 and CHK1 proteins was identified in BC tissue and cell lines. The inhibition of CHK1 induced a greater cell killing and a higher level of replication stress in BC cells expressing RNF126 compared to RNF126 depleted cells.

Conclusions

RNF126 protein is highly expressed in invasive BC tissue. The high expression of RNF126 is an independent predictor of a poor prognosis in invasive BC and is considered a potential biomarker of a cancer’s responsiveness to CHK1 inhibitors. CHK1 inhibition targets BC cells expressing higher levels of RNF126 by enhancing replication stress.

Introduction

Ionizing radiation (IR) and most chemotherapies damage DNA as a major part of their mechanism of action. These remain standard therapies for all types of breast cancers (BC), the most common cancer affecting women and the second most common cause of death due to cancers (1). The choice of radiotherapy (RT) or chemotherapy (CT) for BC is currently made according to clinical factors. However, a subtype of BC may be intrinsically resistant due to an upregulation of the DNA damage response (DDR), a major mechanism antagonizing DNA damage caused by RT/CT and involving cell cycle checkpoints and DNA repair. This would result in a BC patient subtype receiving unnecessary, aggressive treatments with minimal benefit. In addition, resistance to RT/CT may lead to tumor recurrence that can cause considerable morbidity, the dissemination of disease and an increased probability of mortality due to BC (2,3). Thus, there is an important need for identifying patients who are more likely to fail therapy and to improve treatment plans for those patients. By conducting large-scale profiling of cellular survival after exposure to radiation in a diverse collection of 533 genetically annotated human tumor cell lines, including BC cell lines, a recent study demonstrated a broad variation in the response to RT, and perhaps also CT (4), as a result of genetic alterations. Thus, it is critical to identify patients whose BC subtypes are intrinsically resistant to RT/CT and to explore new approaches to target their cancers.

RNF126 is a ring E3 ligase. Recent studies have suggested that RNF126 may have broad functions by targeting a variety of proteins for degradation; these may range from a role in endosomal sorting to the BAG6-dependent quality control of mislocalized proteins (5–10). RNF126 also promotes the proliferation of BC by ubiquitinating CDKN1A/p21 and targeting it for degradation (11). By promoting non-homologous end joining (NHEJ) and homologous recombination (HR) (12,13) RNF126 promotes the repair of DNA double-stranded breaks (DSBs), the most dangerous type of DNA damage and that can be caused by endogenous and exogenous sources such as replication stress, RT and chemotherapeutic drugs. RNF126 promotes NHEJ via the ubiquitination of Ku80 (12). Interestingly, we recently reported that RNF126 facilitates HR by promoting the expression of BRCA1 in a manner independent of its E3 ligase activity but dependent on its interaction with E2F1 (13). A member of the family of E2F transcription factors, E2F1 is required for the expression of genes involved in a wide range of cellular processes, including cell-cycle progression, DNA replication, DNA repair, differentiation, and apoptosis. Consistent with its role in HR and NHEJ, RNF126 expression is associated with resistance to ionizing radiation (IR) and poly (ADP-ribose) polymerase (PARP) inhibition (13) since both pathways are required to repair DSBs caused by IR and/or a PARP inhibitor. Thus, RNF126 appears to be associated with a diverse set of cellular processes in which its E3 ligase activity may or may not be involved. RNF126 has a close relative, BCA2, that shares 46% overall amino acid identity, and 75% identity in RING domains. Although BCA2 is highly expressed and is a prognostic biomarker in BC (14–16), the pattern of RNF126 protein expression and its association with outcomes of BC have not yet been evaluated.

The cell cycle checkpoint kinase, ataxia telangiectasia mutated and Rad3-related kinase (ATR) and its key downstream effector, CHK1, can be activated by RPA (replication protein A)-coated elongated ssDNA. The ATR/CHK1 pathway prevents the entry of cells with damaged or incompletely replicated DNA into mitosis when cells are challenged by DNA damaging agents, such as IR or chemotherapeutic drugs, the major modalities used to treat cancers. This regulation is particularly evident in cells with a defective G1 checkpoint, a common feature of cancer cells, owing to mutations in p53. In addition, ATR and CHK1 suppress replication stress by inhibiting excess origin firing, particularly in cells with activated oncogenes (17–19). Thus, ATR and CHK1 inhibitors have been developed and are currently used either as single agents, or paired with radiotherapy or a variety of genotoxic chemotherapies in preclinical and clinical studies. Although CHK1 inhibitors were initially thought to enhance the effects of radiotherapy and genotoxic drugs, particularly in p53-deficient cells (20–22), recent preclinical studies suggest that CHK1 inhibitors may function as signal agents since cancer cells are more reliant on ATR/CHK1 for survival (18). Indeed, ATR/CHK1 inhibition specifically target cancer cells expressing Myc, Cyclin E, and Ras (19,23–26). In addition, we recently also reported that CHK1 inhibitors, as single agents, have antitumor activity in radioresistant BC by enhancing replication stress (25). Radioresistant BC cells carry high levels of c-Myc/CDC25A/c-Src/H-Ras/E2F1 oncogenes and ATR/CHK1/BRCA1/CtIP DDR proteins, indicating that upregulation of DDR proteins, including cell cycle checkpoint proteins and oncogenes, may be characteristic of being targeted by CHK1 inhibitors (25). Since we have demonstrated that RNF126 binds to E2F1 (13), a transcription regulator that controls the expression of several hundred genes, including oncogenes and CHK1 (27–30), we hypothesize that RNF126 promotes CHK1 expression and that cells expressing high levels of RNF126 may be targeted by CHK1 inhibitors. Thus, the aims of the current study are to investigate the clinical significance of RNF126 expression in BC, and to determine the role of RNF126 in promoting CHK1 expression, with particular attention to its influence on the efficacy of CHK1 inhibitors.

Here, we demonstrate that RNF126 protein is highly expressed in invasive BC and is associated with a poor prognosis. Critically, RNF126 is an independent factor for a poor prognosis. We also reveal that RNF126 controls CHK1 expression via a direct interaction with E2F1. A strong correlation between RNF126 and CHK1 protein expression exists in both BC tissues and cell lines. Treatment with a CHK1 inhibitor led to increased cell killing by enhancing replication stress in cells expressing a higher level of RNF126. Our studies suggest that RNF126 is a potential biomarker for the poor prognosis of invasive BC and for the efficacy of CHK1 inhibitors.

Materials and Methods

Patients and Specimen Collection

Two cohorts of BC samples were included. The first cohort consisted of 110 paraffin-embedded tumor tissues from patients with invasive BC, as well as 78 paired adjacent normal tissues as negative controls. The samples were taken from The First Affiliated Hospital of Sun Yat-sen University from January 1, 2004 to December 31, 2006. The patients in this study all had primary operable invasive BC and were under the care of a single surgeon, Professor Zhefu Ma. The most common histological BC type was invasive ductal carcinoma that comprised 88.18% (97/110) of cases, with the other 13 cases being invasive lobular, medullary or mucinous carcinomas. Treatment included mastectomy or local excision, with or without adjuvant systemic chemotherapy and/or radiotherapy. Follow-up involved clinical reviews at six-monthly intervals for the first 5 years, and then annually. All samples were confirmed histologically by two pathologists. Histological diagnosis was determined according to American Joint Commission on Cancer Staging (AJCC) criteria. Conventional pathological data were collected retrospectively and characteristics of primary BCs are summarized in Table S1. A second cohort that consisted of 67 invasive BC cases was prepared for TMA analysis of the expression of both RNF126 and CHK1. The patients of the second cohort were treated at the same hospital between January 1, 2014 and April 1, 2015.

All patients were female and signed informed consent forms. The protocol was approved by the ethics committee of The First Affiliated Hospital of Sun Yat-sen University (application ID: [2016]060). Inclusive criteria were: (1) All patients had unilateral invasive BC and underwent either a radical mastectomy or modified radical mastectomy. Adjacent normal breast tissues were selected from an area more than 5 cm from the edge of the tumor and were confirmed by two pathologists. (2) Patients who received preoperative radiotherapy, chemotherapy, hormonal therapy, or any other anti-cancer therapy before resection were excluded. (3) Adjuvant treatments such as chemotherapy, radiotherapy, or endocrine therapy were chosen based on the patient's condition after surgery in accordance with the relevant The National Comprehensive Cancer Network guidelines (NCCN). (4) All patients were followed up with medical appointments or by telephone. Cancer recurrence, metastasis, or death were end events. The follow-up deadline was April 18, 2016.

Immunohistochemistry

Full tissue sections of 110 paraffin embedded invasive BC and 78 normal tissues were processed for immunohistochemical staining of RNF126. TMA blocks was constructed containing 67 invasive BC. Serial 4-µm sections were cut from the TMA blocks for both RNF126 and CHK1 staining. Antigen retrieval, blocking procedures, and a modified ImmunoMax method were used as previously described (31). In brief, slides were heated to 60°C and then deparaffinized in xylene. The slides were rehydrated in descending alcohol concentrations. Antigen retrieval was performed by incubating slides in a retrieval solution of citrate buffer. Hydrogen peroxide was added to block endogenous peroxidase activity in order to decrease unwanted background staining. Primary antibody (ab183102, 1:100; Abcam, Cambridge, UK; 25887-1-AP, 1:150; Proteintech, Rosemont, IL, USA) was added at an optimum dilution. Negative controls were performed by the substitution of primary antibody with phosphate buffered saline (PBS). To guarantee consistent immunohistochemical evaluation, slides from a reference tumor previously determined as positive were included in each staining procedure.

Immunohistochemical Scoring

Evaluations of staining reactions were performed in accordance with the immunoreactive score (IRS) proposed by Remmele and Stegner: IRS = staining intensity (SI) × percentage of positive cells (PP). Staining intensity was marked as non-granulated [0]; low grade (light yellow) [1]; moderate (brownish yellow) [2]; or strong (reddish brown) [3]. The PP was scored as negative (<5%) [0]; weak (5–10%) [1]; moderate (11–50%) [2]; strong (51%–80%) [3]; or very strong (>81%) [4]. Specimens scoring beyond 3 were considered positive overexpression (32). All slides were independently evaluated by two pathologists blind to patients and their corresponding clinical information.

Cell lines, Infections, Transfections and CHK1 Inhibitors

MCF7, MDA-MB-231, SKBR3, MDA-MB-361, MCF10A, HCC202, ZR751, T47D, MDA-MB-468, HCC1187, HCC1569, HCC70, BT549, HCC1143, BT474, HCC38, and HCC1954 were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum (FBS; Gibco/Thermo Fisher Scientific, Waltham, MA, USA), in a humidified atmosphere containing 5% CO₂ at 37°C. The shRNA of RNF126 was purchased from Sigma–Aldrich (St Louis, MO, USA). Full-length wild-type RNF126 and RING-domain mutated (C229A/C232A) RNF126 have been described previously (11). The CHK1-promoter reporter was a gift from Dr. Pier Paolo Pandolfi (Beth Israel Deaconess Cancer Center, Boston). All DNA-plasmid transfections were performed using Lipofectamine 2000 according to the manufacturer's recommendations (Invitrogen). Flag-RNF126 construct, the full-length RNF126 fragment, has been described previously (13). Two CHK1 inhibitors were used in this study, including LY2603618 from ApexBio (A8638; Hsinchu, Taiwan) and AZD7762 from Selleckchem (S1532; Houston, TX, USA).

MTT and Colony Formation Assays

For the MTT assay, cells were plated into 96-well plates and incubated overnight. Cells were then exposed to various doses of CHK1 or ATR inhibitors for 72 h. MTT (20 µL of 5 mg/mL) was added to each well and cells incubated for a further 3.5 h in an incubator. MTT solvent was added after removing the medium and the cells in plates were agitated on an orbital shaker for 15 min. The absorbance was read at 590 nm with a reference filter of 620 nm. For clonogenic survival assays, cells plated into petri dishes (60 mm × 15 mm) were exposed to various doses of CHK1 or ATR inhibitors for 24 h, and then replaced with fresh medium. After 13–15 days of incubation at 37°C, the cells were stained using Giemsa. The number of colonies (>50 cells) was counted.

Quantitative Reverse Transcription-Polymerase Chain Reaction

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was conducted as described previously (13). Total RNA was isolated using an RNeasy Kit (Qiagen). Experiments were carried out in triplicate for each data point. Reactions were performed using SYBR Green mix and a MyiQ real-time PCR detection system (Bio-Rad). Relative mRNA levels were calculated using the comparative Ct method (ΔCt).

• GAPDH forward/ reverse primers: 5’-CTCTGCTCCTCCTGTTCGAC-3’/5’-TTAAAAGCAGCCCTGGTGAC-3’

• CHK1 forward/ reverse primers: 5’-CCAGATGCTCAGAGATTCTTCCA-3’/ 5’-TGTTCAACAAACGCTCACGATTA-3’.

• E2F1 forward/ reverse primers: 5’-GTGGACTCTTCGGAGAACTT-3’/5’-TGTTCTCCTCCTCAGAAGTG-3’.

• Cyclin E forward/ reverse primers: 5’-TTTCTTGAGCAACACCCTC-3’/5’-TGTCACATACGCAAACTGG-3’.

• RNF126 forward/reverse primers: 5’-TATCGAGGAGCTTCCGGAAGAGA-3’/5’-AAAGCAAACTGTCCGTAGCCCT-3’

Chromatin Immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) was performed using a Simple ChIP Enzymatic Chromatin IP kit (#9002, Cell Signaling Technology). In brief, 5 × 10⁷ cells were fixed with 1% final concentration of formaldehyde for 10 min at room temperature. The formaldehyde was quenched by adding 125 mM glycine for 5 min at room temperature. Cells were washed with cold PBS containing a protease inhibitor cocktail and were then lysed with cold Buffer A. Collected pellets were resuspended in cold Buffer B and then treated with 7 µL of micrococcal nuclease for 30 min at 37°C. Digested chromatin was sonicated and purified according to kit instructions. Chromatin (10 µg) was incubated with the following antibodies: 2 µg E2F1 (Santa Cruz, sc193), 2 µg H3 (provided by the kit), and 2 µg IgG (provided by the kit). The primers used to amplify the regions containing the putative consensus DNA-binding sites of RNF126 in the CHK1 promoter by PCR were as follows: forward 5′-AGCACTCTGCTTCACCGACT-3, reverse 5′-CTGGGCCCAAATATGAAGTG-3′.

Immunofluorescence Analysis

Immunofluorescence assays were performed as described previously (25). Cells growing on slides were fixed directly in 3–4% paraformaldehyde. For unextractable CDC45 staining, cells were extracted for 5 min on ice with 0.5% Triton X-100 in cytoskeletal (CSK) buffer (10 mM PIPES, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl₂; pH = 6.8) supplemented with 1 mM phenylmethylsulfonyl fluoride, 0.5 mM sodium vanadate and proteasome inhibitor for 10 min at 4°C. Then, extracted cells were fixed with 3–4% paraformaldehyde. The cells were permeabilized for 10 min with PBS containing 0.5% Triton X-100 for 15 min at room temperature, followed by blocking with 1% bovine serum albumin (BSA) and then incubated with primary antibodies. The bound antibodies were revealed with goat anti-mouse IgG Alexa Fluor 594 and chicken anti-rabbit IgG Alexa Fluor 488. Slides were viewed at 1000× magnification with an NIKON 90i fluorescence microscope (photometric cooled mono CCD camera).

Immunoblotting

Cellular extracts were prepared by resuspending cells in RIPA lysis buffer and proteins were resolved by 5%, 12% or 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). For chromatin CDC45 isolation, chromatin-bound proteins were prepared according to a previous publication (33). In brief, 3×10⁶ cells were resuspended in 200 µl of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol, and protease inhibitor mixture (Roche Molecular Biochemicals)). Triton X-100 was added to a final concentration of 0.1%, and the cells were incubated for 10 min on ice. Nuclei were collected in the pellet (P1) by low speed centrifugation (1500×g, 4 min, 4 °C). The supernatant (S1) was further clarified by high speed centrifugation (13,000×g, 10 min, 4 °C) to remove cell debris and insoluble aggregates. Nuclei (P1) were washed once with buffer A and then lysed in 200 µl of buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and protease inhibitor mixture). After a 10-min incubation on ice, soluble nuclear proteins (S2) were separated from chromatin by centrifugation (2000×g, 4 min). Insoluble chromatin (P2) was washed once in buffer B, and centrifuged again under the same conditions. The final chromatin pellet (P3) was resuspended in 30 µl Laemmli buffer and sonicated for 30 s in a sonicator using a microtip at 25% amplitude. The fractioned chromatin-bound protein was denatured by boiling the sample for 5–10 min, and analyzed by immunoprecipitation.

Dual-Luciferase Assays

Dual-luciferase assays were conducted as described previously (34) Cell extracts were prepared according to the instructions of the manufacturer and assayed in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) using the Dual-Luciferase Reporter assay System (E1910, Promega). Briefly, cells were co-transfected with vector control, RNF126-WT, RNF126-Δf or RNF126-C229A/C232A and CHK1-reporter vector in the ratios of 10:1 for 6 h, then replaced with fresh medium and continually cultured for additional 48 h. The cells were washed with PBS, and then lysed with PLB reagent. Lastly, the lysate were detected with a TD-20/20 luminometer according to the manufacturer’s instructions.

Cell-Cycle Analysis

Cell-cycle analyses were conducted as described previously (25). Cells were collected and fixed with cold 70% ethanol. Approximately 10⁶ cells/mL were incubated for 30 min with staining solution containing RNase A (10 µg/mL, Sigma), and propidium iodide (20 µg/mL, Sigma) for 30 min. The DNA content was measured by flow cytometry.

DNA Fiber Assays

DNA fiber assays were performed as published with some modifications (25,35). Cells were pulse-labeled with 50 µM IdU (Sigma–Aldrich, I7125) for 40 min and then pulse-labeled with 200 µM CldU (Sigma–Aldrich #C6891) for 40 min in the presence or absence of CHK1 inhibitor. At the end of the CldU pulse, cell suspensions (2.5 µL) were mixed with 7.5 µL of lysis buffer (0.5% sodium dodecyl sulfate, 200 mM Tris-HCl [pH 7.4], 50 mM EDTA). Each mixture was dropped on the top of an uncoated regular glass slide. Slides were inclined at 25° to spread the suspension on the glass. Once dried, DNA spreads were fixed by incubation for 10 min in a 3:1 solution of methanol-acetic acid. The slides were dried and placed in precooled 70% ethanol at 4°C for at least 1 h. DNA was denatured with 2.5 M HCl for 30 min at 37°C. The slides were blocked in 1% BSA in PBS for 30 min at room temperature and then incubated with mouse anti-BrdU antibody (BD Biosciences, #347580) at a 1:200 dilution and rat anti-CldU antibody (Abcam, #ab6326) at a 1:400 dilution. The slides were incubated with secondary fluorescent antibodies (goat anti-mouse IgG [H+L] Alexa Fluor 594 secondary antibody (A-11032, 1:400; Thermo Fisher Scientific); or chicken anti-rabbit IgG [H+L] Alexa Fluor 488 secondary antibody [A-21441, 1:400]; Thermo Fisher Scientific). Replication fibers were viewed at 1000× magnification on a NIKON 90i fluorescence microscope (photometric cooled mono CCD camera; Nikon, Tokyo, Japan). Signals were measured using Image J software (NCI/NIH), with some modifications made specifically to measure DNA fibers.

Antibodies

Primary antibodies used for western blots were against: BRCA1 (Clone D-9, 1:200; Santa Cruz Technology, Santa Cruz, CA, USA); RPA2 (Clone NA18, 1:100; Calbiochem/EMD Millipore, Billerica, MA, USA); E2F1 (Clone KH95, 1:200; Santa Cruz Technology); β-Actin (Clone AC-74, 1:50000; Sigma–Aldrich); CHK1 (G-4, 1:200; Santa Cruz Technology); phospho-CHK1 antibody (#2344, CHK1-pSer317, 1:500; Cell Signaling Technology); phospho-CHK1 antibody (#133D3, CHK1-p345, 1:500; Cell Signaling Technology, Danvers, MA, USA); E2F1 (clone KH95 sc-251, 1:500; Cell Signaling Technology); Cdc45 (G-12 sc55569, 1:200; Santa Cruz Technology); γ-H2Ax (ser139 JBC301, 1:500; Millipore clone); p-RPA2(S4/S8) (rabbit polyclonal, BL647, 1: 1000; Bethyl Laboratories, Montgomery, TX, USA), CDC25A (clone DCS-120, 1:100; Thermo Fisher Scientific); ORC2 (sc13238, 1:200; Santa Cruz Technology); Cdk2 (610146, 1:200; BD Biosciences, San Jose, CA, USA); and Cyclin E (sc247, 1:200; Santa Cruz Technology); PARP (65995,1:400; BD Biosciences); Caspase 9 (M044232,1:1000; BD Biosciences); cleaved Caspase 9 (D35427, 1:500; Calbiochem); Caspase 8 (M043764,1:250; BD Biosciences); Caspase 7 (SC56063,1:500; Santa Cruz Technology); Caspase 3 (#9665,1:500; Cell Signaling Technology); cleaved Caspase 8 (#2008,1:500; Upstate Biotechnology/Thermo Fisher Scientific); cleaved Caspase 6 (D35426,1:500; Calbiochem); and cleaved Caspase 3 (76658,1:100; BD Biosciences). Secondary antibodies were goat anti-mouse IgG-horseradish peroxidase (HRP) conjugated (#7076S, 1:1000; Cell Signaling Technology), goat anti-rabbit IgG-HRP conjugated (#7074S, 1: 1000; Cell Signaling Technology) and donkey anti-goat IgG-horseradish peroxidase (HRP) conjugated (A2216, 1:1000; Santa Cruz Technology).

The primary antibodies used for immunofluorescence were against: gamma-H2AX (clone JBW301, 1:500; Millipore); RPA32 (S4/S8) (A300-245A, 1:500; Bethyl Laboratories); Cdc45 (H-300 clone, SC20685, 1:50; Santa Cruz Technology); phospho-Histone H3 (Ser10; #9706, 1:100; Cell Signaling Technology); goat anti-mouse IgG (H+L) Alexa Fluor 594 secondary antibody (A-11032, 1:400; Thermo Fisher Scientific); and chicken anti-rabbit IgG (H+L) Alexa Fluor 488 secondary antibody (A-21441, 1:400; Thermo Fisher Scientific).

Cell Line Authentication

MCF7 and MDA-MB-231, the two major cell lines used in this study, were authenticated via Short Tandem Repeat profiling by Genetica DNA Laboratories (a LabCorp brand; Burlington, NC, USA) using a PowerPlex® 16HS amplification kit (Promega Corporation, Madison, WI, USA) and GeneMapper ID v3.2.1 software (Applied Biosystems, Foster City, CA, USA). The authentication of each cell line was confirmed by a 100% match to the reference STR profile of the respective cell lines from the American Type Culture Collection.

Statistical Analysis

Statistical analyses were undertaken using the statistical software package, R version 3.3.4 and stata12.0 (StataCorp, College Station, TX, USA). Comparisons between RNF126 staining and various existing prognostic factors were performed using a chi-squared (χ²) test and logistic regression. Analyses of cumulative survival probability were performed using the Kaplan–Meier method and differences between groups were tested by logrank test. Multivariate analysis was undertaken using the Cox proportional hazard regression model. The effects of CHK1 inhibition on DNA repair recruitment/foci, DSB formation, and replication dynamics were examined using t test (two groups) or ANOVA (more than two groups). Tukey's honest significant difference (HSD) test was further used to compare the difference between groups. Correlation analysis was examined using Spearman's rank correlation.

Results

1. RNF126 is highly expressed in invasive BC and is an independent predictive marker for a poor prognosis

To determine RNF126 protein expression in cases of invasive BC, we collected 110 early-stage operable primary invasive BC specimens and 78 adjacent normal tissues for study. All patients were female. The clinicopathologic features of patients with BC enrolled in this study are shown in Table S1. RNF126 expression was detected by immunohistochemistry (IHC; Fig. 1A, 1B). Because of the lack of any study to define positivity according the expression level of RNF126, we determined RNF126 staining in tissues in accordance with an immunoreactive score (IRS) proposed by Remmele and Stegner (32). Of all samples, 55.45% (61 cases) of tumors were positive for RNF126 staining while 44.55% (49 cases) showed negative staining. In comparison, only 7.69% (6 cases) of adjacent tissue samples showed positive immunoreactivity to RNF126 and 92.31% (72 cases) displayed negative staining. Thus, the difference in RNF126 immunoreactivity between tumor samples and adjacent tissues was significant (χ²= 45.3894, P<0.001; Fig. 1A). Representative RNF126 staining in both normal and tumor tissues is shown in Fig. 1B. RNF126 staining was found in both the nucleus and cytoplasm of cancer cells, a result consistent with a previous report (11) and that of our unpublished data showing that RNF126 is located in both the cytoplasm and nucleus of cultured cancer cells. In addition, RNF126 protein expression was further compared with several clinicopathologic variables in BC, such as age, TNM stage, histological grade, menstruation status and molecular subtypes (Table S2). With regard to cases with luminal A tumors, 68.75% (11/16), as well as 58.49% (31/53) of cases with luminal B tumors, which were both positive for ER/PR, displayed positive RNF126 staining whereas only 42.86% (9/21) of cases with triple negative BC and 50.00% (10/20) of cases with HER2-enriched tumors were positive for RNF126 staining. Nevertheless, differences in RNF126 expression between triple-negative, HER2 and hormone receptor positive tumors were not statistically significant (χ²= 2.9327 P=0.402). In addition, logistic regression analysis was also established to measure the relationship between RNF126 expression and clinicopathological parameters, including patient age, TNM stage, histological grade, menstruation, and molecular sub-types. In this multi-variable regression analysis, the odds ratios (OR) were 1.57, 1.07, 1.03, 0.64, and 0.67, respectively. The P values for all parameters were more than 0.05 (Fig. 1C), indicating that RNF126 expression had no obvious relationship with these well-known clinicopathological factors.

Fig. 1. RNF126 high expression was associated with poor outcomes in patients with BC and was an independent predictive marker for a poor prognosis.

(A) The percentage of invasive BC tumors with RNF126 positive staining was elevated, compared to that of adjacent regions (χ² test, P<0.001). (B) Representative RNF126 staining detected by immunohistochemistry in adjacent normal and invasive BC tissues. Anti-RNF126 antibody (ab183102) was used. Adjacent normal tissues were collected 5 cm away from the edge of tumors. Specimens were surgically removed before patients were exposed to any neoadjuvant treatment. (C) Logistic regression analysis of RNF126 expression and clinicopathological parameters. RNF126 expression had no obvious relationship with the indicated clinicopathological variables. (D) Kaplan–Meier survival analysis in patients with invasive BC. Increased RNF126 expression correlates with a lower probability of cumulative survival. Recurrence, metastasis, or death were the final events (n=110). (E) Kaplan–Meier survival analysis in patients with invasive BC who received adjuvant chemotherapy. RNF126 positive staining was also associated with a poor prognosis in patients with a subtype of invasive breast tumor who received chemotherapy (n=90). (F) Expression of RNF126 was an independent predictor of a poor prognosis. Multivariate analyses of RNF126 expression and clinicopathological parameters in a cox proportional hazards model are indicated. RNF126 positive staining was an independent factor related to patients’ poor outcomes (HR: Hazard Ratio, 95% CI: 95% Confidence Interval).

Next, using recurrence, metastasis, or deaths as end points that reflect a low cumulative survival probability and poor prognosis, Kaplan–Meier plots for negative versus positive RNF126 expression showed that RNF126 positive staining was associated with a poor prognosis (logrank test, P=0.003; Fig. 1D). The median follow-up was 102 months (range 14–145 months). In order to determine whether RNF126 expression was associated with outcomes in the group of patients who received adjuvant therapies, 90 patients who received adjuvant chemotherapy after surgical resection based on RNF126 staining of their tumors were sorted into subgroups. Patients who showed RNF126 positive staining of BC tumors displayed a lower cumulative survival probability compared to patients who had negative RNF126 staining of BC tumors (logrank test, P=0.001; Fig. 1E), indicating that RNF126 positive staining was associated with a poor outcome in the group of patients who received adjuvant chemotherapy. Lastly, we used a Cox proportional hazard model to determine the prognostic value of RNF126. RNF126 immunoreactivity, patient’s age, TNM stage, histological grade, menstruation, and molecular sub-types were chosen as risk variables since all are potential factors affecting a low cumulative survival probability of BC. Hazard ratios are indicated in Fig. 1F. The hazard ratio values for RNF126 immunoreactivity and TNM stage were 7.3 (P=0.009) and 3.8 (P=0.002), respectively. This indicates that in multivariate analyses, RNF126 expression and TNM stage are two independent factors related to a poor outcome in patients with invasive BC (Fig. 1F). Thus, high RNF126 expression is associated with a poor prognosis and is an independent predictor of a poor prognosis in BC.

2. RNF126 facilitates expression of the CHK1 gene via interaction with E2F1

That RNF126 is associated with a poor prognosis highlights the clinical significance of this protein in BC. However, a specific inhibitor of RNF126 is not currently available. Studying the role of RNF126 in the regulation of CHK1 expression will provide new opportunities for therapeutic intervention in BC. RNF126 knockdown by two shRNAs targeting different regions of RNF126 led to decreased CHK1 protein levels in MCF7 (Fig. 2A) and MDA-MB-231 cells (Fig. 2B). Of note, downregulation of RNF126 was tolerated well by MCF7 and MDA-MB-231 cells, without a significant alteration in cell cycle profiles being observed (13) (Fig. S1). Thus, the decreased expression of CHK1 in cells depleted of RNF126 was not caused by cell cycle changes in our experimental conditions. However, it has been suggested that RNF126 knockdown can cause cell arrest (11). This discrepancy may be caused by differences in the magnitude of RNF126 knockdown. RNF126 likely regulates CHK1 at the transcriptional level because RNF126 depletion resulted in a decrease in CHK1 mRNA in both cell lines (Fig. 2C). In accordance with these results, overexpression of Flag-RNF126 led to increased CHK1 protein (Fig. 2D, E) and mRNA levels (Fig. 2F) in both MCF7 and MDA-MB-231 cells. Most importantly, the E3 ligase activity of RNF126 appears to be dispensable for the regulation of CHK1 expression because the expression of a validated RNF126 E3 ligase mutant (RNF126 C229A/C232A) (11) retained the ability to increase CHK1 protein expression in both MCF7 and MDA-MB-231 cells (Fig. 2G, H). This result was consistent with our previous study where E3 ligase activity of RNF126 was found not to be required for BRCA1 expression (13).

Fig. 2. RNF126 facilitated CHK1 expression.

(A, B) RNF126 knockdown by shRNAs led to decreased expression of CHK1 protein in MCF7 (A) and MDA-MB-231 cells (B) (upper panels). Band intensities of RNF126 and CHK1 protein expression in cells, with or without RNF126 depletion, were quantified using ImageJ software, and normalized to β-actin. n=3. (lower panels) (MCF7: P1=0.040, P2=0.014, P3=0.016, P4=0.013; MDA-MB-231: P1=0.012, P2=0.002, P3=0.021, P4=0.024). (C) RNF126 and CHK1 mRNA levels in MCF7 or MDA-MB-231 cells, with or without RNF126 knockdown by shRNAs. n=3. (One-way ANOVA, P1=0.002, P2=0.002, P3=0.012, P4=0.007, P5=0.007, P6=0.005, P7=0.014, P8=0.005). (D, E) Flag-RNF126 overexpression resulted in increased CHK1 protein expression in MCF7 and MDA-MB-231 cells. CHK1 protein band intensities were quantified using ImageJ software, and normalized to β-actin. n=3. (D). One of three independent experiments is presented (E). (F) The level of CHK1 mRNA expression in MCF7 or MDA-MB-231 cells, with or without Flag-RNF126WT overexpression. n=3. (Paired t test). (G, H) The expression of an E3 ligase mutant of RNF126 did not affect CHK1 protein expression. MCF7 or MDA-MB-231 cells were transfected with control vector, Flag-RNF126-WT, or E3 ligase-deficient RNF126 (Flag-RNF126-C229A/C232A) plasmids and levels of CHK1 protein were then detected by western blotting. RNF126 and CHK1 protein band intensities were quantified using ImageJ software, and normalized to β-actin. n=3. (One-way ANOVA, P1=0.037, P2=0.008, P3=0.001, P4=0.001, P5=0.023, P6=0.004, P7=0.008, P8=0.013) (G). One of three independent experiments is presented (H).

We next investigated if interaction between RNF126 and E2F1 was required for controlling CHK1 expression by using a CHK1 promoter–driven luciferase reporter (36), given that promotion of E2F1 mediated-transactivation by RNF126 depends on the direct interaction of these two proteins via a 185–195 (f) region in RNF126. Flag-RNF126 overexpression increased luciferase activity, indicating that RNF126 promotes transactivation of the CHK1 promoter. In contrast, RNF126-Δf overexpression failed to induce a significant increase in luciferase activity but reduced luciferase activity compared to control cells in both MCF7 and MDA-MB-231 cell lines (Fig. S2A), indicating that RNF126-Δf expression may interfere with the function of endogenous RNF126. These results are consistent with our previous report suggesting that a RNF126-Δf mutant lacking an association with E2F1 leads to a loss of function of RNF126 in promoting the E2F1-mediated transactivation of BRCA1; it also has a dominant-negative effect (13). This result was further supported by a chromatin immunoprecipitation (ChIP) assay showing that RNF126 overexpression enhanced the enrichment of E2F1 on the CHK1 promoter; however, RNF126-Δf overexpression reduced the binding of E2F1 protein to the CHK1 promoter (Fig. S2B). Moreover, the decreased expression of CHK1 at both mRNA and protein levels was observed in cells expressing RNF126-Δf compared to control cells, whereas increased CHK1 mRNA (Fig. S2C) and protein expression (Fig. S2D) was found in cells expressing Flag-RNF126-WT. Again, in support of the idea that the E3 ligase activity of RNF126 is dispensable for the regulation of CHK1 expression, the expression of RNF126 C229A/C232A led to increased luciferase activity of CHK1 (Fig. S2A), enrichment of RNF126 at a promoter of CHK1 (Fig. S2B) and elevated CHK1 expression at both mRNA (Fig. S2C) and protein (Fig. S2D) levels, similar to that observed in cells expressing wild-type Flag-RNF126. In addition to CHK1, RNF126 also promoted the expression of Cyclin E, another downstream factor of E2F in both MCF7 and MDA-MB-231 cells (Fig. S3A, B). Thus, we conclude that by interacting with E2F1, RNF126 promoted CHK1 expression at the mRNA transcription level.

3. Correlation of RNF126 and CHK1 protein expression

Next we were interested in determining any association between RNF126 and CHK1 in BC tissues. We assessed immunoreactive staining of these two proteins by analyzing a second cohort of BC cases that consisted of samples from 67 patients with early-stage primary invasive BC prepared as tissue microarrays (TMA; n=67). Both RNF126 and CHK1 staining were determined by IHC using TMA and quantified by IRS scores. Of note, CHK1 immunoreactivity was predominantly located in the cytoplasm and was granular in appearance, although nuclear staining was also observed. CHK1 positive staining was found in 94.59% (35/37) of RNF126 positive staining breast cancer samples. CHK1 staining was negative in 80% (24/30) of RNF126 negative staining breast cancer samples. The expression of RNF126 in tissues was related to that of CHK1 (χ²= 38.82, P<0.001, Cramér's V=0.7612; Fig. 3A). Representative staining of these two proteins is shown in Fig. 3B. Thus, in invasive BC, there was a strong and statistically significant correlation between RNF126 and CHK1 protein expression.

Fig. 3. Correlation of RNF126 and CHK1 protein expression.

(A) Co-expression of RNF126 and CHK1 proteins was analyzed by tissue microarrays (TMAs; n=67). (B) Typical immuno-staining patterns for serial sections of the same tumor for RNF126 and CHK1. TMA immunostaining was visualized with 3,3’-diaminobenzidine substrate following probing with antibodies against RNF126 (ab183102, 1:100, Abcam) and CHK1 (25887-1-AP, 1:150, Proteintech). (C) The expression of RNF126, CHK1 and Cyclin E proteins in a panel of 16 human breast cancer (BC)-derived cell lines by western blotting. Normal primary cultured MCF10A cells were used as a control. Four cell lines chosen for the toxicity assay are labeled either in blue (lower expression) or yellow (higher expression). (D) Band intensities of RNF126 and CHK1 protein expression in BC cell lines were quantified using ImageJ software, and normalized to β-actin. n=3. (E) Positive correlation between RNF126 and CHK1 proteins in BC cell lines (Spearman rank correlation, r=0.682, P=0.004). (F) The mRNA expression of RNF126 and CHK1 in a panel of BC cell lines was detected by quantitative real time PCR. n=3. (G) RNF126 protein levels paralleled CHK1 mRNA levels (Spearman rank correlation, r=0.532, P=0.034). (H) RNF126 protein and mRNA transcripts did not correlate in tested BC cell lines (Spearman rank correlation, r=0.300, P=0.259)

Additionally, we further analyzed the expression of RNF126 and CHK1 proteins in a panel of 16 human mammary carcinoma–derived cell lines that comprised: luminal A, ER+ breast cancer (MCF7, ZR-75-1 and T47D), luminal B (MDA-MB-361, BT474), HER2+ breast cancer (HCC202, SK-BR-3, HCC1569) and triple negative breast cancer (MDA-MB-231, HCC1143, HCC1954, HCC38, HCC1187, HCC70, BT549, MDA-MB-468) cells. We set MCF10A, a normal immortalized breast epithelial cell line, as a control. The expression of RNF126 and CHK1 proteins was determined by western blotting. Band intensities were quantified using ImageJ software, and normalized to β-actin (n=3; Fig. 3C, D). RNF126 expression was increased in a large majority of BC cell lines when compared to a MCF10A cell line used as a control. The highest level of RNF126 protein was found in highly tumorigenic MDA-MB-231 cells (Fig. 3C, D). Correspondingly, CHK1 protein expression was also relatively high in these cells. The cell lines, BT474 and ZR751, showed lower or undetectable levels of RNF126 and CHK1 protein expression compared to MDA-MB-231 cells (Fig. 3C, D). Therefore, a positive correlation between RNF126 and CHK1 protein expression was observed in BC cell lines (Fig. 3E; correlation coefficient of Spearman rank correlation, r=0.682, P=0.004), which is consistent with observations from BC tissues (Fig. 3A). In addition, we also measured RNF126 and CHK1 mRNA by qRT–PCR (n=3; Fig. 3F). Levels of RNF126 protein essentially paralleled mRNA levels of CHK1 (Fig. 3G; Spearman rank correlation, r=0.532, P=0.034). This result is consistent with Fig. 2. S2 showing that RNF126 promoted CHK1 mRNA expression. However, interestingly, RNF126 protein and mRNA transcripts did not correlate in tested BC cell lines (Fig. 3H; Spearman rank correlation, r=0.300, P=0.259), indicating that the high expression of RNF126 may not be a consequence of transcriptional regulation. Similarly, a co-relationship between the expression of RNF126 protein and Cyclin E mRNA existed (Spearman rank correlation, r=0.624, P=0.009; Fig. S3D) that aligns with the result described in Fig. S3A, B where RNF126 facilitated the expression of Cyclin E at both mRNA and protein levels (Fig. S3A, B). Thus, we concluded that RNF126 and CHK1 protein expression positively correlated in both BC tissue and cell lines.

4. CHK1 inhibition by pharmacological CHK1 inhibitors is more effective against BC cells expressing a higher level of RNF126

We chose two pairs of BC cell lines showing higher (MDA-MB-231, and MDA-MB-468), or lower/undetectable RNF126 expression (BT474, ZR751) for a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Fig. 4A). LY2603618, one of the first highly selective and potent CHK1 inhibitors, was used in this study. We found that the two cell lines expressing higher levels of RNF126 were more sensitive to LY2603618 compared to the cells showing lower RNF126 expression, suggesting that RNF126 expression may determine sensitivity to CHK1 inhibitors (Fig. 4A). To confirm these results, we determined the effect of RNF126 knockdown by RNF126 shRNA#1 (Fig. 4B, C) or #2 (Fig. S4A, B) on the efficacy of LY2603618. LY2603618 exposure resulted in more killing of parental cells MCF7 and MDA-MB-231, compared to the corresponding cells with RNF126 knockdown by shRNA in MTT (Fig. 4B, Fig. S4A, B) and/or colony-forming assays (Fig. 4C). Treatment with a second CHK1 inhibitor, AZD7762, also decreased RNF126-expressing cell numbers compared to both MCF7 (Fig. S4C) and MDA-MB-231 (Fig. S4D) cells with knocked down RNF126, as determined by MTT assay. CHK1 inhibition was monitored by measuring protein levels of CHK1-p-S345 and its downstream factor CDC25A (Fig. 4D). Thus, we concluded that RNF126 depletion abrogated CHK1-inhibited cell killing.

Fig. 4. CHK1 inhibition by LY2603618 in parental cells compared with cells depleted of RNF126.

(A) A (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay following CHK1 inhibition by LY2603618 in BC cells with higher RNF126 expression vs. BC cells with lower RNF126 expression. Cells were treated with various concentrations of LY2603618 for 72 h. n=3. (Two-way ANOVA, P_{BT474 VS. MDA-MB-231}<0.001; P _{BT474 VS. MDA-MB-468}<0.001; P_{ZR751 VS. MDA-MB-231} P<0.001; P_{ZR751 VS. MDA-MB-468}<0.001). (B) MTT assay for observing the effect of the CHK1 inhibitor, LY2603618, on MCF7 and MDA-MB-231 cell proliferation. Cells were treated with various concentrations of LY2603618 for 72 h. n=3. (Two-way ANOVA). (C) Clonogenic survival following CHK1 inhibition by LY2603618 in MCF7 and MDA-MB-231 cells. n=3. (Two-way ANOVA). (D) CHK1 inhibition was monitored by measuring levels of CHK1 p-S345 and CDC25A by western blots. Cells were treated with various concentrations of LY2603618 for 8 h. The representative result from three independent experiments is presented.

The reduction in cell viability was accompanied by an increase in CHK1 inhibitor–induced apoptosis as determined by measuring optimal biomarkers of apoptosis, such as cleaved caspases 3, 6, 7, 8, 9 as well as PARP. Cleaved PARP and cleaved caspase 7 increased in LY2603618 treated MCF7 (Fig. S5A) and MDA-MB-231cells (Fig. S5B) whereas an obvious increase was not found in corresponding cells with RNF126 depletion under the same conditions. Interestingly, cleaved caspase 8 was observed in MDA-MB-231 cells with intact RNF126, but a reduced effect on cleaved caspase 8 was observed in RNF126-depleted cells (Fig. S5B). However, cleaved caspase 8 was not seen in MCF7 cells (Fig. S5A). The differences in the response of apoptosis proteins in MCF7 and MDA-MB-231 cells may be due to differences in basal levels of apoptotic proteins. For instance, caspase 3 was absent in MCF7 cells whereas caspase 3 was present in MDA-MB-231 cells (37). Interestingly, according to immunofluorescence (IF) results, we found that as a single agent, CHK1 inhibition by LY2603618 did not increase the rate of mitotic cells in MCF7 cells, with or without RNF126 knockdown. Instead, CHK1 inhibition resulted in a decrease in the proportion of mitotic cells in MCF7 (Fig. S5C) and MDA-MB-231 cell lines (Fig. S5D), but not in cell lines showing RNF126 depletion (representative staining of mitotic cells, as determined by IF of p-histone H3, is shown in Fig. S5E). This may be explained by the fact that when RNF126 is intact, DNA damage induced by CHK1 inhibition triggers ATR activity and G2/M arrest, preventing cells from entering the next stage. However, in cells with depleted RNF126, less DNA damage is induced by CHK1 inhibition. An insufficient amount of DNA damage may trigger a G2/M checkpoint by CHK1 inhibition. Thus, we conclude that CHK1 inhibition was more effective in cells expressing higher levels of RNF126. Of note, a similar result was also seen with an ATR inhibitor. ATR inhibition by AZD6738 was more toxic in MCF7 (Fig. S6A, B) and MDA-MB-231 cells (Fig. S6C, D) with intact RNF126, compared to cells with RNF126 knocked down by RNF126 shRNA #1 (Fig. S6A, C) or #2 (Fig. S6B, D). These results support the notion that ATR has a similar function to that of CHK1 in terms of suppressing oncogenic stress/checkpoints/HR.

5. CHK1 inhibition upregulates replication stress, particularly in cells showing higher expression of RNF126

We next determined the extent of replication stress following CHK1 inhibition in cells, with or without RNF126 knockdown. We analyzed foci of phosphorylated RPA2 (p-RPA2), a marker for replication stress, in response to exogenous DNA damaging agents by immunofluorescence staining. A more profound increase in the proportion of cells with p-RPA2 foci was observed in MCF7 cells compared to cells depleted of RNF126 by RNF126shRNA #1 (Fig. 5A). The greater increase in p-RPA2 foci in LY2603618-treated MCF7 cells were also confirmed by western blot (Fig. 5B). In addition, LY2603618 treatment led to a greater increase in γ-H2AX foci (Fig. 5C) and protein levels (Fig. 5D) in parental MCF-7 cells compared to MCF-7 cells with RNF126 knockdown. Of note, although CHK1 inhibition caused an increase in CHK1-p-S345 in cells depleted of RNF126, the extent was much less than that seen in cells with intact RNF126. This result supported our hypothesis that CHK1 inhibition leads to a reduced amount of DNA damage in RNF126 depleted cells. A similar result was observed in parental MDA-MB-231 cells (Fig. 5E, F), and in MCF7 and MDA-MB-231 cells treated with the second CHK1 inhibitor, AZD7762 (Fig. S7A, B). These results suggest that CHK1 inhibition suppresses the proliferation of BC cells expressing higher levels of RNF126. A similar result was also seen using a second RNF126 shRNA #2 (Fig. S7C, D). RNF126 knockdown by RNF126 #2 abrogated CHK1 inhibition-induced replication stress in both MCF7 and MDA-MB-231 cells.

Fig. 5. CHK1 inhibition enhanced replication stress, particularly in cells with RNF126 expression.

(A, C) The proportion of cells with foci of phosphorylated RPA2 (p-RPA2, green) (A) or γ-H2AX (red) (C) in MCF7 cells, with or without RNF126 knockdown (left panels). Cells were treated with LY2603618 (5 µM) for the indicated times and then subjected to immunofluorescence staining. Representative foci of p-RPA2 or γ-H2AX are indicated (right panels). n=3. (Two-way ANOVA). (B, D) CHK1 inhibition by LY2603618 (5 µM) led to a greater increase in levels of p-RPA2 and γ-H2AX in parental MCF7 cells compared to MCF7 cells with RNF126 knockdown as determined by western blot. (E) CHK1 inhibition by LY2603618 (5 µM) led to a greater increase in the levels of p-RPA2 and γ-H2AX proteins in parental MDA-MB-231 cells, compared to MDA-MB-231 cells with RNF126 knockdown, as determined by western blotting. (F) The proportion of cells with foci of p-RPA2 (left panel) or γ-H2AX (right panel) in MDA-MB-231 cells, with or without RNF126 knockdown, as determined by immunofluorescence. n=3. (Two-way ANOVA).

6. CHK1 inhibition disrupts dynamics of replication forks, particularly in cells expressing higher levels of RNF126

Deregulated origin firing contributes to oncogene-induced replication stress (38). We next determined how treatment with CHK1 inhibitor affects the initiation of DNA replication by analyzing DNA fiber spreads, according to the protocol illustrated in Fig. 6A and our previous publication (25). The percentage of new origins increased when cells were treated with LY2603618 in both parental MCF7 cells and MCF7 cells with RNF126 knockdown (Fig. 6B). However, the magnitude of the increase was greater in parental cells compared to cells with RNF126 knockdown. A similar result was seen in MDA-MB-231 cells (Fig. 6C). CHK1 is involved in controlling replication initiation via regulating CDC45 (39), a protein that is implicated in initiation rather than elongation processes. We next measured the amount of CDC45 in a non-extractable chromatin fraction. LY2603618 treatment caused a remarkable increase in the amount of non-extractable CDC45 protein in control cells compared to RNF126 depleted cells (Fig. 6D), although overall CDC45 levels were comparable (Fig. 6D). The effect of CHK1 inhibition on chromatin loading of CDC45 was further confirmed by IF assay (Fig. 6E). As a result of an alteration in replication initiation, the elongation ratio was decreased when CHK1 activity was inhibited, particularly in MCF7 cells and MDA-MB-231 with intact RNF126 (Fig. 6F, G). Representative DNA fiber staining is presented in Fig. S8. Cumulatively, the results presented in Fig. 6 suggest that CHK1 inhibition led to a greater increase in replication initiation and a decrease in replication speed, particularly in cells with higher RNF126 expression. This result was consistent with the results described in Fig. 5 where CHK1 inhibition caused greater replication stress in cells with RNF126 expression compared to RNF126 depleted cells.

Fig. 6. CHK1 inhibition disrupted dynamics of replication forks, particularly in cells expressing RNF126.

(A) Schematic of DNA fiber analysis (left panel) in MCF7 cells. Red tracks, IdU; Green tracks, CldU. (B, C) CHK1 inhibition by LY2603618 (5 µM) increased the rate of replication initiation, particularly in cells with intact RNF126, compared to cells depleted of RNF126. The frequency of new origins was calculated as the number of green signals (b) divided by the total of green (b) plus green/red signals (a+b; right panel). (B: MCF7; C: MDA-MB-231, n=3, One-way ANOVA). (D) CHK1 inhibition led to an increase in non-extractable CDC45 protein, particularly in parental cells with RNF126 compared to cells with RNF126 knockdown, as determined by western blotting. ORC2 was used as a loading control. (E) Measurement of CDC45 chromatin loading by immunostaining after pre-extraction of cells with detergent. Cells presenting with CDC45 staining were considered positive. n=3. (Two-way ANOVA). (F, G) CHK1 inhibition induced a greater decrease in replication fork speeds in MCF7 (F) and MDA-MB-231 cells (G) compared to corresponding cells with RNF126 knockdown. The CIdu/Idu ratio was used to determine elongation. (n=3, One-way ANOVA). (H) Model for targeting BC cells expressing RNF126 by CHK1 inhibitors.

Discussion

High RNF126 expression and invasive BC

The biological functions of RNF126 have been explored recently (6,8,11–13). However, to date, a report determining RNF126 expression in human cancers is lacking. Our results suggest that RNF126 protein was highly expressed in invasive BC (Fig. 1). Although the mechanism contributing to increased RNF126 expression is not clear, the data obtained from a panel of BC cell lines suggest levels of RNF126 protein and mRNA transcripts are not correlated (Fig. 3). Thus, it is postulated that the increased RNF126 protein measured in BC tissues may not necessarily be a consequence of an alteration in RNF126 mRNA transcripts. In addition, RNF126 positive staining appears to be slightly higher in the ER+ cohort compared to the other cohorts, such as the triple negative subtype. However, this difference did not reach statistical significance. Thus, a further study with a larger number of invasive BC cases is required. The RNF126 gene maps to chromosome 19p13.3, which is a commonly deleted region in ovarian cancer (40–42). Interestingly, a genome-wide study of BC also detected a high frequency of loss of heterozygosity (LOH) in the 19p13 genomic region (43). It is not clear if the LOH of 19p13 in BC led to the decreased RNF126 expression observed. However, it is likely that RNF126 may be a context-dependent signaling molecule and that the expression of RNF126 in cancer may be contingent on the biological context. The high expression of RNF126 in BC suggests that RNF126 may contribute to BC development, although the molecular mechanisms behind this are, as yet, unclear.

In this study, we demonstrate that RNF126 expression is associated with a poor prognosis, such recurrence, metastasis or deaths, in patients with invasive BC (Fig. 1). Nevertheless, the relationship between RNF126 protein expression and each end point of a poor prognosis needs to be investigated further. Our most significant finding is that high RNF126 expression is an independent predictor for a poorer patient prognosis, which is independent from established prognostic markers such as patients’ age, TNM stage, histological grade, menstruation and molecular subtypes (Fig. 1). Further analysis using adjuvant chemotherapy as a stratification criterion suggested that patients with RNF126 positive BC tumors had a significantly lower cumulative survival probability compared to those with RNF126 negative tumors (Fig. 1). Although conclusions from our observations are limited due to the small number of patients who received adjuvant therapies (n = 90), the differences in survival probabilities are striking and suggest that RNF126 expression levels may influence the response to adjuvant therapies. As DSB repair proteins have been suggested to play an important role in the cellular response to chemotherapy as well as to radiotherapy, the role of RNF126 in the repair of DSBs by promoting HR and NHEJ may contribute to its poor prognosis. The association of RNF126 with a poor prognosis in BC highlights the clinical significance of this protein.

Higher expression of RNF126 as a biomarker for determining CHK1 inhibitor use

In our study, we identify a relationship between RNF126 and CHK1 by demonstrating that RNF126 promotes E2F1-mediated expression of CHK1 transcripts (Fig. 2), which is consistent with our previous publication that outlined how RNF126 promoted the activity of the transcriptional factor, E2F1 (13). BC tumors expressing higher levels of RNF126 often show elevated CHK1 protein expression in both BC tissue and cell lines (Fig. 3). Most importantly, a correlation between RNF126 protein levels and CHK1 transcripts in BC cell lines was also observed, supporting our finding that RNF126 promotes CHK1 expression at transcriptional levels (Fig. 2). Nevertheless, the positive relationship between RNF126 protein and CHK1 transcripts needs to be verified in breast tumor tissues in future.

It is well established that ATR/CHK1 suppress oncogene-induced replication stress. Cancer cells often harbor some degree of replication stress due to oncogene activities, which can be lethal to cells. Thus, they often upregulate ATR and CHK1 activity to mediate survival because ATR/CHK1 suppress replication stress to an intolerable level by the suppression of replication initiation and/or promoting HR (25,44,45). In support of this concept, increased ATR/CHK1 expression was frequently observed in a variety of cancer cells, including lung cancer, ovarian cancer, head neck cancer, triple negative breast cancer, neuroblastoma, T-cell acute lymphoblastic leukemia, acute myeloid leukemia and hepatocellular carcinoma. Although the biological significance of the correlation between RNF126 and CHK1 expression remains unknown, it may be related to the inhibition of replication stress by CHK1 in cells expressing high levels of RNF126. Thus, increased CHK1 protein expression in RNF126 positive BC cancer cells is likely related to the suppression of replication stress because RNF126 also promotes oncogene expression such as Cyclin E (Fig. S3), an oncogene that causes replication stress. It is most likely that RNF126 positive BC upregulates oncogenes in addition to CHK1, rendering cells dependent on ATR/CHK1 for survival. Indeed, CHK1 inhibition causes greater killing in cells expressing RNF126, whereas a lesser effect was found in cells with RNF126 depletion. Thus, in addition to Myc, Cyclin E and Ras that have been reported to affect the outcome of CHK1 or ATR inhibitors, RNF126 is also a potential factor determining the efficacy of CHK1 inhibitors (Fig. 6H). Using RNF126 expression as a biomarker for a CHK1 inhibitor has a greater advantage than CHK1 expression alone because the high expression of CHK1 may not be functionally important. Indeed, p-CHK1, instead of CHK1 expression levels, is a biomarker for CHK1 inhibitors (46). We also reported that radioresistant BC cells that carry high levels of oncogene and DDR proteins, including ATR/CHK1, are more sensitive to CHK1 inhibition (25), suggesting that expression of both oncogene and cell cycle checkpoint proteins are features that could be targeted by CHK1 inhibitors. Thus, the role of RNF126 in promoting CHK1 expression, and perhaps also oncogene expression, determine the sensitivity of RNF126 positive BC to CHK1 inhibitors.

The current model for oncogene-induced replication stress is related to deregulated replication initiation, because an excess of ongoing replication forks will consume the limited dNTP pool and cause fork stalling (38). This will generate extensive ssDNA regions that are protected by RPA coating. With a limited supply of RPA, uncoated ssDNA causes DSB. However, ATR/CHK1 can be activated during replication stress, which, in turn, suppresses oncogene-induced replication by targeting CDC25A for degradation. Our studies provide evidence that further support the notion that CHK1 suppresses replication stress by inhibiting replication initiation, particularly in cells expressing RNF126 (Fig. 6). Since ATR/CHK1 can also promote the repair of DSBs by facilitating HR, increased DSBs induced by CHK1 inhibition may also be related to the impaired HR repair of collapsed replication forks. Thus, multiple mechanisms are involved in CHK1 inhibition–induced replication stress in cells expressing RNF126.

We have reported that RNF126 promotes the expression of HR protein BRCA1 at the transcriptional level (13). The probability that BRCA1 affects the efficacy of the CHK1 inhibitor on BC cells expressing relatively high levels of RNF126 is very low. Transient RNF126 overexpression increases mRNA expression of the BRCA1 (13). However, co-expression of these two proteins may not be seen in tumor tissues or cancer cell lines, as it is well-known that the BRCA1 promoter is frequently methylated, leading to low expression (47,48). Even if some high RNF126–expressing cell lines have high BRCA1 protein expression, its effect may be to reduce, rather than increase, the sensitivity to CHK1 inhibitors, as HR defective cells are more sensitive to CHK1/ATR inhibition (49).

Of note, despite the initial hypothesis that CHK1 inhibitors can increase efficacy in combination with IR and chemotherapy drugs, particularly in cells with p53 deficiency, our studies show that RNF126 promotes CHK1 expression and affects sensitivity to CHK1 inhibitors in cells, with or without wild-type p53. Our results are consistent with previous publications showing that ATR/CHK1 inhibition can target cancer cells as single agents independent of p53 (25,50). Thus, acting as single agents and in combination with other chemotherapy drugs/IR, the mechanisms by which CHK1 inhibitors lead to cell death may be distinct.

In summary, we identify that RNF126 is highly expressed in invasive BC and is an independent predictor of a poor outcome for this disease. High RNF126 expression may be used as a potential biomarker for CHK1 inhibitors. Our study provides proof of concept in pre-clinical models for a new paradigm for treating BC expressing high levels of RNF126 by CHK1 inhibitors (Fig. 6H). Identifying BCs with high levels of RNF126 expression that can then be targeted by CHK1 inhibitors will significantly improve the efficacy of such agents. It will be necessary to validate our findings in BC using large randomized clinical trials. This may be done by assessing whether a simple immunohistochemical assay of RNF126 expression performed on routine paraffin-embedded tissue would be able to predict a patient's response to CHK1 inhibitors. We also need to evaluate whether RNF126 positive breast tumors are more responsive to CHK1 or ATR inhibitors.

Translational Relevance.

We have previously reported that RNF126 expression is associated with resistance to radiotherapy and PARP inhibition. However, RNF126 protein expression in human tumors and its association with the outcomes of patients with breast cancer (BC) have not been evaluated. CHK1 inhibitors are currently in clinical trials but a specific target-based biomarker to identify treatment responsive populations does not exist, which may significantly reduce the efficacy of such agents. Our study will be the first step in designing clinical trials that consider RNF126 status in the selection of patients with BC for treatment with CHK1 inhibitor-associated clinical investigations. Also, the identification of biomarker to guide the use of CHK1 inhibitors will significantly improve the efficacy of such agents. Moreover, our results may improve the survival of patients with BC and high RNF126 expression, given this suggests such patients generally have a poor prognosis.