EXPAND1 Loss Triggers Apoptotic Sensitization to PARP Inhibition in Breast Cancer Cells

Abstract

Chromatin remodeling is central to the orchestration of DNA replication, repair, transcription and recombination. EXPAND1 (also known as MUM1) is a chromatin-associated factor that facilitates chromatin relaxation at DNA damage sites via interaction with 53BP1, thereby supporting genome stability under genotoxic stress. In this study, we explored whether EXPAND1 influences sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi) in BRCA2-proficient breast cancer cells. Using RNA interference, EXPAND1 was silenced in MDA-MB-231 and MCF7 cells, with knockdown validated by qPCR and Western blot. Functional assays revealed that EXPAND1 loss sensitized cells to PARPi (olaparib, rucaparib), significantly reducing viability and colony survival, including in MCF7/PIR (PARPi-resistant) clones. Mechanistically, EXPAND1 depletion under PARPi treatment induced cleaved PARP and caspase-3 expression, coupled with elevated p53 and a failure to maintain p21, indicating a shift from checkpoint control to apoptosis. Notably, EXPAND1 protein levels were reduced in a dose-dependent manner upon PARPi treatment in cancer cells, but not in non-transformed RPE1 cells. While these results suggest a role for EXPAND1 in modulating chromatin-linked stress responses to PARPi, translational implications remain preliminary due to experimental limitations. Future studies should aim to validate EXPAND1-related signatures in patient-derived models and explore its utility as a biomarker within defined molecular contexts. Collectively, our findings suggest EXPAND1 as a potential chromatin-regulating determinant of PARPi sensitivity and a candidate for further investigation in refining personalized therapeutic strategies.

Keywords:
EXPAND1; chromatin remodeling; PARP inhibition; breast cancer.

HIGHLIGHTS

• EXPAND1 loss enhances cancer cell sensitivity to PARP inhibitors through apoptosis.

• EXPAND1 loss re-sensitizes olaparib-resistant MCF7 cells to PARP inhibition.

INTRODUCTION

Cancer is defined as a pathological condition characterized by the uncontrolled and abnormal proliferation of cells that escape the normal regulatory mechanisms governing the cell cycle [¹-³]. According to the International Agency for Research on Cancer (IARC), approximately 20 million people worldwide were diagnosed with cancer in 2022, while the disease claimed the lives of more than 10 million individuals [⁴]. With 2,296,840 new cases, breast cancer represents 11.5% of the global cancer burden [⁴]. Despite significant progress in screening and therapeutic interventions, breast cancer remains a major public health concern, particularly due to the heterogeneity of its subtypes [⁵].

Poly(ADP-ribose) polymerases orchestrate single-strand break (SSB) repair through PARylation of chromatin proteins and subsequent autoPARylation-mediated chromatin release. PARPi-induced synthetic lethality arises from transcription-replication conflicts, replication fork collapse, and accumulation of single-stranded DNA gaps that persist into subsequent cell cycles [⁶,⁷]. In BRCA1/2-mutant cells, PARPi induce cytotoxicity through dual mechanisms: catalytic inhibition allows replication-associated DNA damage accumulation, while "PARP-trapping" creates DNA-protein crosslinks that block fork progression [⁸-¹⁰]. BRCA-deficient cells fail to suppress single-stranded DNA gaps and protect reversed forks from nuclease degradation, leading to replication catastrophe [⁹,¹⁰]. This underlies regulatory approval of olaparib, rucaparib, niraparib, and talazoparib for homologous recombination-deficient cancers [¹¹].

Chromatin architecture critically governs replication-associated DNA damage responses [¹²]. EXPAND1, a PWWP domain-containing chromatin factor, interacts with 53BP1 to mediate rapid chromatin decondensation at double-strand break (DSB) sites, with EXPAND1 depletion causing chromatin compaction and genotoxic hypersensitivity [¹³]. While 53BP1 loss confers PARPi resistance in BRCA1-deficient cells by restoring homologous recombination (HR) [¹⁴,¹⁵], whether EXPAND1-as a 53BP1 effector-similarly influences PARPi response remains unexplored. Critically, PARPi cytotoxicity involves persistent chromatin-associated PARP1-DNA complexes requiring chromatin remodeling for resolution [¹⁶], suggesting EXPAND1's chromatin-opening function [¹³] may be essential for tolerating PARPi-induced replication stress.

We hypothesize that EXPAND1 functions as a context-dependent modulator of PARPi sensitivity through chromatin-mediated replication stress tolerance. In HR-proficient cells, EXPAND1 loss may impair chromatin relaxation at stalled forks and PARP-trapping sites, prolonging toxic PARP-DNA complexes and sensitizing cells to PARPi independent of BRCA2 status. In BRCA1-deficient cells, EXPAND1 depletion may either confer resistance by mimicking 53BP1 loss or enhance sensitivity if its chromatin functions are non-redundant. This study tests whether EXPAND1 depletion synergizes with or antagonizes PARPi cytotoxicity across different genetic backgrounds, potentially identifying EXPAND1 as a potential biomarker candidate for expanding PARPi utility beyond BRCA2-mutant tumors.

MATERIAL AND METHODS

Materials

In this study, cell viability and cytotoxicity were assessed using the Cell Proliferation and Cytotoxicity Assay Kit (Boster, Cat. No. AR1156). For gene silencing, a non-targeting control siRNA (siCTRL; Qiagen, Cat. No. 1022076; sequence: 5′-AACGTACGCGGAATACTTCGA-3′) was used. EXPAND1 was targeted using two independent siRNAs, siEXPAND1#4 (Qiagen FlexiTube GeneSolution GS84939, Cat. No. 1027416; sequence: 5′-CTGGGCGTACGTTGTGCCAAA-3′) and siEXPAND1#5 (Qiagen FlexiTube GeneSolution GS84939, Cat. No. 1027416; sequence: 5′-GACGTGCACATCGATCTCGTA-3′), while BRCA2 was silenced using siBRCA2#6 (Qiagen, Cat. No. 1027417; sequence: 5′-CAGGACACAATTACAACTAAA-3′). Lipofectamine™ RNAiMAX Transfection Reagent from Thermo Fisher Scientific (#13778150) was employed for transient transfection. The following primary antibodies were used at 1:1000 dilution: anti-EXPAND1 (Cell Signaling Technology, #13437), anti-cleaved PARP (Cell Signaling Technology, #9532S), anti-BRCA2 (Cell Signaling Technology, #10741), anti-BRCA2 (Calbiochem, #OP95) and anti-α-tubulin (homemade). For signal detection, HRP-conjugated secondary antibodies were used at a 1:5000 dilution, including anti-rabbit (ThermoFisher, #31460), anti-mouse (ThermoFisher, #32430), and anti-rat (ThermoFisher, #31470). All antibodies were diluted in 5% non-fat milk in TBS-T and applied according to the manufacturers’ instructions.

Cell culture

MDA-MB-231, MCF7 and RPE1 cell lines, originally obtained from ATCC, were kindly provided by Dr. Alexander Hergovich (Evotec, France). Cells were maintained in DMEM/F12 (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS), 64 µg/ml penicillin, and 100 µg/ml streptomycin, and cultured at 37°C in a humidified incubator with 5% CO₂. Throughout the study, all cell lines were routinely monitored for mycoplasma contamination using PCR-based detection methods. To minimize the risk of microbial contamination, culture media were supplemented with Antimycotic/Antifungal Solution (Capricorn, AMP-B) and periodically treated with Mycoplasma Removal Reagent (Capricorn, MycoXpert™, MYX-B). All cell culture procedures were performed in accordance with Good Cell Culture Practice (GCCP) guidelines.

Silencing of EXPAND1 and/or BRCA2 expression via siRNA transfection

In this study, siRNA complexes specifically designed to target the open reading frame (ORF) region responsible for the synthesis of EXPAND1 and/or BRCA2 proteins were used to achieve gene silencing. Lyophilized siRNA complexes were reconstituted in nuclease-free water according to the manufacturer's protocol to prepare 10 µM stock solutions, which were stored at -20°C until use. The siRNA/transfection reagent mixture was prepared and applied following the manufacturer’s instructions. The gene-silencing efficiency of EXPAND1-targeting siRNAs (siEXPAND1#5 and siEXPAND1#4) and/or BRCA2-targeting siRNA (siBRCA2#6) was evaluated by comparison with a non-silencing control siRNA (siCTRL), which does not interfere with any known mRNA targets. Pilot experiments were conducted to optimize the concentrations of siRNA and transfection reagent, as well as the duration of transfection. All RNA interference experiments were validated by Western blot analysis. All gene transfer experiments, including siRNA-mediated knockdown procedures, were conducted in accordance with institutional biosafety guidelines and approved under the relevant biosafety committee protocols at Bingol University.

RNA Isolation, cDNA Synthesis, and qPCR Analysis

Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (BioDrop 80-3006-55). For cDNA synthesis, 1 µg of total RNA was reverse transcribed using the iScript™ One-Step RT-PCR Kit (Bio-Rad). Quantitative PCR (qPCR) was performed using SYBR Green chemistry (QuantiTect SYBR Green PCR Kit, Qiagen) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Pre-validated gene-specific primer sets (QuantiTect Primer Assays, Qiagen; catalog numbers QT00083860, QT00060235, QT00062090, QT00199367 for EXPAND1, p53, p21, and 18S rRNA, respectively) were used for target genes, ensuring standardized primer design and amplification efficiency according to the manufacturer’s specifications. Reactions were performed in technical triplicates. 18S rRNA was used as the internal reference gene, and relative gene expression levels were calculated using the 2^-∆∆Ct method [¹⁷].

Western blotting

To assess protein expression levels in cells, total protein extracts were prepared and analyzed using the Western blot technique, as previously described [¹⁸,¹⁹]. Total protein was extracted from cells collected via trypsinization and centrifugation, followed by resuspension in PBS and lysis in ice-cold buffer. After incubation on ice and centrifugation, protein concentrations were determined using the Bradford assay. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked and incubated overnight at 4°C with primary antibodies (diluted in 5% BSA), followed by HRP-conjugated secondary antibodies for 2 hours at room temperature. Protein bands were visualized using an ECL detection system, and densitometric analysis was performed with ImageJ software (NIH, USA).

In vitro WST-1 assay

The WST-1 assay, a colorimetric method used to assess cell viability and proliferation based on metabolic activity, was performed following the protocol described by Graham and coauthors [¹⁹]. Following transfection, cells were seeded into 96-well plates at a density of 5 × 10³ cells per well and incubated at 37°C in a 5% CO₂ atmosphere. After 72 hours post-seeding, 10 µL of tetrazolium salt was added to each well, and cells were incubated for 4 hours under the same conditions. Absorbance was measured at 450 nm with background correction at 650 nm. Data were analyzed after subtracting background and blank control values. Unless otherwise stated, all experiments were performed in triplicate across three independent biological replicates.

In vitro colony survival assay

Colony survival assays were performed as previously described [¹⁸-²¹]. Cells prepared at the appropriate density were transfected with the indicated siRNA and transfection reagent, then seeded into 6-cm plates. For treatment experiments, following an optimized post-transfection interval, cells were exposed to various concentrations of the test agents or inhibitors for durations determined in preliminary studies. The cells were incubated at 37°C in a humidified incubator with 5% CO₂ for 7-15 days to allow colony formation, with medium changes every 3-4 days. Colony growth was monitored microscopically, and experiments were terminated when colonies reached a size of at least 50 cells without overlapping. At the endpoint, dishes were washed with PBS, and cells were fixed using a methanol-acetic acid solution (3:1) for 5 minutes. Colonies were then stained with 0.5% crystal violet for 15 minutes, washed carefully with water, and visualized under a microscope. Only those consisting of ≥50 cells were counted as valid colonies. Colony numbers from treated groups were compared with those of the control groups to evaluate relative survival. Unless otherwise specified, all experiments were conducted in at least duplicate and repeated in three independent biological replicates.

Statistical Analysis

Unless otherwise stated, all experiments were performed with at least three independent biological replicates. Data are presented as mean ± S.E.M., and standard deviations were calculated where appropriate. Graphs and statistical analyses were generated using GraphPad Prism software (GraphPad Software, USA). Statistical significance was assessed using Student’s t-test or one-way analysis of variance (ANOVA), as indicated in the corresponding figure legends. A p-value < 0.05 (*), < 0.01 (**), < 0.001 (***), or < 0.0001 (****) was considered statistically significant.

RESULTS

Dual inhibition of EXPAND1 and PARP synergistically reduces cell viability in breast cancer cells

To assess the basal expression profile of EXPAND1 and confirm the specificity of the commercially obtained anti-EXPAND1 antibody, we performed Western blot analysis using protein lysates from one non-tumorigenic human cell line (RPE1) and three cancer cell lines (MDA-MB-231, HCT116, and OVCA420). As shown in Figure 1-A, EXPAND1 protein was detected in all four cell lines, with varying levels of expression. The antibody recognized a single band at the expected molecular weight (~85 kDa), supporting its specificity and suitability for downstream analyses. To validate the efficiency and duration of siRNA-mediated knockdown of EXPAND1, we transfected MDA-MB-231 cells with siEXPAND1#5 and collected protein lysates at 24-, 48-, and 72-hours post-transfection. As shown in Figure 1-B, Western blot analysis demonstrated a marked decrease in EXPAND1 protein expression beginning at 24 hours and sustained through 72 hours. Densitometric quantification (Figure 1-C) confirmed this trend, revealing that EXPAND1 protein levels were reduced to approximately 20-30% of control levels at all time points following siRNA treatment. Importantly, we also confirmed the efficacy of a second siRNA (siEXPAND1#4) targeting EXPAND1 mRNA, as shown in Figure 1-D, further supporting the specificity of the knockdown approach. These findings verify that the siRNA construct effectively and consistently suppresses EXPAND1 expression over a 72-hour period, supporting its use in downstream functional assays.

Figure 1
Silencing of EXPAND1 sensitizes MDA-MB-231 cells to PARP inhibition. (A) Western blot analysis showing endogenous expression levels of EXPAND1 across a panel of cell lines: non-tumorigenic retinal epithelial cells (RPE1) and cancer cell lines (MDA-MB-231, HCT116, and OVCA420) (n=1). (B) Western blot showing time-course validation of siRNA-mediated knockdown of EXPAND1 (siEXPAND1#5) in MDA-MB-231 cells at 24-, 48-, and 72-hours post-transfection (n=1). (C) Densitometric analysis of EXPAND1 protein levels from panel (B), normalized to tubulin and expressed relative to siCTRL. (D) Western blot showing time-course validation of siRNA-mediated knockdown of EXPAND1 (siEXPAND1#4) in MDA-MB-231 cells at 24-hour post-transfection (n=1). (E) MDA-MB-231 cells transfected with control siRNA (siCTRL), siEXPAND1#5 or siEXPAND1#4 were treated 24 hours later with increasing concentrations of olaparib (0-10 µM) for 72 hours. Cell viability was assessed using WST-1 assay. Data represent mean ± S.E.M. of three independent experiments: *p< 0.05, **p< 0.01, ***p< 0.001 by one-way ANOVA. (n=4) (F) Proliferation assay showing no significant difference in growth rates between siCTRL and siEXPAND1-transfected (both siEXPAND1#5 and siEXPAND1#4) MDA-MB-231 cells in the absence of drug treatment (n=3). (G) Colony survival assay showing comparable colony-forming capacity between siCTRL and siEXPAND1-transfected (both siEXPAND1#5 and siEXPAND1#4) MDA-MB-231 cells without olaparib treatment (n=3). (H) Representative images of colonies stained with crystal violet are shown. Tubulin was used as a loading control. siEXPAND1#5 and siEXPAND1#4: siRNAs targeting EXPAND1. ns = not significant. Olap: Olaparib.

Next, to investigate whether EXPAND1 modulates sensitivity to PARP inhibition, we silenced EXPAND1 expression using two independent siRNA constructs in MDA-MB-231 cells and, 24 hours post-transfection, treated the cells with increasing concentrations of olaparib for 72 hours. Cell viability was assessed using the WST-1 assay, a colorimetric method that reflects mitochondrial metabolic activity and is widely used to quantify the viability of adherent cells [²²]. As shown in Figure 1-E, olaparib treatment alone led to a dose-dependent decrease in viability in both siCTRL- and siEXPAND1-transfected cells. However, cells with EXPAND1 knockdown exhibited significantly greater sensitivity to olaparib, particularly at concentrations of 2 µM and above, where viability was markedly reduced compared to control. These findings suggest that depletion of EXPAND1 enhances the cytotoxic effects of PARP inhibition, supporting its potential role in modulating cellular responsiveness to olaparib in breast cancer cells. Statistical analysis confirmed the significance of these differences (*p < 0.05, **p < 0.01, ***p < 0.001). To rule out the possibility that the enhanced sensitivity to olaparib observed in EXPAND1-depleted cells was due to impaired cell cycle progression or baseline proliferative defects, we performed cell viability and colony survival assays in the absence of drug treatment. As expected, knockdown of EXPAND1 did not significantly affect short-term proliferation, as measured by WST-1 assay, nor long-term clonogenic potential compared to siCTRL-transfected cells (Figure 1-F-H). Quantitative analysis and representative colony images revealed no substantial difference in colony number or size between control and EXPAND1-silenced cells (Figure 1-G, H). These findings indicate that the observed effects upon PARP inhibition are not due to general defects in proliferation or survival resulting from EXPAND1 depletion but rather reflect a specific sensitization to genotoxic stress.

EXPAND1 depletion sensitizes both parental and PARPi-resistant breast cancer cells to PARP inhibition in colony survival assays

To further investigate the impact of EXPAND1 knockdown on long-term survival following PARP inhibition, we performed a colony survival assay in MDA-MB-231 cells treated with increasing concentrations of olaparib. Cells were first transfected with either siCTRL or siEXPAND1 (#5 or #4), and successful knockdown was confirmed by Western blotting. Twenty-four hours after transfection, cells seeded at low density were exposed to olaparib (0, 1, 2.5, 5 µM) for 10-14 days. Representative images show a progressive reduction in colony numbers with increasing olaparib concentration in both groups (Figure 2-A). However, EXPAND1-depleted cells exhibited a markedly stronger suppression of colony survival compared to controls. Quantification of colony numbers (Figure 2-B,C) confirmed that EXPAND1 knockdown significantly enhanced the sensitivity of cells to olaparib treatment. To further validate our findings and confirm that the observed effects were not specific to a single PARPi, we repeated the colony survival assay using rucaparib, a clinically approved alternative to olaparib. As shown in Figure 2-D, EXPAND1-silenced MDA-MB-231 cells exhibited significantly reduced colony-forming capacity following rucaparib treatment compared to siCTRL-transfected cells. To determine whether the EXPAND1-dependent sensitization to PARP inhibition observed in MDA-MB-231 cells is reproducible in other models and selective for cancer cells, we performed WST-1 cell viability assays in MCF7 breast cancer cells and RPE1 non-tumorigenic epithelial cells. As shown in Figure 2-E, EXPAND1 knockdown in MCF7 cells significantly enhanced sensitivity to olaparib, mirroring the results obtained in MDA-MB-231 cells. However, in RPE1 cells, depletion of EXPAND1 had no significant impact on cell viability following olaparib treatment (Figure 2-F). These findings indicate that the synergistic effect of EXPAND1 loss and PARP inhibition is cancer cell-specific and does not compromise the viability of normal cells, thereby underscoring the therapeutic potential and selectivity of targeting EXPAND1 in malignant contexts.

Figure 2
EXPAND1 depletion sensitizes breast cancer cells to PARP inhibition in long-term survival. (A) Colony survival assay of MDA-MB-231 cells showing the long-term effects of EXPAND1 knockdown on cellular sensitivity to increasing concentrations of olaparib (0, 1, 2.5, 5 µM) for 10-14 days. Cells were fixed, stained with crystal violet and imaged. (B,C) Quantification of colony survival following olaparib treatment (0, 1, 2.5, 5 µM for 10-14 days) in control (siCTRL) and EXPAND1-depleted (siEXPAND1#5, siEXPAND1#4) MDA-MB-231 cells. Graph depicts mean survival fraction ± S.E.M. from three independent experiments (n=3). (D) Quantification of colony survival following rucaparib treatment (0, 1, 2.5, 5 µM for 10-14 days) in control (siCTRL) and EXPAND1-depleted (siEXPAND1#5) MDA-MB-231 cells. Graph shows mean survival fraction ± S.E.M. from three independent experiments (n=3). (E) Quantification of colony survival following olaparib treatment (0, 1, 2.5, 5 µM for 10-14 days) in control (siCTRL) and EXPAND1-depleted (siEXPAND1#5) MCF7 cells. Graph shows mean survival fraction ± S.E.M. from three independent experiments (n=3). (F) RPE1 cells transfected with siCTRL or siEXPAND1#5 were treated 24 hours later with increasing concentrations of olaparib (0-5 µM) for 72 hours. Cell viability was assessed using WST-1 assay. Data represent mean ± S.E.M. of three independent experiments (n=3). *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical significance between siCTRL and siEXPAND1 groups, as determined by Student’s t-test analysis. ns = not significant. siEXPAND1#5 and siEXPAND1#4: siRNAs targeting EXPAND1. Olap: Olaparib, Rucap: Rucaparib.

To further investigate the functional contribution of EXPAND1 to acquired drug resistance, we examined its role in an olaparib-resistant MCF7/PIR model, which we generated through a stepwise dose-escalation protocol [²³]. Parental MCF7 cells were exposed to progressively increasing concentrations of olaparib every other week over a 4-month period, and resistant clones arising at each dose level were isolated and cryopreserved (Figure 3-A). We confirmed the establishment of resistance using a colony survival assay, and as shown in Figure 3-B,C, MCF7/PIR clones exhibited markedly higher survival under olaparib treatment compared with the parental MCF7 cells (*p <0.05, **p <0.01, ***p <0.001), validating the resistant phenotype for downstream studies. Colony survival assays revealed that silencing EXPAND1 in MCF7/PIR cells significantly re-sensitized them to olaparib treatment (Figure 3-D). Compared to siCTRL, EXPAND1-depleted cells displayed a marked decrease in colony number, particularly at higher olaparib concentrations. Quantitative analysis confirmed this observation, with a statistically significant drop in colony survival upon EXPAND1 knockdown (Figure 3-E) (**p < 0.01). These results indicate that EXPAND1 contributes to the maintenance of PARPi resistance and suggest that targeting EXPAND1 may help overcome acquired resistance in HR-proficient breast cancer cells.

Figure 3
Generation and characterization of MCF7/PIR cells and impact of EXPAND1 depletion on PARPi sensitivity. (A) Schematic overview of the stepwise olaparib dose-escalation protocol used to generate MCF7/PIR model. Parental MCF7 cells were exposed to gradually increasing concentrations of olaparib every other week for 4 months, and resistant clones arising at each step were isolated and cryopreserved. (B) Representative colony survival assay of parental MCF7 and resistant MCF7/PIR cells treated with increasing doses of olaparib (0, 2.5, 5 µM) for 10-14 days. (C) Quantification of colony survival following olaparib treatment in (B) (n=3). (D) Representative colony survival images of MCF7/PIR cells transfected with siCTRL or siEXPAND1#5 and subsequently treated with increasing concentrations of olaparib (0, 1, 2.5, 5 µM) for 10-14 days. (E) Quantification of colony survival following olaparib treatment in (D) (n=3). Data represent mean ± S.E.M. of three independent experiments (n=3). *p <0.05, **p < 0.01, ***p < 0.001 indicate statistical significance between siCTRL and siEXPAND1#5 groups, as determined by Student’s t-test analysis. siEXPAND1#5: siRNA targeting EXPAND1. PIR: PARPi-resistant. Olap: Olaparib

EXPAND1 depletion promotes apoptotic elimination of cancer cells following PARP inhibition

To gain mechanistic insight into how EXPAND1 depletion sensitizes cells to PARP inhibition, we examined the expression of key regulators of DNA damage response and apoptosis-p53, p21, and cleaved PARP (cPARP)-in MDA-MB-231 cells treated with a fixed dose of olaparib for 72 hours. Protein levels of all three markers were assessed by Western blot, while qPCR analysis was performed to measure p53, p21, and EXPAND1 mRNA expression. Since cPARP is a post-translational cleavage product, it was analyzed only at the protein level. In the case of EXPAND1, qPCR was also used alongside Western blot to confirm efficient gene silencing following siRNA transfection. This combined approach allowed us to characterize the molecular consequences of EXPAND1 loss under PARPi treatment at multiple levels of regulation. Western blot analysis (Figure 4-A) revealed a dramatic accumulation in p53 protein levels in EXPAND1-depleted cells upon olaparib treatment, compared to control (siCTRL). This induction was consistent with the upregulation of p53 mRNA detected by qPCR (Figure 4-B, C). However, it is critical to note that MDA-MB-231 cells harbor a mutant p53 [²⁴] that is stabilized in cells and lacks canonical transcriptional activity toward many wild-type p53 targets [²⁵]. Therefore, the observed upregulation most likely reflects accumulation of dysfunctional p53 due to impaired degradation rather than activation of a wild-type DNA damage response. This interpretation is consistent with prior literature showing [²⁶,²⁷] that mutant p53 proteins are often highly stable and can be further induced by genotoxic stress without eliciting normal checkpoint responses. Accordingly, p21 expression displayed a complex pattern: although p21 mRNA showed a transient increase at 48 h in EXPAND1-depleted cells (Figure 4-D), it declined at later time points and was not sustained at the protein level (Figure 4-A). Given the p53 mutation, this decoupling should not be interpreted as a failure of a wild-type p53-p21 axis, but rather as a reflection of an aberrant, context-dependent regulation of p21 that does not effectively enforce cell cycle arrest in response to dual EXPAND1-PARP inhibition-induced damage. To validate the pro-apoptotic effect of EXPAND1 silencing under PARP inhibition, we assessed cleaved PARP (cPARP) and cleaved caspase-3 levels by Western blot in both MCF7 (p53 wild-type) [²⁸] and MDA-MB-231 (p53 mutant) [²⁴] cells. In MCF7 cells treated with olaparib, EXPAND1 knockdown resulted in a marked increase in cPARP levels compared to siCTRL-treated cells (Figure 4-G,I), indicating enhanced apoptosis. Similarly, in MDA-MB-231 cells, a comparable increase in cPARP expression was observed following EXPAND1 depletion and PARPi treatment (Figure 4-E,F). To further confirm apoptotic induction, we examined cleaved caspase-3 levels. Both cell lines showed strong caspase-3 cleavage upon combined EXPAND1 knockdown and PARPi treatment (Figure 4-G,H). These results confirm that EXPAND1 silencing promotes apoptosis following PARPi exposure in both p53 wild-type and mutant contexts, as evidenced by activation of the caspase cascade and PARP cleavage.

Figure 4
Mechanistic evaluation of EXPAND1 knockdown under PARPi treatment. (A) Western blot analysis of MDA-MB-231 cells treated with a fixed dose of olaparib (5 µM) for 24-, 48- and 72-hours, showing expression of EXPAND1, p53 and p21 in siCTRL and siEXPAND1#5 conditions (n=2). (B-D) Quantitative PCR (qPCR) analysis of gene expression levels of (B) EXPAND1, (C) p53, and (D) p21 upon EXPAND1 depletion. Bars represent mean ± S.E.M. of three biological replicates each with three technical repeats. ns = not significant; *p < 0.05; **p < 0.01; ***p < 0.001. (E) Western blot analysis of cPARP expression following 72-hour treatment with olaparib (+, 5 µM) or vehicle (-, DMSO) in siCTRL and siEXPAND1#5 MDA-MB-231 cells (n=3). (F) Western blot analysis of cPARP expression in MDA-MB-231 cells treated with 72-hour rucaparib (+, 5 µM) or vehicle (-, DMSO), with or without EXPAND1 knockdown (n=2). (G) Western blot analysis of cPARP expression following 72-hour treatment with olaparib (+, 5 µM) in siCTRL and siEXPAND1#5 MCF7 cells (n=2). (H) Western blot analysis of cleaved caspase-3 expression following 72-hour treatment with olaparib (+, 5 µM) in siCTRL and siEXPAND1#5 MDA-MB-231 and MCF7 cells (n=2). (I) Western blot analysis of cPARP expression following 72-hour treatment with olaparib (+, 5 µM) or vehicle (-, DMSO) in siCTRL and siEXPAND1#4 MCF7 cells (n=2). (J) Western blot analysis of EXPAND1 protein levels in MDA-MB-231 and RPE1 cells treated with increasing concentrations of olaparib (0-10 µM) for 72 hours (n=2). (K) Western blot showing EXPAND1 protein levels in MDA-MB-231 cells treated with increasing concentrations of rucaparib (0-10 µM) for 72 hours (n=2). Tubulin was used as a loading control. siEXPAND1#5 and siEXPAND1#4: siRNAs targeting EXPAND1. Olap: Olaparib. Rucap: Rucaparib. cPARP: cleaved PARP.

Finally, to investigate whether EXPAND1 protein levels are modulated in response to PARP inhibition, we treated MDA-MB-231 cells with increasing concentrations of olaparib and rucaparib and assessed EXPAND1 expression by Western blotting. Interestingly, EXPAND1 protein levels exhibited a gradual and dose-dependent decrease following treatment with both PARPi (Figure 4-J, K). This trend suggests that PARPi exposure-particularly in cancer cells-may directly or indirectly destabilize EXPAND1, adding a new layer to the interplay between chromatin remodeling factors and DNA repair inhibition. In contrast, in non-cancerous RPE1 cells, EXPAND1 levels remained largely unchanged even under high-dose olaparib treatment (Figure 4-J, right panel), indicating that this response is not a universal cellular reaction, but may reflect an underlying vulnerability in cancer-specific or DNA-repair-deficient contexts.

EXPAND1 depletion does not enhance PARPi sensitivity in BRCA2-deficient cancer cells

To determine whether EXPAND1 depletion could further sensitize cells already deficient in homologous recombination repair, we investigated the impact of dual silencing of BRCA2 and EXPAND1 on the cytotoxic response to olaparib. First, we validated the knockdown efficiency of BRCA2 by transfecting MDA-MB-231 cells with siBRCA2 and harvesting lysates at 24-, 48-, 72- and 96-hours post-transfection. As shown in Figure 5-A, Western blotting confirmed a progressive reduction in BRCA2 protein levels over time, which was further supported by densitometric quantification (Figure 5-B), verifying the efficiency and durability of siRNA-mediated knockdown. Next, we examined whether EXPAND1 depletion enhances olaparib sensitivity in BRCA2-deficient cells. Cells were co-transfected with siRNAs targeting BRCA2, EXPAND1, or both, followed by olaparib treatment and colony survival analysis. As expected, BRCA2-deficient cells showed a significant reduction in colony survival upon olaparib exposure, consistent with the well-established role of BRCA2 in homologous recombination-mediated DNA repair (Figure 5-C, D). However, although EXPAND1 knockdown alone sensitized cells towards olaparib as expected, co-depletion of EXPAND1 and BRCA2 did not lead to further reduction in colony survival beyond that observed with BRCA2 knockdown alone.

Figure 5
EXPAND1 depletion does not enhance PARPi sensitivity in BRCA2-deficient cells. (A) Western blot showing time-course silencing efficiency of BRCA2 using siBRCA2#6 in MDA-MB-231 cells transfected for 24-, 48-, 72-and 96-hours. (B) Densitometric quantification of BRCA2 protein levels normalized to tubulin from blot shown in (A), confirming knockdown efficiency (n=1). (C) Western blot analysis showing BRCA2 and EXPAND1 protein levels following single or dual siRNA transfection (siBRCA2#6 and/or siEXPAND1#5) in MDA-MB-231 cells. (D) Colony survival assay results from cells transfected with siCTRL, siBRCA2#6, siEXPAND1#5, or the combination of siBRCA2#6 + siEXPAND1#5 and subsequently treated with/without olaparib (0 or 2 µM for 10-14 days). Bar graph shows the percentage of colony survival normalized to untreated siCTRL group (n=3). ns = not significant; *p < 0.05; **p < 0.01. Tubulin was used as a loading control. siEXPAND1#5: siRNA targeting EXPAND1. siBRCA2#6: siRNA targeting BRCA2.

DISCUSSION

The expanding clinical application of PARPi has transformed the therapeutic landscape for homologous recombination-deficient (HRD) cancers, particularly those with BRCA1/2 mutations. However, the efficacy of PARPi in BRCA-proficient tumors, including a substantial subset of triple-negative breast cancers (TNBCs), remains limited due to inherent or acquired resistance mechanisms [¹⁵,²⁹-³⁴]. Understanding the chromatin context and the auxiliary regulators of DNA damage response is therefore essential for refining the therapeutic window of PARP inhibition beyond classical HR-deficient models [³⁵-³⁷]. In this study, we identify EXPAND1, a chromatin-binding protein recruited to sites of DNA damage via 53BP1 and γH2AX [¹³], as a potential modulator of PARPi sensitivity in BRCA2 wild-type breast cancer cells.

Chromatin architecture plays a pivotal role in orchestrating the cellular response to DNA damage, acting as both a physical barrier and a regulatory platform for repair factors. The study by Huen et al. [2010] identifies EXPAND1 as a chromatin-binding protein recruited to sites of DNA damage through its interaction with 53BP1 and γH2AX [¹³]. Through its PWWP domain, EXPAND1 anchors to nucleosomes and facilitates chromatin decondensation, a necessary step for the access and coordination of DNA repair machinery. Notably, depletion of EXPAND1 results in chromatin compaction, accumulation of unrepaired damage, and heightened sensitivity to genotoxic stress, despite intact checkpoint signaling [¹³]. These findings underscore the essential role of chromatin remodeling factors in enabling DNA repair and maintaining genomic stability. In the context of PARP inhibition, where DNA lesions persist due to inhibition of single strand break repair and trapping of PARP on chromatin, impaired chromatin relaxation caused by EXPAND1 loss could further exacerbate DNA damage accumulation. This suggests that the efficacy of PARPi may be amplified in tumors with compromised chromatin remodeling capacity, providing a rationale for targeting structural chromatin regulators like EXPAND1 to enhance therapeutic response.

Mounting evidence underscores the importance of chromatin dynamics in shaping the cellular response to DNA damage, particularly in the context of therapeutic agents that disrupt repair fidelity. In this study, we demonstrate that EXPAND1, a PWWP domain-containing chromatin-associated factor, plays a key role in maintaining cancer cell viability under PARPi stress. Consistent with previous work identifying EXPAND1 as a 53BP1-interacting protein that facilitates DNA damage-induced chromatin relaxation [¹³], our data reveal that depletion of EXPAND1 markedly sensitizes BRCA-proficient breast cancer cells to PARPi, including both olaparib and rucaparib. This synthetic lethal interaction is notably absent in non-transformed RPE1 cells, indicating a cancer-selective vulnerability and suggesting that EXPAND1 supports survival specifically in chromatin-stressed, repair-competent cells. While these findings are promising, we acknowledge that experiments were conducted in only two breast cancer cell lines, which limits translational generalization. Therefore, our claims regarding clinical relevance remain hypothesis-generating and require broader validation in additional cancer models.

Mechanistically, we found that EXPAND1 knockdown under PARPi treatment triggers robust apoptotic signaling, as evidenced by a significant increase in cleaved PARP (cPARP) [³⁸,³⁹] and sustained activation of p53 [⁴⁰-⁴²]. Interestingly, although p53 was strongly upregulated at both transcript and protein levels, p21 -a key downstream effector mediating p53-driven cell cycle arrest [⁴³]-exhibited only transient induction, with expression ultimately declining below control levels. This decoupling between p53 and p21 [⁴⁴] suggests that EXPAND1 may play a non-redundant role in sustaining transcriptional programs downstream of DNA damage, likely through chromatin-based mechanisms that influence promoter accessibility or transcriptional elongation. The functional consequence of this uncoupling appears to be a shift in cellular fate: in the absence of sustained p21, p53 activation no longer promotes cytostasis but instead facilitates apoptotic elimination. However, it is important to consider that MDA-MB-231 cells harbor a mutant p53 [²⁴] that compromises DNA-binding capacity. Thus, our findings of p53 protein induction must be interpreted cautiously, as this may reflect transcriptionally inactive mutant accumulation. The observed increase in caspase-3 and cPARP in both MDA-MB-231 and p53-wild-type MCF7 cells supports the involvement of p53-independent apoptosis as well.

This model is further reinforced by our observation that EXPAND1 protein levels decrease in a dose-dependent manner upon PARPi treatment, specifically in MDA-MB-231 cells but not in RPE1 cells, which was reported previously for BRCA1 protein. In a study using lymphoblastoid cell lines, BRCA1 heterozygous cells exhibited suppressed BRCA1 protein levels following olaparib treatment, along with increased apoptosis and radiation hypersensitivity, suggesting that PARP inhibition can destabilize DNA repair proteins in a context-dependent manner [⁴⁵]. Similarly, in hormone receptor-positive breast cancer cells (MCF7 and ZR-75-1), prolonged exposure to olaparib resulted in reduced intracellular levels of APE1 and PARP1 proteins [⁴⁶]. Another study in C4-2 prostate cancer cells demonstrated that olaparib significantly decreased PLK1 expression at both mRNA and protein levels, implying that PARP inhibition may broadly impact the expression of proteins involved in DNA repair and cell cycle regulation [⁴⁷]. These findings are in line with our data and support a model in which PARP inhibition disrupts not only repair activity but also the stability of key chromatin-associated factors like EXPAND1, potentially via altered transcription, post-translational modification, or proteasomal degradation. Several mechanisms could potentially explain this observation. One possibility is that PARPi-induced chromatin stress alters the transcriptional regulation of EXPAND1, thereby reducing EXPAND1 protein expression. Alternatively, PARP inhibition may affect the stability of chromatin-associated proteins through post-translational mechanisms such as ubiquitin-mediated proteasomal degradation. Another plausible explanation is that PARPi-induced changes in chromatin structure impair EXPAND1 recruitment or retention at DNA damage sites, leading to its destabilization in cells that rely on high levels of chromatin remodeling for survival-such as aggressive, mesenchymal-like breast cancer cells. The absence of this regulation in RPE1 cells suggests that EXPAND1 modulation by PARPi is context-dependent and may reflect differences in the epigenetic or mutational landscape between normal and cancer cells.

To further explore the interplay between EXPAND1 and homologous recombination, we assessed whether EXPAND1 depletion could enhance PARPi sensitivity in BRCA2-deficient cells. As expected [⁹,¹⁰] BRCA2 knockdown alone rendered cells highly sensitive to olaparib, but co-depletion of EXPAND1 did not produce additive effects, suggesting that once homologous recombination is disabled, EXPAND1’s contribution becomes dispensable. This supports the concept that EXPAND1’s function is most critical in BRCA2-wild-type settings, where cells still rely on organized chromatin remodeling to mediate checkpoint signaling and facilitate repair through available HR pathways. In contrast, in HR-deficient cells, the apoptotic threshold may already be maximally triggered by unrepaired DNA lesions, and further perturbation of chromatin architecture yields no additional cytotoxicity. Future work should aim to dissect the molecular dependencies between EXPAND1 and HR factors more precisely-for example, by evaluating its role in alternative end-joining or replication fork stability, and by determining whether EXPAND1 loss alters sensitivity in other HR-impaired genetic backgrounds beyond BRCA2 deficiency.

In summary, our findings identify EXPAND1 as a promising chromatin-associated factor that modulates the response to PARP inhibition in BRCA-proficient breast cancer cells. Its loss sensitizes cells to PARPi via impaired chromatin remodeling, uncoupling of p53-p21 signaling, and activation of apoptosis, as evidenced by increased caspase-3 and cPARP levels in both p53-wild-type and mutant backgrounds. However, limitations-including the absence of detailed apoptotic analysis, lack of phenotype rescue and reliance on two cell lines-necessitate further validation. In order to position EXPAND1 as a potential PARPi biomarker, future studies should also aim to validate EXPAND1-linked signatures in TCGA datasets and patient-derived organoids, develop IHC/IF- or RNA-based biomarker tools with defined expression thresholds and explore combination strategies that exploit chromatin remodeling defects to enhance PARPi efficacy.

CONCLUSION

In this study, we identify EXPAND1 as a chromatin-associated protein that modulates cellular sensitivity to PARP inhibition in BRCA2-proficient breast cancer cells. Its depletion significantly reduced cell viability and colony survival upon PARPi treatment, while exerting minimal effects in non-transformed or BRCA2-deficient cells. Mechanistically, EXPAND1 knockdown triggered a strong apoptotic response, marked by cleaved PARP and caspase-3 activation, alongside elevated p53 expression and a failure to sustain p21-mediated cell cycle arrest-shifting the damage response toward apoptosis rather than cytostasis. Notably, EXPAND1 protein levels were downregulated in a dose-dependent manner by PARPi in cancer cells, but not in normal cells, suggesting a selective vulnerability linked to chromatin remodeling stress. Collectively, these findings suggest that EXPAND1 may function as a chromatin regulator influencing PARPi responsiveness and warrant further investigation of its potential as a candidate determinant of PARPi responsiveness that warrants further validation in BRCA2-proficient cancer contexts.

Funding:

This research was funded by the Scientific and Technological Research Council of Türkiye (TUBITAK), grant number 218S877.

Institutional Review Board Statement:

Not applicable.

Informed Consent Statement:

Not applicable.

Acknowledgments:

The authors thank Ronahi Askan, a TUBITAK STAR scholar, as well as all members of the Bingol University Cancer Research Laboratory for their insightful contributions to this study.

Author Declarations:

All authors have agreed to the Declaration of Responsibility and Licence Agreement, confirming their acceptance of publication under the Creative Commons Attribution license. The authors declare that all sources of financial support and any potential conflicts of interest have been fully disclosed. This study was conducted in accordance with the ethical standards and regulatory procedures of the Research Ethics Commission of the authors' institution. As no human participants were involved, informed consent was not applicable. Should future use of patient data or clinical material arise, informed consent will be obtained and archived in compliance with the journal’s policies.

Use of Generative Artificial Intelligence:

The authors declare that large language models and other generative artificial intelligence (AI) or AI-assisted technologies cannot be credited as authors and have not been listed as authors of this paper.

The authors declare that no generative artificial intelligence (AI) or AI-assisted technologies were used to generate or modify the scientific content of this manuscript, including the conception of the study, data collection, data analysis, interpretation of results, or creation of original text, figures, tables or graphical abstracts, apart from routine tools for spelling, grammar checking and reference management that do not create original scholarly content.

Data Availability Statement:

Research data are only available upon request from the corresponding author.

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Editor-in-Chief:

Paulo Vitor Farago

Associate Editor:

Jaiesa Zych

*

Correspondence: [e-mail] Tel.: [phone] (R.G.).

Conflicts of Interest:

The authors declare no conflict of interest.

Publication Dates

• Publication in this collection
  06 Nov 2026
• Date of issue
  2026

History

• Received
  13 Dec 2025
• Accepted
  05 July 2026