Upregulated PARP1 confers breast cancer resistance to CDK4/6 inhibitors via YB-1 phosphorylation

SK-BR-3, MCF7, MDA-MB-231, HCC1937, T47D and HCC1806 cells were obtained from ATCC. MCF7AR was generated from the parent MCF7 cell by treating abemaciclib continuously. All cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum and 1% antibiotic (Gibco) at 37 °C with 5% CO2. Cells were used for no longer than 12 months before being replaced. Abemaciclib and olaparib were purchased from TargetMol, USA, and LJI308 was purchased from Med Chem Express, USA. The inhibitors were dissolved in DMSO.

MCF7 abemaciclib-resistant cells (MCF7AR) were derived by treating MCF7 cells with increasing concentrations of abemaciclib starting at 0.1 μM, followed by a stepwise dose up to a final abemaciclib concentration of 20 μM. On the 1st day, the MCF7 cells were exposed to 0.1 μM abemaciclib for 3 days, and then the cells were grown in medium without drugs. The density of the remaining cells returned to 80% after approximately 4 days. On the 8th day, the cells were cultured in medium with 0.3 μM abemaciclib for 3 days and recovered to 80% confluence in the next 7 days. On the 18th day, we increased the drug concentration to 1 μM for 5 days of treatment and recovered to 80% density in the next 7 days. On the 30th day, we exposed the cells to 1 μM abemaciclib in media for up to 30 days, and the cells grew steadily in media with 1 μM abemaciclib. On the 60th day, we increased the abemaciclib concentration to 5 μM for approximately 3 days, and the cells recovered to 80% confluence after withdrawing the drug in the next 7 days. We exposed the cells to 5 μM abemaciclib in media for the next 20 days. On the 90th day, the cells continued to grow in 10 μM abemaciclib for approximately 30 days. On the 120th day, the cells were exposed to 20 μM abemaciclib in media for up to 60 days. MCF7AR cells can grow stably in medium with abemaciclib at 20 μM. Cells were washed with PBS 3 times after withdrawing the drugs and replacing them with fresh drug and media. The resistant cell lines established in this manner were then maintained in drug-free medium or with 1 μM abemaciclib within 3 generations after thawing from storage in liquid nitrogen.

Paraffin-embedded breast cancer tumor specimens from 90 patients were collected at the First Affiliated Hospital of Shenzhen University (Shenzhen, China). The study was approved by the Institutional Review Board of First Affiliated Hospital of Shenzhen University; human sample collection procedures were in accordance with the established guidelines. All human breast cancer sample acquisitions were approved by the Committee on Ethics of Shenzhen Second People’s Hospital, Shenzhen University. The pathological information of clinical breast cancer patients is listed in Additional file 1: Table S1.

CCK-8 assays were used to test the drug efficiency on the cell lines. The breast cancer cells were laid on a 96-well dish starting at 5 × 10³ cells per well for the CCK-8 assay. Thereafter, 10 μL CCK-8 working solution (Beyotime) was added at the indicated time points after the cells were treated with the inhibitors. The plates were incubated at 37 °C for 2 h. Next, the cell viability was measured by a microplate reader (Thermo Fisher) with the optical density of each well at 450 nm. A cell crystal violet staining assay was employed to detect cell viability based on the amounts of cell stains. Cells were laid on a 12-well dish starting at 5 × 10⁴ cells/well, and 3 parallel dishes were used to assess the experimental accuracy. The cells were all stained with crystal violet solution and then rinsed for 5 min. Next, pictures were captured under a microscope.

WB and IHC assays were performed as previously reported by our laboratory [20]. Briefly, total intracellular protein was extracted using RIPA lysis buffer (Beyotime, China) for WB assays, protease inhibitor cocktail (Beyotime) and 0.1 mM PMSF (Beyotime) were added, and a BCA assay kit (Beyotime) was used to assess protein concentrations. Thereafter, protein loading, electrophoresis and membrane transfer were performed according to the manufacturer’s instructions. Next, milk blocking, membrane washing and primary antibody incubation overnight were performed in sequence. Finally, after incubation with the secondary antibody for 2 h, the target proteins on the membrane were detected by the Beyo ECL Star Kit (Beyotime), and Bio-Rad Quantity One Software was used for quantification. The IHC assay was performed to detect PARP1 and p-YB-1 expression in human breast cancer sections and adjacent tissues. The slides were soaked in xylol for 10 min and washed in a series of alcohols with decreasing concentrations. Antigen retrieval was performed when the sections were soaked in citrate buffer in a microwave for 5 min after tissue deparaffinization. Thereafter, the sections were blocked in 1% peroxidase and then incubated in goat serum. Primary antibodies against PARP1 and YB-1 were incubated for 12 h. The biotinylated rabbit anti-mouse antibody (Vector, Germany) was used for signal amplification. Subsequently, the slides were stained with diaminobenzidine (DAB) (Sigma, USA) to label PARP1/p-YB-1 and counterstained with hematoxylin to label nuclei. To increase the accuracy of the results, the staining results came from the same position of successive slices. The antibody information for Western blot and immunohistochemistry are listed in Additional file 2: Table S2.

Construction of the PARP1 and WT/S102A/S102E YB-1 plasmids were designed and synthesized by the Yunzhou Biotechnology Company in China. The wild-type and mutant YB-1 plasmids were transfected into MCF7 cells using Lipofectamine 3000 reagent (Gibco) according to the manufacturer’s instructions. The siRNAs targeting PARP1 and YB-1 were purchased from Tsingke Company (Beijing, China), and the construction of shYB-1 stable cell lines was completed by laboratory colleagues previously [20]_. The siRNAs targeting PARP1 and YB-1 were transfected into MCF7 and MCF7AR cells using Lipofectamine 3000 reagent (Gibco). The primer information is listed in Additional file 3: Table S3.

The ChIP samples from MCF7AR-siYB-1 cells were fixed with 37% formaldehyde (final concentration 1%) and then decrosslinked by adding 10 × 1.25 M glycine. DNA‒protein complexes were obtained under ultrasonication and were used for subsequent DNA purification steps. The ChIP assay (Beyotime P2080S) protocol can guide the follow-up experimental procedure according to the manufacturer’s instructions. Thereafter, a primary antibody targeting YB-1 was coincubated with the lysate of the DNA‒protein complex for immunoprecipitation, and an IgG antibody was used as a negative control. The immunoprecipitated DNA was treated with RNase A and proteinase K, followed by purification using phenol‒chloroform extraction and ethanol precipitation. The content determination of targeted DNA was analyzed by quantitative real-time PCR. The primer sequences and antibodies used in this study are listed in Additional files 2, 3: Tables S2, S3.

Construction of the reporter plasmid pGL3-Basic containing the promoter region 2100 bp of PARP1 was conducted by Tsingke in China. Renilla luciferase reference plasmid pRL-TK, pGL3-Basic and WT/S102A/S102E-YB-1 plasmid were simultaneously transfected into cells for 48 h, and empty plasmid pGL3-Basic was used as a control. PLB buffer was added for cell lysis, and then 10 μL of supernatant was transferred to 96-well wells. Next, 100 μL luciferase assay reagent was added, and the intensity of the fluorescein reaction was detected in a dark environment. Thereafter, the luciferase reaction intensity of Renilla pRL-TK was detected after adding 100 μL Stop and Glo Reagent. Three parallel wells were used to reduce errors, RLU1 represented firefly fluorescein plum intensity, and RLU2 represented Renilla luciferase intensity. The ratio of RLU1/RLU2 was further analyzed.

Relevant experimental steps were carried out according to the procedure described in this paper [37]_. For the immunofluorescence assay, on Day 1, the breast cancer cells were grown on glass coverslips in 6-well plates overnight. On Day 2, we washed the slides with PBS for approximately 5 min three times, and the climbing tablet was fixed with 4% paraformaldehyde for 15 min. Then, the slides were permeabilized for 20 min with 0.5% Triton X-100, and goat serum was used for blocking at room temperature for 30 min. Finally, the first antibody was incubated overnight. On Day 3, fluorescent secondary antibody was added after 3 rinses with PBST(PBS-Tween20) in a darker environment, and then DAPI was added to restain the cell nucleus for 5 min. Finally, after the sealing solution containing the fluorescence quencher was sealed on slides, pictures were captured under a fluorescence microscope. For multiple immunofluorescence staining, an iterative staining method was used. Three rounds of staining were as follows: round 1, primary Ab-secondary Ab HRP-conjugated -TSA-fluorophore (FITC, Cy3, Cy5.5) was performed according to the three-step staining protocol. Round 2 was followed by an Ab denaturation (stripping) step and, in turn, by the next round of staining with another primary antibody. The primary and HRP-conjugated secondary Abs of the previous round were removed in the denaturation step, the remaining TSA fluorophore was guaranteed for sequential staining steps, and nuclei were visualized with DAPI. The antibody information for the immunofluorescence assay is listed in Additional file 2 Table S2.

EdU, a thymidine analog, can incorporate replicated DNA molecules by replacing T-thymine during cell proliferation. A stable triazole ring is formed by a fluorescently labeled covalent reaction. The effects of inhibitors such as olaparib and LJI308 on DNA replication can be analyzed quantitatively. Then, 100 μL of 50 μM EdU solution was added to the cells in the 96-well plate for 2 h of incubation and washed with PBS solution 3 times for 5 min each time. Thereafter, 50 μL glycine was coincubated with cells in each well for 5 min and then washed with PBS 3 times. Next, 100 μL of 0.5% Triton X-100 was added to enhance cell membrane permeability. Finally, 100 μL Hoechst 33,342 solution was coincubated with cells for DNA staining. All stained cells and pictures were captured by fluorescence microscopy.

The distribution of cells at every G1/S/G2 stage was analyzed by flow cytometry. The sample DNA was labeled by the nucleic acid dye PI, which is a fluorescent nucleic acid dye. PI can bind selectively between the bases of nucleic acid DNA and RNA double helices. The binding amount is positively correlated with DNA content. MCF7AR cells were collected for the detection of the apoptosis ratio by staining with Annexin V-FITC and PI, and the operation was performed according to the instructions of the Apoptosis Detection Kit I (Beyotime, Shanghai, China). For cell cycle analysis, MCF7AR cells were fixed in 70% ethanol overnight and then stained with 20 μg/mL propidium iodide (PI) (Thermo Fisher Scientific) for 30 min. Fluorescence detection was performed by flow cytometry, and the cells were suspended before loading. The data were recorded after the signal was stabilized, and the maximum PI excitation wavelength was 535 nm. All stained cells were analyzed on a BD Flow Cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed by FlowJo software (Treestar Inc.).

The nude mice were purchased from Beijing Huafukang Company. MDA-MB-231 or MCF7AR cell mixtures with 100 μL serum-free DMEM (Gibco) were subcutaneously injected into 4 week-old BALB/c nude mice (six mice/group). The cell number for inoculation was calculated to be approximately 2 × 10⁶. The tumor volumes (mm³) were calculated according to the following formula: V = 1/2 ab², where a represents the largest tumor diameters and b represents the shortest. Abemaciclib and olaparib were dissolved in carboxymethylcellulose sodium (CMC-Na) solution, and LJI308 was dissolved in 5% DMSO + 30% PEG300 + 10% Tween 80 solution. Abemaciclib and olaparib were administered intragastrically with 100 mg/kg injection per nude mouse, and LJI308 was administered intraperitoneally with 10 mg/kg injection. These drugs were administered three times a week. All in vivo experiments were approved by the Institutional Animal Care and Use Committee of Shenzhen University (SZU-IACUC-2022–00136) and followed the Guide for the Care and Use of Laboratory Animals.

The results are expressed as the mean ± SEM and were analyzed using a two-tailed Student’s t test, two-way analyses of variance and ANOVA. ANOVAs were performed as previously reported [38‐40]. The synergistic effects of both drugs involved in Fig. 2D–G were calculated by CompuSyn software, and the smaller the composite index (CI) values were, the better the combination effect. Pearson correlation coefficient analysis was performed using GraphPad Prism V5.0. Differences were considered statistically significant when P < 0.05.

To further explore what molecules were involved in the development of CDK4/6i resistance, we analyzed genomic alterations in 348 ER + breast cancers treated with CDK4/6i from the open literature published in 2018 CancerCells [15] and found that the protein and RNA of PARP1 were upregulated in some patients (Fig. 1A). In addition, PARP1 was highly expressed in breast cancer samples included in the GEPIA database (Fig. 1B–C). To better explore the underlying CDK4/6i resistance mechanism, we generated CDK4/6i Abemaciclib resistant cell lines (AR, Abemaciclib resistant), and the construction process of drug-resistant strains is shown (Fig. 1D). Western blot results indicated that PARP1 was significantly upregulated in AR cell lines compared with parent MCF7 cells (Fig. 1E). MCF7AR cells were confirmed to be resistant to abemaciclib with an IC₅₀ of 50 μM compared with MCF7 cells with an IC₅₀ of 1 μM (Fig. 1F). The results of transcriptome sequencing showed that PARP1 was upregulated significantly, with a 40-fold change in MCF7AR cells compared with MCF7 cells, and there was slight upregulation of the YBX-1 gene, which is considered to be the key regulator of PARP1 (Fig. 1G). Thus, high PARP1 protein levels may be related to CDK4/6i resistance in breast cancer. To confirm the relationship between PARP1 and CDK4/6i resistance over a wider range, we obtained the IC₅₀ for the CDK4/6i palbociclib in 30 breast cancer cell lines from the GDSC database and paired them with corresponding PARP1 mRNA level data gained from CCLE. The 30 breast cancer cell lines were divided into two groups based on the level of PARP1 mRNA, and those with a high level of PARP1 demonstrated a tendency to have a higher palbociclib IC₅₀ (Fig. 1H). Furthermore, Abemaciclib treatment led to PARP1 elevation in 3 breast cancer cell lines (Fig. 1I).

PARP1 inhibition may help us determine whether PARP1 plays a crucial role in abemaciclib resistance. We used three siRNA sequences to silence the PARP1 gene in MCF7AR cells. Western blot analysis showed that PARP1 knockdown was successful, and the MTT assay indicated that PARP1 silencing restored abemaciclib sensitivity (Fig. 2A–B). Overexpression of PARP1 in MCF7 cells can enhance abemaciclib resistance (Fig. 2C). Olaparib, the FDA-approved drug targeting PARP1, can restore CDK4/6i sensitivity. The MTT assay indicated that olaparib can significantly decrease cell viability when combined with abemaciclib in 4 breast cancer cell lines, and the smaller the composite index (CI) values were, the better the synergistic effect. (Fig. 2D–G). The same result was obtained according to the crystal violet staining assay (Fig. 2H–I). The cell cycle assay showed that G1 phase increased remarkably when treated with combined therapy, which indicated that the cell cycle was blocked in the G1 phase (Fig. 2J). The flow cytometry results showed that the combined therapy significantly induced cell apoptosis (Fig. 2K–L). The combined therapy of abemaciclib and olaparib synergistically suppressed tumor growth in the xenograft model, and samples derived from animal tumors were used to confirm the key role of PARP1 in CDK4/6i resistance by immunohistochemical assay (Fig. 6A–C, G). The fluorescence level of p-H2AX, a DNA-damage marker molecule, was upregulated simultaneously with DNA repair protein PARP1 levels in MCF7 cells when treated with abemaciclib. The results showed that DNA damage and repair occurred synchronously when treated with abemaciclib, and the upregulation and nuclear localization of YB-1 may be correlated with abemaciclib resistance (Fig. 2M–N). The results showed that olaparib treatment induced DNA replication reduction, which may be attributed to cell cycle arrest. The combined treatment of abemaciclib and olaparib led to DNA replication stagnation (Fig. 2O–P). The DNA repair pathway was enriched when MCF7 cells were treated with abemaciclib (Fig. 2Q).

We investigated the reason that the PARP1 protein level was elevated in the MCF7AR cell lines or abemaciclib treatment. Interestingly, we discovered that p-YB-1 was elevated in MCF7-AR cells compared with parental MCF7 cells, and p-YB-1 and PARP1 were positively correlated based on western blot results (Fig. 3A). The expression of p-YB-1/PARP1 and the abemaciclib IC₅₀ were positively correlated in seven breast cancer cell lines based on the quantitative analysis results of the protein level of p-YB-1/PARP1 and IC₅₀ detection (Fig. 3B–C). Comparative analysis suggested that the YB-1 mRNA level was positively correlated with the CDK4/6i IC₅₀ (Fig. 3D). Previous reports have suggested that LJI308 is a YB-1 inhibitor based on the conclusion that RSK2 can directly regulate YB-1 phosphorylation [41]. PARP1 was markedly reduced when p-YB-1 was inhibited by LJI308 (Fig. 3E). In addition, the PARP1 protein level decreased significantly when YB-1 was knocked down (Fig. 3F–H). YB-1 silencing can enhance CDK4/6i sensitivity, and the inhibitory effect was confirmed by siYB-1 (Fig. 3H–I). The MTT assay confirmed that YB-1 overexpression increased CDK4/6i resistance in MCF7AR cells. Western blot data showed that YB-1 overexpression was successful (Fig. 3J–K) and that PARP1 may be regulated by YB-1 phosphorylation. In contrast, p-YB-1 and PARP1 increased consistently when breast cancer cell lines were treated with abemaciclib (Fig. 3L). The fluorescence levels of p-H2AX and p-YB-1 were upregulated simultaneously in MCF7 cells treated with abemaciclib, and the fluorescence intensity of p-H2AX and p-YB-1 reached its maximum when abemaciclib treatment was 10 μM. p-YB-1 may be correlated with DNA damage repair, which may cause CDK4/6i resistance (Fig. 3M–N). Considering that YB-1 is a transcription factor, we designed three sequence primers for the promoter region of PARP1, and a ChIP assay was employed to evaluate whether YB-1 can bind to the promoter region of PARP1. The data demonstrated that YB-1 binds to the P2 primer in the promoter region of PARP1, which may be transcriptionally activated by YB-1 (Fig. 3O).

We focused on whether YB-1 inhibition can enhance sensitivity to CDK4/6i. Western blot data showed that PARP1 and p-YB-1 were decreased simultaneously when treated with abemaciclib and LJI308 (Fig. 4A). As shown by the MTT assay, combined therapy with abemaciclib and LJI308 led to a cytotoxic killing effect in the four cell lines (Fig. 4B). The inhibitory effect of abemaciclib in MCF7 cells was decreased by overexpression of YB-1 compared with the control group (Fig. 4C–D). The sensitivity of Abemaciclib was augmented significantly when MCF7AR cells were treated with LJI308, based on crystal violet staining (Fig. 4E–F). These results suggested that PARP1 and p-YB-1 change simultaneously. To explore the changes in PARP1 and p-YB-1 levels during the construction of resistant strains, we used a stepwise dose-increasing (from 0.1 to 20 μM) drug treatment for 6 months. The initial drug concentration of abemaciclib was 0.1 μM. We selected abemaciclib-treated MCF7 cells from six time points during the AR screening process, and elevated PARP1 and p-YB-1 were observed almost simultaneously as MCF7 cells gradually gained resistance to abemaciclib (Fig. 4G). The cell cycle assay showed that G1 phase increased remarkably when treated with the combined therapy of abemaciclib and olaparib (Fig. 4H). Flow cytometry showed that combination therapy with abemaciclib and LJI308 was effective (Fig. 4I–J). We discovered that the PARP1 and p-YB-1 proteins were colocalized in the nucleus under abemaciclib treatment by immunofluorescence assay, and the protein level of PARP1 gradually increased in a dose-dependent manner, which occurred at the same time as the fluorescence intensity of p-YB-1 increased (Fig. 4K–L). The results of the EdU assay showed that LJI308 treatment induced DNA replication reduction, which may be attributed to cell cycle arrest. The lower fluorescence intensity represented attenuated DNA replication, and DNA replication stagnation was induced by the combined treatment of abemaciclib and LJI308 (Fig. 4M–N).

We found that YB-1 was phosphorylated at S102, was critical for PARP1 transcriptional activation and was associated with CDK4/6i resistance. The mutant plasmids YB-1-S102A and YB-1-S102E were constructed to study the importance of YB-1 phosphorylation. The plasmids YB-1-S102A and YB-1-S102E were used to simulate dephosphorylation and hyperphosphorylation, respectively. The two plasmids were transfected separately into MCF7 cells in which YB-1 was expressed at low levels. There was no increase in the protein level of PARP1 in the YB-1-S102A group compared to the wild-type YB-1 group; in contrast, the PARP1 level was significantly increased in the YB-1-S102E group (Fig. 5A). The MTT assay indicated that YB-1-S102A transfection did not lead to CDK4/6i resistance. Furthermore, YB-1-S102E transfection greatly enhanced CDK4/6i resistance (Fig. 5B). Considering the low level of YB-1 expression in MCF7 cells, we transfected two mutant YB-1 plasmids into MCF7-shYB-1 cells. We discovered that YB-1 activity correlated positively with PARP1, and the PARP1 level in the shYB-1 group was lower than that in the control group (Fig. 5C). Moreover, the inhibition efficiency of cell viability for the YB-1-S102A-MCF7-shYB-1 group reached approximately 13% when transfected with the mutant YB-1 plasmid (Fig. 5D–F).

We found that PARP1 and p-YB-1 were colocalized in the nucleus (Fig. 4G). The protein level of p-YB-1 gradually increased under abemaciclib treatment, which was determined by immunofluorescence assay. The phenomenon of YB-1 nuclear translocalization was barely observed in the YB-1-S102A transfection group when treated with abemaciclib. In contrast, YB-1 nuclear translocation was clearly observed in the YB-1-WT and YB-1-S102E groups, which conferred CDK4/6i resistance. These results reminded us that YB-1 nuclear translocation may be closely correlated with CDK4/6i resistance and may be the driving and critical factor for the development of drug resistance (Fig. 5G–H). To further determine the effect of mutant plasmid transfection on PARP1 transcription, we constructed an upstream 2000 bp fragment in the promoter of PARP1 into a luciferin vector plasmid for a double fluorescein reporter gene assay. The change in fluorescence intensity can be used as the basis of judgment of the effect on PARP1 transcription under transfection with the YB-1-WT/S102A/S102E plasmids. The fluorescence intensity in the YB-1-WT and YB-1-S102E groups was higher than that in the YB-1-S102A group, which barely worked. PARP1 transcription may be influenced by YB-1 phosphorylation at S102. (Fig. 5I). Taking the previously mentioned results together, we can conclude that YB-1 phosphorylation can promote its nuclear translocation and in turn lead to transcriptional activation of PARP1, eventually leading to the occurrence of CDK4/6i resistance.

The ‘‘YB-1-PARP1’’ loop was proven to play a crucial role in the CDK4/6i resistance of breast cancer, and blocking the factor in the ‘‘YB-1-PARP1’’ loop can enhance the sensitivity of CDK4/6i efficiency. MDA-MB-231 and MCF7AR cells were used for the nude mouse model. The mice were randomly divided into vehicle, abemaciclib, olaparib and combined groups when the tumor size reached 150 mm³. At least 6 nude mice were included in each group, and the nude mice were intragastrically injected with 100 mg/kg abemaciclib or olaparib and intraperitoneally injected with 10 mg/kg LJI308 three times a week. The combined therapy of abemaciclib and olaparib synergistically suppressed tumor growth in the xenograft model (Fig. 6A–C). In addition, the YB-1 inhibitor LJI308 combined with abemaciclib exerted a synergistic antitumor effect, causing the xenograft tumor to shrink rapidly. Abemaciclib or LJI308 was intraperitoneally injected i.p. 3 times a week with a 100 mg/kg dose (Fig. 6D–F). The sample derived from the animal tumor tissue in the previously mentioned Experiment A was used to detect c-caspase3, Ki67, and PARP1 expression in the 4 groups (Fig. 6G). The IHC results of c-c3, Ki67, PARP1 and p-YB-1 in Experiment B are displayed for the effects on PARP1 expression induced by YB-1 inhibition (Fig. 6H). Simultaneously, we discovered that YB-1 knockdown slowed the rate of tumor growth under abemaciclib treatment (Fig. 6I–K).

Although abemaciclib has shown efficacy in HR + /HER2- breast cancer patients, many patients demonstrate intrinsic or acquired resistance to CDK4/6i and disease progression [42]. We collected paraffin sections from breast cancer patients from the First Affiliated Hospital of Shengzhen University. Previous research has revealed the relationship of p-YB-1 and PARP1 in cell line experiments, but the expression and colocalization of p-YB-1/PARP1 in breast cancer tissues are unknown. The data from multiple immunofluorescence (MIF) assays indicated that p-YB-1 and PARP1 were coexpressed and colocalized in four representative breast cancer patients. The codistribution of p-YB-1 and PARP1 has obvious characteristics of regional consistency (Fig. 7A). Generally, the patients expressing HR + /HER2- tended to be sensitive to CDK4/6i, so that most of this group can benefit potentially from CDK4/6i therapy. The IHC results showed lower ‘‘p-YB-1/PARP1’’ expression in patients with HR + /HER2- status than in patients with other HR-/HER2-, HR-/HER2 + and HR + /HER2 + statuses. The HR-/HER2- group, as the triple-negative breast cancer type, was considered resistant to CDK4/6i with higher ‘‘p-YB-1/PARP1’’ expression. To increase the accuracy of the results, the staining results came from the same position of successive slices, and typical images are shown (Fig. 7B–C). By further analyzing the correlation of p-YB-1 and PARP1 in HR + /HER2- and non-HR + /HER2- group patients, the data suggested that p-YB-1 was positively correlated with PARP1 in both the HR + /HER2- and non-HR + /HER2- groups and discovered that there were great differences in p-YB-1/PARP1 expression between the HR + /HER2- and non-HR + /HER2- groups (Fig. 7D–E). ‘‘p-YB-1/PARP1’’ expression remained consistently high in HR-/HER2-and low in HR + /HER2- patients. According to the expression of p-YB-1 and PARP1, we divided samples into four groups: p-YB-1^L + PARP1^L, p-YB-1^L + PARP1^H, p-YB-1^H + PARP1^L, and p-YB-1^H + PARP1^H. Surprisingly, the percentage of the high PARP1 group accounted for 100% of the high p-YB-1 group, which indicated that PARP1 was almost highly expressed when p-YB-1 was highly expressed (Fig. 7F–G). Furthermore, the scatter plots of the data clearly indicated that PARP1 positively correlated with p-YB-1 in the BC (n = 90, r = 0.6234, p < 0.0001***), HR + /HER2- (n = 52, r = 0.3556, p = 0.0097**) and HR-/HER2- (n = 13, r = 0.7673, p = 0.0022**) groups of patients (Fig. 7H–J). CDK4/6i-sensitive clinical patients mainly included the p-YB-1^L + PARP1^L and p-YB-1^L + PARP1^H groups, which accounted for approximately 91%. In contrast, the patients with HR-/HER2- status were all ascribed to the p-YB-1^H + PARP1^L and p-YB-1^H + PARP1^H groups, which accounted for approximately 77% (Fig. 7K–L). Based on these results, we concluded that low ‘‘p-YB-1/PARP1’’ was linked to CDK4/6i sensitivity. The PARP1 level positively correlated with p-YB-1. Thus, taking the level of ‘‘p-YB-1/PARP1’’ into consideration is feasible when assessing the benefit of CDK4/6i therapy for patients.

To better understand the molecular mechanism of this paper, we have drawn a schematic model summarizing how PARP1 orchestrates CDK4/6i resistance by promoting DNA damage repair and driving the cell cycle G1/S phase transition. Elevated p-YB-1 enhances the binding of YB-1 to the promoter region of PARP1 when CDK4/6i treatment occurs, and PARP1 is transcriptionally activated by YB-1 phosphorylation. High levels of PARP1 are closely associated with CDK4/6i resistance, and phosphorylated YB-1 can control cell cycle progression by strengthening PARP1-mediated DNA damage repair. YB-1 phosphorylation may remove the blocking effect of CDK4/6i on the G1/S phase transition and lead to the occurrence of CDK4/6i resistance in breast cancer. The role of bypass activation of the cell cycle in PARP1 upregulation and YB-1 phosphorylation was crucial for CDK4/6i resistance. Fig. 8.