CENPF Knockdown Inhibits Adriamycin Chemoresistance In Triple Negative Breast Cancer Mediated By Rb-E2F1 Axis

doi:10.21203/rs.3.rs-1853542/v2

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CENPF Knockdown Inhibits Adriamycin Chemoresistance In Triple Negative Breast Cancer Mediated By Rb-E2F1 Axis

https://doi.org/10.21203/rs.3.rs-1853542/v2

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published 31 Jan, 2023

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Background:

Frequent developed resistance leads to early relapse and short survival in Triple-negative breast cancer (TNBC). Targeting DNA damage response (DDR) has become an effective strategy for overcoming TNBC chemoresistance. CENPF (centromere protein) is a key regulator of cell cycle progression, but its role in TNBC chemotherapy resistance remains unclear.

Methods:

We Used bioinformatics, immunohistochemical staining and real-time quantitative PCR analysis to explore the expression of CENPF in triple negative breast cancer tissues and cells. Then, we analyze the relationship between CENPF expression with clinicopathological parameters and survival prognosis of patients. The effects of CENPF on the proliferation, apoptosis, cycle, and drug sensitivity of triple negative breast cancer cells were detected in vitro. The chemoresistance mechanisms of CENPF in TNBC cells was evaluated by immunoblotting, immunofluorescence co-localization, and co-immunoprecipitation .

Results:

Here we found CENPF, highly expressed in TNBC, is associated with poor prognosis in patients receiving chemotherapy. And in vitro CENPF knockdown significantly increased adriamycin (ADR)-induced cytotoxicity in MDA-MB-231 and its ADR-resistant strains (MDA-MB-231/ADR). Then we demonstrated that CENPF targets Chk1-mediated G2/M phase arrest and binds to Rb for competing with E2F1 in TNBC. Collectively Considering crucial role of E2F1 in DNA damage response and DNA repair, a novel mechanism for CENPF to regulate Rb-E2F1 axis will provide new horizons to finally overcome chemotherapy resistance in TNBC.

Conclusion:

CENPF binds to Rb for competing with E2F1 and promotes Chk1 expression through the Rb/E2F1 axis to enhance chemoresistance to doxorubicin in TNBC.

Biological sciences/Cancer/Breast cancer

Biological sciences/Cancer/Cancer therapy

Triple Negative Breast Cancer

Chemoresistance

CENPF

Chk1

Breast cancer is the second most common cancer worldwide after lung cancer, and the leading cause of cancer death in women[1]. TNBC represents 10–15% of breast cancers which is the most lethal subtype due to high heterogeneity, aggressive nature, and lack of treatment options[2]. Chemotherapy remains the mainstay of treatment for TNBC. Unfortunately, frequent developed resistance is a major cause of early relapse and short survival in TNBC[3].

Dysregulation of cell cycle control mechanisms grants TNBC the continuous malignant proliferation. DNA replication, a central event for cancer cells proliferation, can be destroyed by anthracyclines via inhibiting topoisomerase II[4]. Exogenous DNA-damaging agents trigger cancer cells to activate their DDR including cell cycle arrest and DNA repair[5]. Obviously, these protective DDRs confer tumor chemoresistance. Targeting DDRs has become an effective strategy for overcoming TNBC chemoresistance[6, 7].

In our previous research on the mechanism of Chk1 in TNBC chemotherapy resistance, we found that CENPF acts as the upstream regulatory molecule of Chk1[8]. CENPF is an evolutionarily highly conserved protein related to kinetochore structure and function. The centromere-kinetochore complex mediates the binding of spindle filaments to chromosomes and is the structural basis for the orderly pairing and segregation of chromosomes[9, 10]. CENPF has been found to be overexpressed in various malignant tumors such as prostate cancer, breast cancer, and nasopharyngeal carcinoma, and activate the signaling pathways related to malignant tumor progression to promote the proliferation and metastasis of cancer cells[11–13]. Recent studies have shown that abnormal expression of CENPF is also closely related to chemotherapy resistance in ovarian cancer and lung adenocarcinoma [14, 15]. At present, the mechanism of CENPF involved in TNBC chemotherapy resistance has not been elucidated.

Based on the previous study, we further explored the mechanism of CENPF involved in TNBC chemotherapy resistance through a series of bioinformatics analysis and molecular biology experiments. Our current study revealed the role of CENPF in TNBC chemotherapy resistance, and CENPF targets Chk1-mediated G2/M phase arrest and binds to Rb for competing with E2F1. Considering crucial role of E2F1 in DNA damage response and DNA repair, we hope this study will provide a new intervention target to overcome chemotherapy resistance in TNBC.

Bioinformatics analysis

We used the following bioinformatics analysis strategies and tools at each experimental stage. We measured CENPF expression in TNBC and adjacent peritumoral tissues from the The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression Program (GTEx) databases using the Gene Expression Profiling Interactive Analysis (GEPIA). And GSE86374 and GSE58135 from the Gene Expression Omnibus (GEO) databases also used for that. We also performed survival analysis of CENPF in breast cancer, especially in the patient with neoadjuvant chemotherapy, using the Kaplan Meier Plotter with the best-performing threshold as a cutoff. We downloaded data of TNBC from GSE25066 and used Sangerbox to analyze the relationship between the expression level of CENPF and patient survival prognosis with the best-performing threshold as a cutoff.

Patient Samples And Ihc

TNBC tissue blocks were collected from 32 patients operated at Qilu Hospital and The Second Hospital of Shandong University between 2013 and 2020. All the 32-patient received at least four cycles of neoadjuvant chemotherapy before surgical resection. Tumor grade and MP grade as previously described [16]. According to MP grading system, grade 3 to grade 5 tumors were classified into chemotherapy-sensitive group; and grade 1 and grade 2 tumors were regarded as chemotherapy-resistant. IHC and evaluation of immunohistochemical (IHC) staining were performed as previously described [16].

Cell Lines And Cell Culture

We purchased the human breast cancer cell line MDA-MB-231 from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cell authentication was verified by short tandem repeat profiling. The cell line of MDA-MB-231 with adriamycin-resistant (MDA-MB-231/ADR) was induced by long-term adriamycin treatment at the laboratory in the early stage, and was tested for adriamycin resistance[17]. We cultured these cell lines in Leibovitz’s L15 medium (MDA-MB-231 and MDA-MB-231/ADR) supplemented with 10% fetal bovine serum at 37°C with 5% CO2. All cell lines were tested as free from mycoplasma contamination by the mycoplasma PCR testing and no more than 20 generations of growth for any experiment.

Antibodies And Reagents

Anti-Phospho-Chk1-Ser317 antibody (1:1000,12302), anti-Phospho-Chk1-Ser345 antibody (1:10002348), anti-Rb antibody (1:500,9309) were purchased from Cell Signaling Technology (CST, Danvers, Massachusetts, US). Anti-CENPF antibody (1:1000, DF2310), anti-Chk1 antibody (1:1000, AF6007), anti-E2F1 antibody (1:500, DF6797) and anti-GAPDH antibody (1:3000, AF7021) were purchased from Affinity Biotech (Cincinnati, Ohio, US). Anti-CENPF antibody (1:1000, ab5) was purchased from Abcam, (Cambridge, UK).

Cell Transfection And Sirnas

Both si-CENPF and si-Control were synthesized by GenePharma (Shanghai, China) according to the sequence verified by SigmaAldrich. We used Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) and transfected MDA-MB-231, MDA-MB-231/ADR with siRNA (50 nM) according to the manufacturer's protocol. We confirmed the knockdown efficiency of CENPF by RT-qPCR and western blotting 48 hours after cell transfection. All siRNA sequences are shown in Supplementary Table S1

Quantitative Real-time Pcr (Qrt-pcr)

Total RNA and RT-qPCR were performed as previously described[18]. All primer sequences are presented in Supplementary Table S2

Western Blotting

Cells were lysed on ice for 20 min in RIPA lysis buffer (Beyotime, Beijing, China) supplemented with 1% phenylmethanesulfonyl fluoride (Beyotime, Beijing, China) and 1% ProtLytic Phosphatase Inhibitor Cocktail (New Cell and Molecular Biotech, Suzhou, China). The solution was then centrifuged at 12 000 G for 10 min and the supernatants were collected. Next, total protein concentration was measured using the BCA kit (Invitrogen, Carlsbad, CA, USA). After denaturation at 100°C for 8 minutes, 20 µg protein samples were separated on 7.5% SDS/PAGE gels and then transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA).

Drug Sensitivity Assay

5,000 cells were seeded in 96-well plates. After 24 h, changed medium containing adriamycin (MB1087, Dalian Meilun Bio, Dalian, China), and the culture was continued for 72 h at drug concentrations of 0, 0.1, 0.5, 1, 2, 5, 10 and 20 µM. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; K1018A, PExBIO, Houston, TX, USA) following the manufacturer's instructions. After 2 h incubation at 37°C, absorbance was read at 450 nm.

Cell Proliferation Assay

3,000 cells were seeded in 96-well plates. Cell viability was assessed using the Cell Counting Kit-8 kit (CCK-8; K1018A, PExBIO, Houston, TX, USA) following the manufacturer's instructions at 24, 48, 72, and 96 h. After 2 h incubation at 37°C, absorbance was read at 450 nm. Furthermore, EdU cell proliferation assays were further confirmed as previously described[19].

Cell Apoptosis Assay

We collected MDA-MB-231 and MDA-MB-231/ ADR cells transfected with si-CENPF-1, si-CENPF-2 or si-control after 48 h’ exposure to ADR at a concentration of 1 or 5 µM, which was consistent with the process for the ADR-free group. Annexin staining V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining were performed using the Annexin V-FITC/PI Apoptosis Assay Kit (BestBio, Shanghai, China) according to the manufacturer's instructions. Apoptosis was then detected immediately by flow cytometry (Cytoflex S, Beckman Coulter, California, US).

Cell Cycle Analysis

We collected MDA-MB-231 cells transfected with si-CENPF-1, si-CENPF-2 or si-control after 48 h’ exposure to ADR at a concentration of 1 µM, which was consistent with the process for the ADR-free group. Staining was performed using a Cell Cycle Detection Kit (BestBio, Shanghai, China) according to the manufacturer's instructions, and flow cytometry (Cytoflex S, Beckman Coulter, CA, USA) was performed to determine the effects of CENPF inhibition on cell cycle distribution with or without 48 hours action of 1µM adriamycin in MDA-MB-231 and using Histograms to statistical analysis the cell cycle distribution.

Immunofluorescence

Cells were cultured on glass slides and washed twice with PBS before fixation. Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. To reduce nonspecific binding of antibodies, cells were incubated in PBS containing 1% BSA for 1 hour at room temperature. Primary antibodies (anti-CENPF and anti-Rb) were diluted in PBS containing 1% BSA, dropped on cells and incubated overnight at 4°C. Next, the samples were incubated with secondary antibodies Dylight488 and Dylight594 (Beyotime, Beijing, China) which diluted in PBS containing 1% BSA for 1 hour at room temperature. Then dropwise added Antifade Mounting Medium with DAPI (Beyotime, Beijing, China), and the images were observed and collected under a fluorescence microscope.

Co-immunoprecipitation

Cell lysis and protein concentration determination were as described above. For immunoprecipitation, equal amounts of lysate were incubated with protein A/G magnetic beads (Beyotime, Beijing, China) and anti-CENPF antibody overnight at 4°C. Thereafter, the beads were washed 3 times with cell lysis buffer and immunoprecipitated proteins were analyzed by western blotting.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software version 5 ( GraphPad Software, Inc., San Diego, California, US) and SPSS 16.0 (SPSS Inc, IL, USA), and t-test was used to detect the difference between two independent samples. One-way ANOVA was used to analyze the statistical differences between three or more independent samples. Analysis of the relationship between CENPF expression and neoadjuvant chemotherapy grade and clinicopathological parameters using chi-square test and Fisher's exact test. We used The Kaplan-Meier method and log-rank t test to determine the significance of the survival curve. Analysis of correlations between co-expressed genes in the TCGA dataset in breast cancer using Pearson's correlation coefficient; immunofluorescence co-localization of CENPF and Rb proteins analyzed using Pearson's correlation coefficient, R values > 0.3 or < -0.3 were considered statistically significant. Each experiment was repeated at least 3times, P < 0.05 was considered statistically significant.

1. CENPF is highly expressed in triple-negative breast cancer

The published data from TCGA showed that CENPF expression in breast cancer tissues was significantly higher than that in adjacent peritumoral tissues. (Fig. 1A, n = 1 376, P < 0.05). And survival prognostic analysis using Kaplan Meier Plotter tool showed that low levels of CENPF expression predicted better overall survival (Fig. 1B, n = 1 879, logrank P = 1.3e − 07) and recurrence-free survival (Fig. 1C, n = 4929, logrank P < 1e − 15) in breast cancer. Moreover, based on published data from TCGA and GEO (GSE58135 and GSE86374), we compared CENPF expression between Non-TNBC and TNBC. It suggested that high CENPF expression was more pronounced in TNBC (Fig. 1D-F).

2. Cenpf Increases Adr Chemosensitivity Of Tnbc

Kaplan Meier Plotter data showed that high CENPF expression portended worse recurrence-free survival in breast cancer patients receiving neoadjuvant chemotherapy (NACT) (Fig. 2A, n = 403, logrank P = 0.035). Chemotherapy is the main strategy for the treatment of TNBC, but its frequent resistance to chemotherapy is a significant clinical challenge. Survival prognostic analysis suggested TNBC with high CENPF expression had a worse prognosis after treatment with neoadjuvant chemotherapy (Fig. 2B, n = 200, logrank P = 0.031). This point was also buttressed by the published data from GSE25066 (Fig. 2C, n = 112, P = 0.02). To further confirm the relationship between CENPF and chemotherapy resistance, we collected puncture specimens from 32 cases of TNBC before neoadjuvant chemotherapy in recent years. And they were further stratified by assessment of chemotherapy response according to Miller and Payne (MP) grading system. According to the reported studies[20], patients rated as Miller-Payne regression grade 1 and 2 were assigned to the chemo-resistant group, and the Miller-Payne regression grade 3–5 were considered chemotherapy-sensitive group. The results of immunohistochemical staining indicated that chemo-resistant group displayed a significantly higher CENPF expression compared to chemotherapy-sensitive group (Fig. 2D). As shown in Table 1, the results of the analysis grouped according to CENPF expression also showed that high CENPF expression was associated with chemotherapy resistance.

Consistent with assay results from tissues, CENPF expression is significantly increased in TNBC cell lines (MDA-MB-231 and MDA-MB-468) compared to non-TNBC cell lines (MCF-7 and T47D) (Fig. 2E). MDA-MB-231 and its adriamycin-resistant strains (MDA-MB-231/ADR) were used for the following research on mechanism of CENPF involved in chemotherapy resistance in TNBC. Establishment of MDA-MB-231/ADR has been accomplished in our previous study[8]. The adriamycin resistance assays of two cells were shown in the Fig. 2F. Two specific interference sequences were designed to inhibit CENPF. And interference efficiency of si-CENPF-1 and si-CENPF-2 were analyzed via western blot after 48 h of transfection (Supplementary Fig. 1A, B). CENPF knockdown potentiated ADR-induced cytotoxicity in MDA-MB-231 (Fig. 2G). And using flow cytometry, we found that silencing CENPF increased ADR-induced apoptosis rates of MDA-MB-231(Fig. 2H). As shown in Fig. 2I and J, these findings were in agreement with those in MDA-MB-231/ADR.

3. Cenpf Regulates Adr-induced G2/m Phase Arrest Through Chk1

CENPF is strictly expressed in the cell cycle-dependent manner and it starts to accumulate in S phase and peaks in G2/M phase. To reveal the mechanism of CENPF promoting chemotherapy resistance, we used flow cytometry to observe the effects of CENPF inhibition on cell cycle distribution. Adriamycin induced significant G2/M arrest which was reversed by CENPF inhibition. As shown in Fig. 3A, CENPF knockdown significantly decreased the number of cells in G2/M phase and increased those in G0/G1 and S phase under Adriamycin. Interestingly, our findings suggested that CENPF lost control of cell cycle in TNBC without exogenous stress (Fig. 3A). This was also confirmed by cell proliferation assay using CCK-8 (Fig. 3B, C) and EdU (Fig. 3D). The above suggested that CENPF was required to cope with adriamycin-induced DNA damage stress in TNBC.

Our previous study showed that, under DNA damage stress, Chk1 is activated in a phosphorylated form to facilitate DNA repair by inducing G2/M phase arrest in TNBC[8]. In DNA damage response (DDR), Chk1-induced cell cycle arrest buys sufficient time for DNA repair to prevent DNA damage from causing apoptosis[21]. Results of RT-qPCR (Fig. 3E, F) and western blot (Fig. 3G) showed that CENPF knockdown inhibited Chk1 expression and thus impaired Chk1 activation in MDA-MB-231 and MDA-MB-231/ADR exposed to adriamycin. And finally, CENPF regulates ADR-induced G2/M phase arrest through Chk1 to participate in ADR chemosensitivity of TNBC

4. Cenpf Target The Transcriptional Activity And Expression Of E2f1

According to reports, CENPF orthologs LEK1 and CMF1 function in muscle differentiation through interaction with Rb. And they bind Rb in vitro through a region homologous to that of E2F1[22]. Bargiela-Iparraguirre J et al found Chk1 expression is controlled by Rb/E2F1 at the transcriptional level[23]. Considering crucial role of E2F1 in DNA damage response and DNA repair[24, 25], we assumed that CENPF is likely to target Rb-E2F1 axis to regulate TNBC chemoresistance. Confocal images showed colocalization of CENPF with Rb in MDA-MB-231 and MDA-MB-231/ADR (Fig. 4A). To further validate the interaction of CENPF and Rb, we performed the Co-Immunoprecipitation assay which showed that immunoprecipitated endogenous CENPF binds to endogenous Rb (Fig. 4B). And immunoprecipitation of endogenous Rb led to the co-precipitation of CENPF (Fig. 4C). Moreover, as shown in Fig. 4D, CENPF knockdown increased binding of Rb to E2F1, thereby inhibiting E2F1 release. In addition, CENPF knockdown significantly downregulated E2F1 expression MDA-MB-231 (Fig. 4E) and MDA-MB-231/ADR (Fig. 4F).

Cell cycle arrest and DNA repair are essential components of DDR which is required to maintain genomic integrity of cancer cells, especially exposed in exogenous DNA-damaging agents[26, 27]. These protective DDRs become direct drivers of tumor chemoresistance. TNBC often develops primary or acquired chemoresistance, leading to chemotherapy failure, recurrence and even greatly shortening patient survival. In this study, we found Rb-E2F1 axis mediates regulation of CENPF on Chk1 expression and CENPF promotes adriamycin chemoresistance by regulating Chk1-mediated G2/M phase arrest in TNBC. CENPF is a new potential intervention target to overcome chemoresistance in TNBC.

At present, the mechanism of CENPF in chemotherapy resistance of TNBC remains unclear. Our study shows that CENPF, as the upstream of Chk1, targets and regulates ADR-induced G2/M phase arrest. Chk1 is one of the key proteins of G2/M phase checkpoint. It is involved in the DNA damage response caused by endogenous and exogenous factors, and is also critical for genome stability and cell survival[28]. In DNA damage stress, transient cell cycle arrest is a favorable event for cell survival because it provides sufficient time and space for DNA repair[29]. Once DNA repair is disrupted, cells will turn from cell cycle arrest to apoptosis[30]. Our previous studies have shown that Chk1 inhibition enhances adriamycin chemosensitivity of TNBC by eliminating cell cycle arrest and inducing cell apoptosis[8]. TNBC often has abnormal cell checkpoints in G1/S phase, so it is more dependent on G2/M phase checkpoints for DNA repair and maintenance of chromosomal integrity. Therefore, our results suggest CENPF knockdown ultimately increases adriamycin-induced cell apoptosis as G2/M phase arrest is counteracted.

Then we found CENPF binds to Rb and competes with E2F1 binding site. Rb binding to E2F1 and inhibiting the transcriptional activity of E2F1 to regulate cell cycle has been extensively studied[31]. As our results showed, CENPF knockdown increases binding of Rb to E2F1, thereby inhibiting E2F1 release. Transcription factor E2F1, a member of the E2F family, plays an important role in regulation of cell cycle progression, DNA repair, apoptosis and drug resistance[25, 32]. Verlinden L et al confirmed that E2F1 transcription factor aggregates Chk1 promoter and upregulate Chk1 expression in TNBC[33]. J. Bargiela-Iparraguirre et al demonstrated that Chk1 overexpression specifically increases resistance to chemo/radiotherapy in gastric cancer and Chk1 expression is controlled by Rb/E2F1 at the transcriptional level[34]. In summary, Rb-E2F1 axis mediates regulation of CENPF on Chk1 expression which contributes to chemotherapy resistance in TNBC.

In addition, the oncogenic role of E2F1 in mediating cancer chemoresistance has been confirmed by multiple previous studies[35, 36]. The transcription factor E2F1 has major roles in cell cycle control, checkpoint response and DNA replication and repair which leads to DNA damage tolerance of tumor cells[24, 25]. Our findings about the regulation of CENPF on transcriptional activity and expression of E2F1 revealed a new mechanism by which CENPF is involved in TNBC chemoresistance. Notably, although CENPF knockdown increased binding of Rb to E2F1, it had no effect on cell cycle progression and cell proliferation under normal growth conditions. Here we focused on Igor Shats et al.’ studies which demonstrated E2F1-mediated cell fate depends on its expression level, and Low levels of E2F1 facilitate proliferation, high levels facilitate apoptosis[37]. Moreover, the expression peak of CENPF is in the G2/M phase where [38]. Above factors are likely to contribute to the result that CENPF knockdown has no effect on cell proliferation under normal growth conditions.

Due to time and technical limitations, there are several shortcomings in the present study. In order to ensure that paraffin sections meet the requirements of IHC detection, most of the specimens we collected are from recent cases, so the follow-up results cannot be presented. Considering this issue, we utilized the TCGA and GEO database for prognostic analysis in Figs. 1 and 2. Then the molecular weight of CENPF, as high as more than 360 KD, is too large to perform in vitro CENPF overexpression assays.

Previous studies on CENPF have mainly focused on kinetochore function, tumor proliferation and metastasis [39–41]. Here, we demonstrate a novel mechanism of CENPF to regulate TNBC chemoresistance through Rb-E2F1-Chk1 axis. Our findings initially suggested CENPF as a potential target for intervention to overcome chemotherapy resistance in TNBC. And a more definitive conclusion will be discussed in the follow-up animal experiments. We believe that further research on the mechanism of CENPF will provide new ideas for revealing TNBC chemoresistance.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers: 81572594 and 81671605].

Competing Interests

The authors declare that they have no competing interests in this section.

Authors' contributions

KM and QRY conceptualized and supervised the research. DPW and WX performed experiments and was responsible for extracting data, interpreting results and updating reference lists. DPW conducted experimental data and bioinformatics analyses. MHH, WM and YLL contributed to technical guidance, extract data and interpret results. XCZ was responsible for collecting paraffin specimens of breast cancer. WX prepared the manuscript. K.M. ang QRY contributed to critical reading of the manuscript.

Data Availability

Publicly available datasets were analyzed in this study. TCGA data can be found here: [https://www.cbioportal.org. /Breast Invasive Carcinoma (TCGA, PanCancer Atlas)]. Data sets from each of the GEO databases can be downloaded from the following sites and access numbers: [https://www.ncbi.nlm.nih.gov/geo. / GSE58135/GSE86374/GSE25066].

Ethics approval

- Approval of the research protocol by an Institutional Reviewer Board: Ethics Committee of School of Basic Medical Sciences, Shandong University. All experiments were performed in accordance with relevant guidelines and regulations.

- Registry and the Registration No. of the study/trial: N/A

- Animal Studies: N/A

Consent to participate

- Informed consent was obtained from all individual participants included in the study.

Acknowledgments

We thank The Second Hospital of Shandong University provides paraffin specimens from selected TNBC patients.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

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CENPF Knockdown Inhibits Adriamycin Chemoresistance In Triple Negative Breast Cancer Mediated By Rb-E2F1 Axis

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Abstract

Background:

Methods:

Results:

Conclusion:

Figures

Background

Material And Methods

Bioinformatics analysis

Patient Samples And Ihc

Cell Lines And Cell Culture

Antibodies And Reagents

Cell Transfection And Sirnas

Quantitative Real-time Pcr (Qrt-pcr)

Western Blotting

Drug Sensitivity Assay

Cell Proliferation Assay

Cell Apoptosis Assay

Cell Cycle Analysis

Immunofluorescence

Co-immunoprecipitation

Statistical analysis

Results

1. CENPF is highly expressed in triple-negative breast cancer

2. Cenpf Increases Adr Chemosensitivity Of Tnbc

3. Cenpf Regulates Adr-induced G2/m Phase Arrest Through Chk1

4. Cenpf Target The Transcriptional Activity And Expression Of E2f1

Discussion

Conclusions

Declarations

References

table

Additional Declarations

Supplementary Files

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Journal Publication

Version 2