Down-regulation of ESRP2 inhibits breast cancer cell proliferation via inhibiting cyclinD1

doi:10.21203/rs.3.rs-4438254/v1

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Research Article

Down-regulation of ESRP2 inhibits breast cancer cell proliferation via inhibiting cyclinD1

https://doi.org/10.21203/rs.3.rs-4438254/v1

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Background

Epithelial splicing regulatory protein 2 (ESRP2),an important alternative splicing protein of mRNA, is reported to have a dual role in tumors, which can promote or inhibit the occurrence and development of tumors. However, the function and mechanism of ESRP2 in breast cancer (BC) remain unclear.

Methods

The distribution of ESRP2 expression in breast cancer and the correlation between ESRP2 expression and the overall survival rate were detected by The Cancer Genome Atlas (TCGA) database. Gene Ontology(GO)analysis, containing biological process, cellular components, and molecular function,was utilized to evaluate the potential mechanism of ESRP2 in breast cancer. The ESRP2 expression in breast cancer cell lines was detected by real-time quantitative PCR analysis (RT-qPCR) and western blotting. Cell clone was performed to examine the proliferation of ESRP2 knockdown in MCF-7 cells. The cell cycle was measured by flow cytometry assays. The role of ESRP2 knockdown in synergistic effect with chemotherapeutic agents was also determined by MTT assay.

Results

Bioinformatics analysis demonstrated that the ESRP2 gene was elevated in breast cancer cells and its overexpression was strongly correlated with shorter overall survival. GO analysis revealed that ESRP2 expression was related to cell proliferation. ESRP2 mRNA and protein expression were elevated in breast cancer cell lines, compared to the normal human breast cell line MCF-10A. Dwon-regulation of ESRP2 inhibited cell proliferation and promoted the sensitivity of chemotherapy drug, Cisplatin(DDP) and Paclitaxel (TAXOL), in MCF-7 cells.Additionally,ESRP2 knockdown obstructed the cell cycle at the G1 phase and caused a decrease in cyclinD1 protein expression.

Conclusions

These findings reveal that ESRP2 is highly expressed in breast cancer and is correlated with poor prognosis in breast cancer patients. ESRP2 knockdown can inhibit MCF-7 cell proliferation by arresting the cell cycle at the G1 phase and promoting the sensitivity of chemotherapy drugs (DDP and TAXOL)in MCF-7 cells. ESRP2 may be required for the regulation of breast cancer progression, as well as a critical target for the clinical treatment of breast cancer.

ESRP2

breast cancer

cell proliferation

critical target

According to global cancer data in recent years, BC has been the biggest threat to women's health, which even had became the highest incidence of cancer in the world in 2020^1,2. Despite the continuous development of treatment methods³, Recent years have seen a significant improvement in the survival rate of BC. For instance, non-invasive BC has a 5-year survival rate of 99%, whereas invasive BC with lymph node dissemination has an 85% survival rate.⁴ Yet, when distant body parts are also affected, the survival rate falls to 27%⁵. Metastatic breast cancer (MBC) is currently considered a fatal disease due to its high rate of bone metastases in BC ⁶. The mutation of specific genes increases the likelihood of developing breast cancer and may influence the severity of the disease ⁷. Therefore, it is essential to find new therapeutic targets or molecular markers to reduce the mortality of BC and improve patients’ quality of life ⁸.

Epithelial splicing regulatory protein 2 (ESRP2) ⁹ is a member of the RNA binding protein hnRNP family (including ESRP1 and ESRP2), which promotes epithelial-type splicing and interacts with exon or intron splicing inhibitors to promote exon hopping, thus affecting the post-transcriptional regulation of pre mRNA gene expression¹⁰. Tumors may use alternative splicing to gain growth and survival advantages, affecting tumors' occurrence and development ¹¹. ESRP2 and its family members also participate in the regulation of epithelial-to-mesenchymal transition (EMT). EMT is the process of transforming epithelial cells into migrating cells and invasive cells¹². In addition to changes in transcriptional regulation during EMT, the splicing of variable genes generated after transcription can also cause cell migration or invasion ¹³, which has been proven to be related to the occurrence and development of tumors.

Recently, ESRP2 has been recorded to be dysregulated in various tumors, such as breast cancer, prostate cancer, Wilms tumor, head and neck cancer, hepatocellular carcinoma, laryngeal cancer^14–19, and so on. However, the function of ESRP2 in BC, besides participating in EMT, is still unclear.

We examined the ESRP2 expression in BC and how its down-regulation affects BC cells proliferation in this study. Additionally, it has been confirmed that down-regulation of ESRP2 can synergically inhibit the proliferation of BC cells with chemotherapy drugs in our study. We postulate that ESRP2 in BC may suggest a new therapeutic target..

2.1. GEPIA Analysis

GEPIA2.0(http://gepia2.cancer-pku.cn/)is a web-based tool, based on TCGA and GTEx data, that provides interactive and user-defined function analysis of gene expression, including differential expression analysis, contour map drawing, correlation analysis, and dimension reduction analysis ²⁰. Using this database, we can get the expression of ESRP2 in different cancer tissues and normal tissues, and the correlation between ESRP2 and cyclinD1 mRNA levels in breast cancer and normal breast tissues.

2.2. Analysis of ESRP2 protein expression in normal breast samples and breast cancer samples

The full name of HPA is Human Protein Atlas (www.proteinatlas. org). It can be used to summarize the overall expression of human proteins through immunohistochemistry and immunocytochemistry²¹. The database can be used to compare the protein expression of ESRP2 in normal breast samples and breast cancer samples.

2.3. University of Alabama Cancer Database Analysis

UALCAN database(http://ualcan.path.uab.edu)is used for verification of target genes and screening of tumor subgroup-specific biomarkers, mainly through in-depth analysis of TCGA data, including Level 3 RNA seq and clinical data from 31 cancer types²². In this study, the UALCAN database was used to analyze the relationship between the expression of ESRP2 and breast cancer classification, patient race, and disease grade.

2.4. Survival Analysis

Based on GEO, EGA, and TCGA data, Kaplan Meier Plotter can analyze the correlation between specific genes and cancer prognosis by integrating gene expression and clinical data²³. Through Kaplan Meier Plotter analysis, we can get the expression of ESRP2 and the total survival analysis of BC patients, and we can also get the survival analysis formula of ESRP2 in different subtypes and different grades of BC patients.

2.5. GO analysis

LinkedOmics database (http://www.linkedomics.org) includes 32 species of TCGA and 11158 patients with multiple omics data, and clinical data, and can carry on the correlation analysis and the function to the cancer gene enrichment analysis ²⁴. By selecting GO analysis and setting corresponding parameters, the role function of ESRP2 in biological processes, cellular components, and molecular function can be obtained.

2.6. Cell culture

Human normal breast epithelial cells (MCF-10A) and breast cancer cell lines (MCF-7, T47D, MDA-MB-468) were obtained from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences. The composite culture of MCF-10A cells used DMEM/F12 media supplemented with insulin (10 µg/mL), cholera toxin (100 ng/mL) (Sigma-Aldrich), EGF (20 ng/mL) (R&D systems, Wiesbaden, Germany), hydrocortisone (500 ng/mL), and L-glutamine (Procell, Wuhan, China). MCF-7 and T47D cells were cultured in RPMI Medium 1640 with 10% FBS (A31160801, Gibco, Carlsbad, CA). MDA-MB-468 cells were cultured in DMEM/HIGH GLUCOSE (Gibco, Carlsbad, CA) with 10% FBS. The growth condition for all cells was 37°C at 5% concentration of CO₂. SiRNA of human ESRP2 and control (siNC) were ordered from Guangzhou RiboBioCo, Ltd. To knockdown ESRP2 in vitro, ESRP2-siRNA was transfected into MCF-7 cells using Lipofectamine 2000 (11668-019, Invitrogen) under the manufacturer’s recommendations.

2.7. RNA extraction and real-time quantitative PCR analysis

Total RNA was extracted from cultured cells using Trizol (15596-018, Invitrogen, CA, USA). The RNA quality was assessed using a NanoDrop 2000 spectrophotometer to determine the 260/280 ratio. Gene expression analysis was conducted on samples with a ratio ranging from 1.8 to 2.0. Using the PrimeScript RT Reagent Kit with gDNA Eraser (AG11705, Accurate Biology, Hunan, China), one microgram of mRNA was reverse-transcribed into complementary DNA by the manufacturer's instructions. Assays for alternative splicing based on polymerase chain reaction (PCR) were conducted using Invitrogen's Platinum II Hot-Start Green PCR Master Mix. Real-time quantitative PCR (RT-qPCR) was performed using SYBR Green (AG11701, Accurate Biology, Hunan, China). The relative expression levels of mRNA were evaluated by using the 2 − ΔΔCt method. The following primer sequences were used for PT-qPCR analysis: ESRP2, forward 5’-TGC CAC AGA GGA TGA CTT TG-3’ and reverse 5’- ATT GAC TGC TGG GCT CTT TG-3’; GAPDH, forward 5’-CGA GAT CCC TCC AAA ATC AA-3’ and reverse 5’- GGC AGA GAT GAT GAC CCT TT-3’.

2.8. Western blotting

Cells were lysed in RIPA lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) containing phenylmethylsulfonyl fluoride (PMSF) (Beyotime) on ice for 30 min and centrifuged at 12,000 rpm for 20 min at 4°C. The total protein concentration was measured with a bicinchoninic acid (BCA) protein assay kit (P0010, Beyotime Biotechnology, Shanghai, China). Protein lysates were resolved by SDS-PAGE and immunoblotted with the indicated primary antibodies and their corresponding HRP-conjugated secondary antibodies. The antibodies used are the following: Esrp2 (1:1000, Thermo Fisher, #PA5-21110), CyclinD1(1:1000, Abcam, ab226977) and GAPDH (1:2000, Proteintech, 60004-1-Ig). The appropriate secondary antibody (1:5000, Anti-Mouse IgG, Cell Signaling Tech, #14709; 1:5000, Anti-Rabbit IgG, Cell Signaling Tech, #7074s) coupled to horseradish peroxidase (HRP) was then incubated on the membranes for 1 hour at 37°C. Using an electrochemiluminescence (ECL) kit from Tanon in Shanghai, China, immunoreactions were seen and scanned using a Bio-Rad ChemiDocTM Touch Imaging System. The relative integrated density values (IDVs) based on the internal control GAPDH were analyzed with Image J software.

2.9. Cell clone formation assay

MCF-7 cells were digested by 0.25% trypsin/0.02% EDTA solution (SM-2003, Sigma-Aldrich®) at the logarithmic phase to make a single-cell suspension with culture medium. Then, a cell counting chamber was used to calculate the number of cells in a 10 µL single-cell suspension under an inverted microscope. The cells were then delivered into six-well culture plates containing a sterile glass coverslip in each well, with about 200 cells added to each well. The medium was refreshed every 3 days until cell clones were measured by crystal violet (C0121-100ml, Beyotime Biotechnology, Shanghai, China).

2.10. Flow cytometry assays of the cell cycle

The cells were cultured and transfected with the si-ESRP2 or siNC for 48 h. The cells were digested and then collected by centrifugation, the supernatant was discarded, and the cells were washed twice with ice-cold PBS. Pre-chilled 70% ethanol was added and stored fixed at 4°C. Cells were collected and incubated for 30 min at 37°C protected from light after Propidium Iodide (PI) staining (ST511, Beyotime Biotechnology, Shanghai, China). Flow experiments were performed within 5 hours. Finally, cell cycle distribution was determined using a flow cytometer (Beckman Coulter, Brea, CA, USA).

2.11. Statistical analysis

Statistical analysis was performed with SPSS software version 25.0. Data are expressed as mean ± SD. Differences between the two groups were evaluated by Student’s t-test. P < 0.05 was considered statistically significant. Clinical data analysis of survival and relevant correlations were performed with GraphPad Prism.

3.1. ESRP2 is differentially expressed in human cancers and up-regulated in BC

To investigate ESRP2 expression profiles in different types of human cancers, we analyzed the Interactive Analysis of Gene Expression Profiles (GEPIA) dataset. As shown in Fig. 1, ESRP2 expression levels are downregulated in esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), testicular germ cell tumors (TGCT), and thyroid carcinoma (THCA), etc, compared with normal tissue samples. However, the expression level of ESRP2 was upregulated in bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma (CESC), colon adenocarcinoma (COAD), acute myeloid leukemia (LAML), ovarian cancer (OV), rectum adenocarcinoma (READ), thymoma (THYM), uterine corpus endometrial carcinoma (UCEC), uterine carcinosarcoma (UCS) etc. What is more, Heilmann K et al. ²⁵ reported that high expression of ESRP2 was observed in estrogen receptor-positive MCF-7 breast cancer cells by RT-qPCR. These results suggest that ESRP2 has a dual role in multiple tumors.

3.2. The up-regulation of ESRP2 expression is closely related to BC

Immunohistochemical results of ESRP2 were checked by Human protein atlas, and the overall expression of ESRP2 in normal breast samples and BC samples was compared. It was found that the ESRP2 protein was highly expressed in BC tissues (Fig. 2A). To verify whether ESRP2 is highly expressed in BC samples compared to normal samples, breast cancer cells were validated. Compared to MCF-10A, the mRNA and protein expression level of ESRP2 in T-47D, MCF-7, and MDA-MB-468 cells was upregulated (Fig. 2B, 2C). The above results confirm that ESRP2 is highly expressed in BC.

3.3. ESRP2 expression pattern analysis in BC patients with different clinical features

The previous database analysis and experimental results showed that the expression level of ESRP2 was significantly up-regulated in BC, so we used the UALCAN database again to analyze the degree of ESRP2 expression pattern in BC patients with different clinical characteristics. As shown in Fig. 3A, ESRP2 mRNA expression was higher in the total BC samples than in normal breast tissues. As shown in Fig. 3B, significantly high expression of ESRP2 was observed in different subtypes of BC, such as Luminal, Her-2 positive, and triple-negative subtypes. In addition, the expression of ESRP2 in BC tissues of different races was significantly higher than that in normal breast tissues (Fig. 3C). The expression of ESRP2 in breast cancer tissues of different stages was also significantly higher than that in normal breast tissues (Fig. 3D). Thus, significant differences in different tumor subclasses, races, and disease grades, suggest that ESRP2 may play a key role in the occurrence and progression of BC.

Figure 3. ESRP2 expression pattern analysis in BC patients with different clinical features. The UALCAN analysis showed that ESRP2 mRNA levels were significantly higher than normal breast tissue in BC patients with different clinicopathological features, including (A) total sample type, (B) different subtypes, (C) ethnicity, and (D) individual cancer stages. *P < 0.05, ** P < 0.01, ***P < 0.001.

3.4. The high expression of ESRP2 is closely associated with poor prognosis in BC patients

We further evaluated the prognostic value of ESRP2 in BC. Kaplan-Meier Plotter database analysis showed that breast cancer patients with high ESRP2 expression had shorter overall survival. (HR = 1.7, P = 1.3e-6) (Fig. 4A). Analysis of different BC subtypes shows that, especially in the analysis of different grades of BC, high expression of ESRP2 mRNA is found to be significantly correlated with Grade2 (HR = 2.13, P = 0.00058) and Grade3 (HR = 1.81, P = 0.00049) of breast cancer stages. However, the overexpression of ESRP2 is not significantly correlated with the survival rate in Grade 1 (HR = 2.52, P = 0.045, close to 0.05). It may be that the sample size of the Grade 1 rent analysis is too small to analyze the difference in survival between the high-expression and the low-expression group. In different BC subtypes, high expression of ESRP2 significantly shortened the overall survival of patients with positive Luminal A, Luminal B, and Her-2, with P < 0.05. The Kaplan-Meier Plotter database analysis revealed a significant association between elevated levels of ESRP2 mRNA and decreased relapse-free survival in breast cancer patients.

3.5. ESRP2 is related to cell proliferation in breast cancer

To further explore the mechanism of ESRP2 in breast cancer, GO analysis were carried out on the linked omics database to predict the biological function of ESRP2 in breast cancer (Fig. 5). The biological processes related to ESRP2 and breast cancer may include biological regulation, metabolic process, stress response, developmental process, cellular component organization, and cell proliferation, etc. Cell components associated with breast cancer may include cell membrane, nucleus, membrane closed cavity, and protein complex, etc. Molecular functions related to breast cancer may consist of protein binding, ion binding, nucleic acid binding, etc. The results of GO analysis confirm that ESRP2 is related to cell proliferation in BC.

3.6. Down-regulation of ESRP2 inhibits MCF-7 cell proliferation by targeting CyclinD1

The up-regulation of the ESRP2 gene in BC suggests a potential association between elevated ESRP2 levels and the development and advancement of the disease.We then determined whether the down-regulation of ESRP2 could affect cancer cell proliferation. MCF-7 cells were transiently transfected with siESRP2 and the protein level of ESRP2 decreased (Fig. 6A). The ability of cells to form a cloned cell mass was significantly lower after transfection with siESRP2 compared with the negative control (siNC) by cloning assay, which indicated that interference with siESRP2 inhibited the cloning ability of MCF-7 cells (Fig. 6B). To assess whether the effect of ESRP2 on breast cancer cell proliferation was mediated by arresting the cell cycle, we performed a flow cytometry assay to investigate the distribution of specific phases of the cell cycle in breast cancer cell lines. In MCF-7 cells, ESRP2-down-expressed cells were strikingly blocked at the G1 phase of the cell cycle as compared to the control group (Fig. 6C). Furthermore, the correlation analysis of GEPIA Analysis showed that ESRP2 was significantly positively correlated with CCND1(Cyclin D1, cell cycle key regulator, and the marker for G1 phase) (Fig. 6D). Beyond that, the down-expression of ESRP2 decreased the protein level of Cyclin D1(Fig. 6E). These results illustrate that the knockdown of ESRP2 may arrest the cell cycle at the G1 phase by targeting CyclinD1 in breast cancer cells.

3.7. Down-regulation of ESRP2 enhances the DDP and TAXOL-mediated inhibition of proliferation

Our previous experiment confirmed that down-regulation of ESRP2 can inhibit the proliferation of BC cells. We then investigated whether the down-regulation of ESRP2 and the combination of DDP/TAXOL have a synergistic effect on cell proliferation after ESRP2 knockdown for 48 hours. Compared with the blank control group treated with DDP/TAXOL alone or DDP/TAXOL combined with siNC, the combination of down-regulation ESRP2 and DDP/TAXOL reduced cell viability (Fig. 7A, B). In addition, Fig. 7C, D showed the CI plots for a combination of down-regulation ESRP2 and DDP/TAXOL in MCF-7 cells generated by CompuSyn software (version 1.0; ComboSyn, Paramus, NJ, USA, for each drug combination, CI < 1, CI = 1, and CI > 1 indicate synergism, additive effect, and antagonism, respectively), which plotted CI against the inhibitory effect. The plots revealed a synergistic pattern (CI < 1) and showed that the synergistic effect was heightened with the increase in the dose of chemotherapy drugs. In conclusion, these results suggest that down-regulation of ESRP2 enhances the antiproliferation effect of DDP/TAXOL-mediated breast cancer cells.

BC is a common malignant tumor that threatens the health of women all over the world and the treatment method has gradually moved from traditional treatment to precise and targeted treatment^{26, 27}. Effective biomarkers can be used to predict patients' progress and formulate treatment plans, which is the key to accelerating the research process of precision treatment of BC²⁸. Previous studies have shown that ESRP2 can directly or indirectly participate in the progression of various tumors by affecting splicing variation, EMT process, or interacting with upstream and downstream molecules^29–33. Most of the reports about ESRP2 in BC only studied the relationship between ESRP2 and EMT, which affects the occurrence and development of BC, yet did not further analyze the role of ESRP2 expression and the related molecular mechanism^{16, 25, 33}. In light of this, we explored its function in BC.

Horvath A et al.³³sequenced RNA of three negative breast cancer (TNBC), non-TBNC, and HER2 + breast cancer subtypes, and they validated a subset of novel variants of cancer-related genes in BC, including ESRP2. Coincidentally, our research results are consistent with theirs. In our study, we found that ESRP2 was highly expressed in BC tissue compared with normal breast tissue. The high expression of ESRP2 in BC was related to breast cancer classification, patient race, and disease grade. We also found that the overall expression level of ESRP2 in BC patients increased by comparing the immunohistochemical maps in the Human Protein Atlas. In addition, survival analysis demonstrated the prognostic value of ESRP2 in BC. High expression of ESRP2 is associated with poor prognosis in BC patients. We found that ESRP2 was highly expressed in human BC cells MCF-7, T-47D, and MDA-MB-468 at mRNA and protein levels compared with human normal breast cells MCF-10A. Therefore, these results proved that ESRP2 levels could potentially serve as a specific oncogene involved in breast carcinoma occurrence and progression.

After discovering that ESRP2 is overexpressed in BC, we explored the role and mechanism of ESRP2. GO analysis in the Linkedomics database showed that ESRP2 was related to cell proliferation in BC. Interestingly and importantly, we proved that the down-regulation of ESRP2 inhibited the cloning ability of MCF-7 cells. To further investigate the mechanism of ESRP2 affecting BC cell proliferation, we found that ESRP2 knockdown could block the cell cycle in the G1 phase. In addition, our results showed that the down-regulated ESRP2 and chemotherapy drugs synergically inhibited the proliferation of BC cells. In summary, the evidence above confirms that ESRP2 knockdown inhibited the proliferation of BC cells, which suggests that ESRP2 may be a potential therapeutic target for BC.

In the future, if more clinical samples and reproducible experiments are used to verify the correlation between ESRP2 expression and BC, and more upstream and downstream protein-related pathways and drug resistance pathways are explored, it will provide more meaningful value for the real application of ESRP2 in the diagnosis and clinical treatment. Future work should aim to determine how ESRP2 interacts with other proteins that could influence cell migration and invasion of BC.

In conclusion, our research demonstrated that ESRP2 was highly expressed in breast cancer and was correlated with poor prognosis in BC patients. Knockdown of ESRP2 expression hampered cell proliferation of BC cells and also resulted in the synergistic anticancer effect with chemotherapeutic agents. Mechanically, down-regulation of ESRP2 could inhibit BC cell growth by blocking the cell cycle at the G1 phase. Our study indicates that ESRP2 is essential in the regulation of BC progression, suggesting an experimental basis for being a biomarker as well as expanding its potential clinical value.

Funding: This work was supported by the general project of the Natural Science Foundation of Guangdong Province (No2022A1515010877 and No2023A1515010454); the key popular science research project of Science Communication Professional Committee of Chinese Pharmaceutical Association (NoCMEI2024KPYJ00113)

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author contribution statement

All authors were involved in the conception and design of the data, or analysis and interpretation, as well as the drafting and revision of the paper. All authors agree to take responsibility for all aspects of their work

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Down-regulation of ESRP2 inhibits breast cancer cell proliferation via inhibiting cyclinD1

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Abstract

Background

Methods

Results

Conclusions

Figures

1. Introduction

2. Materials and methods

2.1. GEPIA Analysis

2.2. Analysis of ESRP2 protein expression in normal breast samples and breast cancer samples

2.3. University of Alabama Cancer Database Analysis

2.4. Survival Analysis

2.5. GO analysis

2.6. Cell culture

2.7. RNA extraction and real-time quantitative PCR analysis

2.8. Western blotting

2.9. Cell clone formation assay

2.10. Flow cytometry assays of the cell cycle

2.11. Statistical analysis

3. Results

3.1. ESRP2 is differentially expressed in human cancers and up-regulated in BC

3.2. The up-regulation of ESRP2 expression is closely related to BC

3.3. ESRP2 expression pattern analysis in BC patients with different clinical features

3.4. The high expression of ESRP2 is closely associated with poor prognosis in BC patients

3.5. ESRP2 is related to cell proliferation in breast cancer

3.6. Down-regulation of ESRP2 inhibits MCF-7 cell proliferation by targeting CyclinD1

3.7. Down-regulation of ESRP2 enhances the DDP and TAXOL-mediated inhibition of proliferation

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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