Overexpressed CFLAR drives 5-fluorouracil sensitivity and immune response in breast cancer by regulating reactive oxygen species

doi:10.21203/rs.3.rs-2753916/v1

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Overexpressed CFLAR drives 5-fluorouracil sensitivity and immune response in breast cancer by regulating reactive oxygen species

https://doi.org/10.21203/rs.3.rs-2753916/v1

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Breast cancer (BRCA), the most frequent malignancy in women, is a highly heterogeneous disease. Chemoresistant is the main factor for the BRCA treatment failure. However, the mechanism underlying chemoresistant is still unclear. In this study, we performed comprehensive bioinformatics to verify differentially expressed cell death genes between BRCA and normal tissues. CFLAR was down-regulated in BRCA patients and high expression level of CFLAR exhibited improved prognosis in BRCA patients. CFLAR played a key role in regulating tumor growth. However, the role of CFLAR in different BRCA subtype and its influence in drug resistance and immune therapy need further clarification. CFLAR was decreased in Ki67 positive patients and increased in ER and PR positive patients. CFLAR was significantly down-regulated in Luminal and TNBC than normal breast tissue. CFALR is higher in drug respond patients than drug non-respond patients. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that aberrantly expressed CFLAR was potentially linked with oxidative phosphorylation, T cell receptor signaling pathway, NADH dehydrogenase (ubiquinone) activity, NADH dehydrogenase (quinone) activity, oxidoreductase activity and oxidoreductase-driven active transmembrane transporter activity. Through cell colony formation experiment, overexpression of CFLAR could inhibit reactive oxygen species (ROS) upon 5-FU treatment and promote 5-FU sensitivity in BT-549 and MDA-MB-231cells. In addition, we verified that CFLAR expression was positively correlated with Tcm, T helper cells, Tem, T cells, B cells NK CD56 bright cells and Th2 cells, especially CD8 + T cells. CFALR was higher in CAR-T response patients, anti-PD-L1 response patients and anti-PD1 patients. In summary, CFLAR could regulated ROS homeostasis and ameliorates 5-FU sensitivity of breast cancer cell. CFLAR may be a novel therapeutic target in BRCA.

Breast cancer

CFALR

ROS

Immune infiltration

5-Fluorouracil

Breast cancer (BRCA), the most frequent malignancy in women, is a highly heterogeneous disease. It is the fifth leading cause of cancer mortality, with 685,000 deaths and an estimated 2.3 million new cases worldwide(.Sung et al. 2021). The risk factors of BRCA include postponement of childbearing, having fewer children, greater levels of excess body weight and physical inactivity(.Escala-Garcia et al. 2020). Its molecular features include epidermal growth factor receptor 2 (HER2), hormone receptors (oestrogen receptor and progesterone receptor) and/or BRCA mutations. Tumors with no expression of ER, PR or HER2 are triple negative breast cancer (TNBC)(.Harbeck et al. 2019). TNBC is also defined as basal-like breast cancer (BLBC) according to gene expression profiling analysis classification. The treatment strategies and prognosis of various molecular subtype differ a lot. Therapy approaches include locoregional and systemic therapy approaches which contain endocrine therapy, chemotherapy, anti-HER2 therapy, bone stabilizing agents, poly (ADP-ribose) polymerase inhibitors for BRCA mutation carriers and immunotherapy(.Goldhirsch et al. 2013). The death rates also vary among subtypes of BRCA with highest death rate of TNBC, followed by HER2-positive and luminal A, luminal B subtypes(.Ren et al. 2019). 15–20% of all BRCA patients are TNBC and TNBC has a mortality rate of 40% within the first 5 years after diagnosis. The average time to relapse in non-TNBC patients is 35–67 months, while that in TNBC patients is only 19–40 months. The mortality rate of TNBC patients within 3 months after recurrence is as high as 75%(.Fremd et al. 2019, Yin et al. 2020). The poor prognosis of TNBC mainly due to molecular subtype and increasing prevalence of drug resistance. In the current, scientists make effort to achieve the goal of individualized therapy. It is urgent to explore promising new therapeutic targets to improve the clinical outcomes of BRCA patients.

Caspase 8 and Fas-associated protein with death domain-like apoptosis regulator (CFLAR) is a key anti-apoptotic regulator for resistance to apoptosis via mediating Fas and TRAIL. CFLAR plays a critical role in autophagy, necroptosis, apoptosis and T lymphocytes(.He and He 2013).It also plays a key role in regulating tumor growth(.Li et al. 2023). Increasing researches show that CFLAR participates in inflammation. CFLAR regulates virous diseases including cerebral ischaemia-reperfusion injury(.Xiaohong et al. 2019), renal cancer(.Jang et al. 2021), lung cancer(.Das et al. 2021, Li et al. 2019), and hepatic steatosis(.Zhai et al. 2020). In Non-alcoholic steatohepatitis, CFLAR could inhibit the phosphorylation of c-Jun N-terminal kinase (JNK) and participate in glucolipid metabolic disorder, oxidative stress(.Wang et al. 2017). In liver cancer, CFLAR was up-regulated and was a target of MiR-20a resulting in facilitating activation of caspase-8 and caspase-3, and enhancing sensitivity of HepG2 cells towards TRAIL-induced apoptosis (.Wang et al. 2017). However, the mechanism of CFLAR in BRCA progression is unclear. Cell death, including apoptosis, necrosis, oncosis, pyrosis, and autophagy, is a programmed death type. Scientists make deep understanding of interrupting cell death for cancer treatment(.Bertheloot et al. 2021). Cancer cells are characterized by evasion of cell death(.Seyrek et al. 2022). In BRCA, caspase-3 is correlated to the expression of apoptosis-associated proteins and a poor prognosis. Recent research showed that flubendazole could regulate autophagy and apoptosis by targeting EVA1A resulting in affecting tumor cell proliferation and migration in TNBC(.Zhen et al. 2020). Apoptosis is closely related to hypoxia and they interact with each other in the process of tumorigenesis in BRCA(.Ren et al. 2022). Therefore, the exploration of cell death in BRCAs is urgent.

Increasing researches demonstrate that the immune system is important to the occurrence and development of BRCA, and immunotherapy may improve the clinical results of BRCA(.Emens 2018, Gonzalez et al. 2018). In current, researches focus on novel immune regulation therapy, such as myeloid suppressor cells and regulatory T cells(.Diaz-Montero et al. 2009, Gabrilovich and Nagaraj 2009, Yan et al. 2022). Notably, blocking immune checkpoints and targeting programmed cell death-1/programmed death ligand-1 (PD-1/PD-L1) have a survival benefit in part of BRCA patients including metastatic TNBC patients(.Chen et al. 2022, So et al. 2022). At present, it is a challenge that identifying novel biomarkers to predict the potential response to immunotherapy. Increasing researches confirm that tumor cells acquire apoptosis resistance and participate in immune escape(.Carneiro and El-Deiry 2020). In our research, we performed a comprehensive bioinformatic platforms and found CFLAR was significantly down-regulated and its high level indicated an improved prognosis in BRCA. CFLAR overexpression increased the 5-fluorouracil sensitivity in BRCA cells by regulating reactive oxygen species (ROS)homeostasis. Aberrantly expressed CFLAR was potentially linked with several cancer-associated signaling pathways including oxidative phosphorylation, T cell receptor signaling pathway and NADH dehydrogenase activity. CFLAR was linked with CD8 + T cells and immune therapy in BRCA. These results have indicated that CFLAR may be a novel biomarker for BRCA prognosis and 5-FU sensitivity. Furthermore, CFLAR can regulate the immune response and ROS homeostasis in BRCA, it also influenced the response of immune therapy in different BRCA subtypes.

Data collection and Gene Expression validation

We obtained two breast cancer related datasets including GSE20437 and GSE21422 through using the GEO database, based on the screening criteria of p < 0.05 and |logFC| > 0.5. Then we screened genes with significant differential expression genes (DEGs). The extracted DEGs were also intersected with the set of cell death genes using Venn diagrams, and the co-differentially expressed genes (co-DGE) were obtained. In two datasets GSE20437 and GSE21422, data from GEO2R were utilized to analyze the levels of CFLAR expression in the tumor and normal groups.

Bioinformatics Analyses

The expression of co-DEGs and the prognosis of BRCA patients were assessed using the Kaplan-Meier plotter and BEST database, including BRCA overall survival (OS) and recurrence free survival (RFS). We also performed the progress free interval (PRI) of III/IV BRCA patient by using Xiantao tools. Through Kaplan-Meier plotter, we analyzed the effect of CFLAR expression on OS in TNBC patients. By utilizing Xiantao tool, we analyzed the CFLAR expression between normal tissues and tumor tissues. We also confirm the expression levels of CFLAR in normal and tumor sample from TNM plot and the GEPIA2 database. UALCAN had also been used to analyze CFLAR protein expression in normal and tumor tissue. The expression of CFLAR in Normal, Luminal, HER2 positive, Triple negative patients were performed by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. Using BEST database, the CFLAR expression level in different subtype including Ki67, ER and PR were analyzed. The relation of CFLAR with the marker of TNBC were analyzed by TIMER database. Using BEST, we analyzed the CFLAR expression level in various therapy strategy. Kaplan-Meier plotter were performed to analyze the patients’ prognosis with multiple therapy. Correlation analysis containing GO and KEGG were assessed by Biomarker Exploration of Solid Tumors (BEST) database. The correlation of CFLAR expression with several immune features in BRCA and therapy response were assessed using the Tumor Immune Estimation Resource 2.0 (TIMER2.0) and by scRNA-seq database Tumor Immune Single-cell Hub 2 (TISCH2).

Cell Transfection

According to previous studies(.Chen et al. 2021), two TNBC cell lines (BT-549 and MDA-MB-231) were established and cultured. We construct a CFLAR overexpression plasmid through cloning human CFLAR cDNA into the plasmid pcDNA3.1 (pc3.1-CFLAR) (TsingkeBiotechnologyCo.,Ltd.). The empty plasmid pcDNA3.1 served as the vector control. Cells at a density of ×10⁵ were seeded in six-well plates, and we performed the transfection through using Lipofectamine 3000 reagent (L3000015, Invitrogen, USA) according to the manufacturer’s protocol.

Western Blot

We harvested the BT-549 and MDA-MB-231cells and obtained total protein lysates of cells by incubation on ice with pre-chilled PBS. The collected cells were lysed in lysis buffer and the supernatant were obtained after centrifugation at 12,000g for 15 min at 4℃. Sample of 35 µg cell protein lysates was prepared for SDS-PAGE, and the separated protein was transferred to PVDF membrane by electro blotting. After being blocked in 5% nonfat dried milk at 25°C for 2 h, the membrane was, respectively, incubated with the primary antibodies of CFLAR (EM1708-94, HUABIO) and β-actin (sc-47778, Santa Cruz) at 4°C overnight and then exposed to the corresponding secondary antibodies at 25°C for 1 h. The target bands were detected by enhanced chemiluminescence (ECL) and quantified by Image Lab Software (Bio-Rad) according to the manufacturer’s protocol.

Measurement of Reactive Oxygen Species (ROS)

We detect the cellular ROS level via utilizing the peroxide-sensitive fluorescent probe DCFDA/ H2DCFDA. Cells were inoculated in six-well plates and treated with plasmid and 5-FU (HY-90006, MedChemExpress). The level of ROS was determined by a flow cytometer.

Cell Colony Formation

According to previous studies (.Song et al. 2022), we performing the cell colony formation assay. BT-549 and MDA-MB-231cells were transfected as previously described and then seeded in 6-well plates (1,000 cells per well). After incubating for 24 h, the cells were treated or untreated with 5-FU. After approximately 14 days, colonies were counted by stained with 0.3% w/v crystal violet/methanol for 20 min at 25℃.

Statistical Analysis

All experiments in this study were conducted at least three times with mean ± standard deviations (SD). We analyzed the expression, ROS levels, cell colony formation, and cell survival rates by adopting the student’s t-test. We assessed the prognosis for BRCA using Kaplan-Meier analysis. We evaluate the relation between CFLAR expression and clinicopathological variables through utilizing Wilcoxon rank sum test or Kruskal–Wallis rank test. Furthermore, we analyzed the correlations between CFLAR and immune cells, medicine response, tumor markers and BRCA subtype by using Spearman’s correlation coefficient. We considered as significant differences at *, P < 0.05; **, P < 0.01; ***, P < 0.001. for all tests.

CFLAR was down-regulated in BRCA patients

To determine the co-DEGs, we screen the GEO database and cell death database. As shown in Fig. 1A, there are three genes including two up-regulated genes SLC11A2 and CASP6, one down-regulated genes CFLAR. Subsequently, we valided the expression level of three genes based on GEO database, CASP6 and SLC11A2 were significantly increased in datasets GSE21422 and GSE20437, CFLAR was significantly decreased in datasets GSE21422 and GSE20437(Fig. 1B-G).

High Expression Level of CFLAR Exhibited Better Prognosis in BRCA Patients

Based on Kaplan-Meier portal, we assessed the roles of CFLAR in BRCA patients’ prognosis. In the 208485_x_at, 209508_x_at, 210564_x_at, 211317_x_at, 211862_x_at and 214486_x_at probe, patients with higher CFLAR conferred an improved OS in BRCA (Fig. 2A-F). In the 208485_x_at, 209508_x_at, 210564_x_at,211316_x_at, 211317_x_at, 211862_x_at, 214486_x_at and 224261_x_at probe, patients with higher CFLAR conferred an improved RFS in BRCA (Fig. 2G-N). In the GSE9893, patients with higher CFLAR conferred an improved OS in BRCA (Fig. 2O). In the online Xiantao tool, a comprehensive bioinformatics analysis portal, patients with higher CFLAR conferred an improved OS in BRCA of stage III and IV(Fig. 2P).

Gene Expression Analysis and the correlation of CFLAR with different BRCA subtype

By analyzing the TCGA_GTEx-BRCA dataset, we found CFLAR expression was down-regulated in BRCA tissues (Fig. 3A-B). Furthermore, the differential expression of CFLAR between BRCA and its corresponding non-cancer tissues can be confirmed by TNM plot and GEPIA2 (Fig. 3C-D). CFLAR protein expression was significantly down-regulated in primary tumor by analyzing the CPTAC database (Fig. 3E). The CFLAR was decreased in Ki67 and increased in ER and PR positive patients in GSE21653 through searching BSET database (Fig. 3F, H and I). The expression of CFLAR in BRCA based on breast cancer subclasses in CPTAC database showed that CFLAR was significantly down-regulated in Luminal and Triple negative breast cancer(Fig. 3G).Subsequently, the relationships between CFLAR expression level and patients’ ER, PR and HER2 were analyzed by exploring the TIMER database (Fig. 3J-L). In TNBC, the high CFLAR expression level conferred a better OS in 209508_x_at, 209939_x_at, and 210563_x_at probe (Fig. 3M-O).

The CFLAR expression correlated with drug sensitivity in BRCA patients

As shown in Fig. 4A-E, the gene expression of CFALR was higher in Trastuzumab, Taxanes, Anthracycline, CMF (cyclophosphamide, methotrexate, and fluorouracil), FEC (fluorouracil, epirubicin and cyclophosphamide) responder patients. In the patients treated with tamoxifen only, the CFLAR in high expression conferred a better OS in 2084985_x_at, 211316_x_at probe (Fig. 4F-G). Figure 4H showed that CFLAR was positively correlated with 5-fluorouracil sensitivity.

Overexpression of CFLAR Inhibited cell survival and Promoted 5-FU Sensitivity in BRCA Cells

To better understand the biological significance of CFLAR in BRCA progression, The KEGG pathway was explored using BEST database. As shown in Fig. 5A-B, the enriched pathways regulated by CFLAR include oxidative phosphorylation, T cell receptor signaling pathway, NADH dehydrogenase (ubiquinone) activity, NADH dehydrogenase (quinone) activity, oxidoreductase activity and oxidoreductase-driven active transmembrane transporter activity. We explore the function of CFLAR on TNBC and the 5-FU resistance by culturing BT-549 and MDA-MB-231cells lines. Overexpression strategy was applied to promote the expression of CFLAR in TNBC cells (Fig. 5C). However, cells clone formation rate and ROS levels were not significantly alter upon CFLAR overexpression (Fig. 5D-E, and 5H-I). While Overexpression of CFLAR could inhibit clone formation rate and ROS upon treatment with the 5-FU in BT-549 and MDA-MB-231cells (Fig. 5F-G, 5J-K). These results demonstrate that high expression levels of CFLAR may increase the 5-FU sensitivity through regulating ROS level in BRCA cells.

Correlation Analysis and the role of CFLAR in Immune infiltration and therapy.

Current researches have verified that cell death may participate in the immune response in BRCA cells(.Chu et al. 2022). Through searching the TCGA-cohort through Spearman correlation, the potential function of CFLAR in regulating the immune cell infiltration was analyzed. The results showed that CFALR expression was positively correlated with Tcm, T helper cells, Tem, T cells, B cells and negatively correlated with NK CD56bright cells and Th2 cells (Fig. 6A-C). By searching the BEST-cohort and TIMER database through Spearman correlation, CFLAR was positively correlated with CD8 + T cells in GSE9893 as shown in Fig. 6D (R = 0.552 P = 9.6e-14), Fig. 6E (R = 0.547 P = 3.13e-77) and Fig. 6F (R = 0.403 P = 3.85e-06). Single cell sequencing analysis of CFLAR in BRCA showed that it was positively related to immune cells (Fig. 5G-I), especially CD8 + T cells (J-K). Figure 6L showed that CFLAR was higher in CAR-T response patients in Lauss 2017 cohort (Fig. 6L), anti-PD-L1 response patients in Wolf 2021 cohort (Fig. 6M), and Anti-PD1 patients (Fig. 6N). Furthermore, in BRCA, the high CFLAR expression level conferred an improved OS with anti-PD1 and anti-PD-L1treatment (Fig. 6O-P).

In the present study, by analyzing multiple bioinformatics platforms, we explored the potential target participating in BRCA progression and the drug sensitivity in TNBC. We found three overlapping genes among two GEO datasets and cell death genes, containing two up-regulated genes (SLC11A2 and CASP6), and one down-regulated genes CFLAR. Furthermore, in our study only CFLAR exhibited potential prognostic values in BRCA patients. Additionally, our experiment and bioinformatic prediction demonstrated that the down-regulated CFLAR was critical to BRCA pathological progression and 5-FU sensitivity.

Recent researches focus on cell death which could result in various forms of cancer. Intracellular ROS participates in inflammation, ferroptosis, apoptosis, DNA damage and plays key roles in cellular signaling and homeostasis, ROS level is strictly regulated by Keap1-Nrf2 pathway and cytoprotective antioxidant response (.Du et al. 2008, Edwards et al. 2014, Kansanen et al. 2013). Apoptosis has a number of subtypes of and that overlap exists between apoptosis, autophagy and necrosis(.D'Arcy 2019). Ferroptosis, characterized by lipid peroxidation, is an iron-dependent form of non-apoptotic cell death. The lethality to cancer cells was enhances via a combination of ferroptosis and apoptosis(.Lee et al. 2018). In human lung cancer cells, CFLAR is down-regulated in parthenolide induced apoptosis and parthenolide is reported to induce ROS which can trigger ER stress response(.Zhao et al. 2014). In pancreatic cancer, FBW7 increased cytotoxic effect of gemcitabine through activating ferroptosis and apoptosis, functionally, FBW7 could negatively regulated glucose metabolism(.Ye et al. 2021). Ketoprofen suppresses TNBC cell growth by inducing apoptosis and inhibiting autophagy(.Patra et al. 2023). However, the detailed roles of cell death genes CFLAR in BRCA prognosis and medicine resistance need further exploration. In our research, we firstly verified that CFLAR, a cell death releated gene, was a novel prognostic and therapeutic target in BRCA. In our study, we found that CFLAR was down-regulated in BRCA and its low expression conferred poor prognosis values in BRCA patients. In BRCA, miR-27a ameliorates chemoresistance of BRCA cells by disruption of ROS homeostasis(.Ueda et al. 2020).In our study, overexpression of CFLAR attenuated the 5-FU resistance by down-regulating ROS level and oxidative stress response.

Surgery, radiotherapy, endocrine therapy, chemotherapy, targeted therapy and immune therapy are the conventional treatment strategy for BRCA. The prognosis of various subtype of BRAC have vast differences TNBC is an invasive phenotype with undesirable clinical features, poor prognosis, and therapy resistance(.Vagia et al. 2020). The luminal A, ER and PR positive subtype, have improved prognosis. In our study, we verify that the expression of CFLAR differ from various cancer subtype. CFLAR was significantly down-regulated in Luminal, HER2 positive and TNBC. The gene expression of CFALR is higher in Trastuzumab, Taxanes, Anthracycline, CMF, FEC responder patients. In the patients treated with tamoxifen only, the CFLAR in high expression conferred an improved OS. These results indicated that CFLAR may be a good prognosis and therapy target.

Tumor immune microenvironment (TIME) is highly correlated with the clinical prognosis of cancer patients. T cells are the main immune cells involved in anti-tumor immunity. In tumor cells, CD8 + T cells activated by immunotherapy could enhance ferroptosis, and increased ferroptosis in turn participating in anti-tumor efficacy of immunotherapy(.Zou et al. 2016). Nivolumab therapy resulting in clinical benefits was correlated with reduced expression of SLC3A2 and increased IFNγ and CD8. T cell is a novel anti-tumor mechanism via promoting tumor ferroptosis(.Yang et al. 2014). In B16 subcutaneous melanoma model, PD-L1 blockade increased lipid ROS in CD45- tumor cells and reduced tumor weight(.Wang et al. 2019). In BRCA, there are controversial conclusions of the prognostic prediction of CD8 tumor-infiltrating T cells. A systematic meta-analysis showed that high levels of CD8 + T cell infiltration conferred better prognosis, containing OS and DFS, in BRCA(.Sun et al. 2023). Our results showed that CFLAR was associated with various immune cells containing Tcm, T helper cells, Tem, T cells, B cells, NK CD56bright cells and Th2 cells. CFLAR was positively correlated with CD 8 + T cells and is correlated with the response of anti-PD1 and anti-PD-L1. These results demonstrated that CFLAR may play a vital role in BRCA microenvironments and potentially function as an immunotherapeutic target.

In conclusion, our study demonstrated that CFLAR was significantly down-regulated in BRCA and its low expression displayed a poor prognosis value. CFLAR overexpression could potentiate the promoting effect of 5-FU and sensitize BRCA cell to 5-FU. Overexpression of CFLAR resulted in decreased ROS after 5-FU treatment. Therefore, our study elucidates that cell death associated gene CFLAR may be a potential target in the prognosis and therapy in BRCA through regulating ROS homeostasis and immune infiltration.

BRCA，Breast cancer；HER2，epidermal growth factor receptor 2；TNBC，triple negative breast cancer；BLBC，basal-like breast cancer；CFLAR，Caspase 8 and Fas-associated protein with death domain-like apoptosis regulator；JNK，c-Jun N-terminal kinase；PD-1, programmed cell death-1; PD-L1, programmed death ligand-1; ROS, reactive oxygen species; DEGs, differential expression genes; OS, Overall survival; RFS, Recurrence free survival; PRI, Progress free interval; ESR1, Estrogen Receptor 1; PRs, progesterone receptors; SD, standard deviations; c-Jun N-terminal kinase (JNK) >ECL, enhanced chemiluminescence

AUTHOR CONTRIBUTIONS

Acquisition of Data: SL and WC. Analysis and Interpretation of Data: SL and TX. Conception and Design: SL and HJ. Data Curation: ZL. Development of Methodology: SL. Performing the experiment: SL and HJ Writing the Manuscript: SL. All authors contributed to the article and approved the submitted version.

FUNDING

This study is supported by grants from the Research Fund of The Third Xiangya Hospital.

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

ACKNOWLEDGMENTS

All authors thank the GEO and TCGA databases for providing the useful data, and all the bioinformatics tools for data analysis.

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No competing interests reported.

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Overexpressed CFLAR drives 5-fluorouracil sensitivity and immune response in breast cancer by regulating reactive oxygen species

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Abstract

Figures

Introduction

Materials And Methods

Data collection and Gene Expression validation

Bioinformatics Analyses

Cell Transfection

Western Blot

Measurement of Reactive Oxygen Species (ROS)

Cell Colony Formation

Statistical Analysis

Results

CFLAR was down-regulated in BRCA patients

High Expression Level of CFLAR Exhibited Better Prognosis in BRCA Patients

Gene Expression Analysis and the correlation of CFLAR with different BRCA subtype

The CFLAR expression correlated with drug sensitivity in BRCA patients

Overexpression of CFLAR Inhibited cell survival and Promoted 5-FU Sensitivity in BRCA Cells

Correlation Analysis and the role of CFLAR in Immune infiltration and therapy.

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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