Low GPR81 in ER+ breast cancer cells drives tamoxifen resistance through inducing PPARα-mediated fatty acid oxidation.

AIMS: The intricate molecular mechanisms underlying estrogen receptor-positive (ER+) breast carcinogenesis and resistance to endocrine therapy remain elusive. In this study, we elucidate the pivotal role of GPR81, a G protein-coupled receptor, in ER+ breast cancer (BC) by demonstrating low expression of GPR81 in tamoxifen (TAM)-resistant ER+ BC cell lines and tumor samples, along with the underlying molecular mechanisms. MAIN METHODS: Fatty acid oxidation (FAO) levels and lipid accumulation were explored using MDA and FAßO assay, BODIPY 493/503 staining, and Lipid TOX staining. Autophagy levels were assayed using CYTO-ID detection and Western blotting. The impact of GPR81 on TAM resistance in BC was investigated through CCK8 assay, colony formation assay and a xenograft mice model. RESULTS: Aberrantly low GPR81 expression in TAM-resistant BC cells disrupts the Rap1 pathway, leading to the upregulation of PPARα and CPT1. This elevation in PPARα/CPT1 enhances FAO, impedes lipid accumulation and lipid droplet (LD) formation, and subsequently inhibits cell autophagy, ultimately promoting TAM-resistant BC cell growth. Moreover, targeting GPR81 and FAO emerges as a promising therapeutic strategy, as the GPR81 agonist and the CPT1 inhibitor etomoxir effectively inhibit ER+ BC cell and tumor growth in vivo, re-sensitizing TAM-resistant ER+ cells to TAM treatment. CONCLUSION: Our data highlight the critical and functionally significant role of GPR81 in promoting ER+ breast tumorigenesis and resistance to endocrine therapy. GPR81 and FAO levels show potential as diagnostic biomarkers and therapeutic targets in clinical settings for TAM-resistant ER+ BC.

Family roles in informed consent from the perspective of young Chinese doctors: a questionnaire study.

BACKGROUND: Based on the principle of informed consent, doctors are required to fully inform patients and respect their medical decisions. In China, however, family members usually play a special role in the patient's informed consent, which creates a unique "doctor-family-patient" model of the physician-patient relationship. Our study targets young doctors to investigate the ethical dilemmas they may encounter in such a model, as well as their attitudes to the family roles in informed consent. METHODS: A questionnaire was developed including general demographic characteristics, the fulfillment of the obligation to fully inform, who will be informed, and the ethical dilemmas in decision-making. We recruited a total of 421 doctors to complete this questionnaire, of which 368 met the age requirements for this study. Cross tabulation and Pearson's chi-squared test were used to analyze the differences between types of patients for categorical variables, and a p-value < 0.05 was considered statistically significant. RESULTS: Our data shows that only 20 doctors (5.40%) stated "informing the patient alone is sufficient" when it comes to informing patients of their serious conditions. The rest of the participants would ensure that the family was informed. When facing elderly patients with decision-making capacity, the data was statistically different (3.8%; P < 0.001) The primary reason for ensuring that family members be informed differs among the participants. In addition, when family members asked doctors to conceal the patient's medical condition for the best interests of patients, 270 doctors (73.4%) would agree and cooperate with the family. A similar proportion (79.6%) would do so when it comes to elderly patients. CONCLUSIONS: (1) Chinese doctors pay extra attention to informing the patient's family, which may not be in the patient's best interests. (2) Chinese doctors treat adult (but not elderly) patients and elderly patients differently when it comes to informing family members. (3) When family members request that doctors withhold information from patients "in the best interest of the patient," the majority choose to comply with the request, although this may cause them distress.

EGR3 and estrone are involved in the tamoxifen resistance and progression of breast cancer.

BACKGROUND: Tamoxifen (Tam) is an effective treatment for estrogen receptor (ER) positive breast cancer. However, a significant proportion of patients develop resistance under treatment, presenting a therapeutic challenge. The study aims to determine the role of early growth response protein (EGR) 3 in tamoxifen resistance (TamR) and elucidate its molecular mechanism. METHODS: TamR cell models were established and NGS was used to screening signaling alternation. Western blot and qRT-PCR were used to analysis the expression of ERα, EGR3, MCL1 and factors associated with apoptosis. CCK8, colony formation and apoptosis assay were used to analysis resistance to Tam. Immunofluorescence, chromatin immunoprecipitation, and dual luciferase assays were used to investigate mechanism of regulation. RESULTS: We observed that EGR3, a deeply rooted ERα response factor, showed increased upregulation in response to both estrone (E1) and Tam in TamR cells with elevated level of E1 and ERα expression, indicating a potential connection between EGR3 and TamR. Mechanically, manipulating EGR3 expression revealed that it imparted resistance to Tam through increased expression of the downstream molecule MCL1 (apoptosis suppressor gene) that it regulated. Mechanismly, EGR3 directly binds to the promoter of the anti-apoptotic factor MCL1 gene, facilitating its transcription. Furthermore, apoptosis assays revealed that E1 reduces Tam induced apoptosis by upregulating EGR3 expression. Importantly, clinical public database confirmed the high expression of EGR3 in breast cancer tissue and in Tam-treated patients. CONCLUSIONS: These findings shed light on the novel estrogen/EGR3/MCL1 axis and its role in inducing TamR in ER positive breast cancer. EGR3 emerges as a promising target to overcome TamR. The elucidation of this mechanism holds potential for the development of new therapeutic modalities to overcome endocrine therapy resistance in clinical settings.

MCT4/Lactate Promotes PD-L1 Glycosylation in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) has the highest percentage of lymphocytic infiltration among breast cancer subtypes, and TNBC patients may benefit from anti-PD-1/PD-L1 immunotherapy. However, some cases whether the immune checkpoint blockade (ICB) shows low targeting efficiency have occurred and effective synergistic targets need to be found, which inspired our exploration of the co-expression analysis of MCT4 (SLC16A3) and PD-L1 (CD274) and their potential regulatory mechanisms. After bioinformatic analysis of the relationship between MCT4 and PD-L1, we validated their positive co-expression relationship in triple-negative breast cancer through multiple immunohistochemical staining (mIHC), CRISPR/Cas9, and lentiviral transduction for MCT4 knockout (sgMCT4/231 KO) or overexpression (pEGFP-N1-MCT4/231). We examined the effect of lactate treatment on PD-L1 expression in triple-negative breast cancer cells by qRT-PCR and Western blot. Combined with our results, we found that MCT4 positively regulated PD-L1 expression through discharging lactate and stabilized PD-L1 through promoting its glycosylation by the classic WNT pathway in MDA-MB-231 cells. More importantly, the high co-expression of MCT4 and PD-L1 appears to predict more effective targets for treating TNBC, which would improve immune checkpoint therapy for TNBC.

The red packet phenomenon from the perspective of young Chinese doctors: a questionnaire study.

BACKGROUND: In China, informal payments in the medical profession, which workers in the public health care system receive from patients in the course of performing profession-related activities, are usually referred to as "red packets" (Hongbao ). The phenomenon of red packets is widespread and has become one of the most negative factors affecting the doctor-patient relationship in China. Our study aims to explore the situation concerning the phenomenon of red packets in China after the "Red Packet Ban". METHODS: A questionnaire was developed including general demographic characteristics, asking whether they had ever been offered red packets, whether they had ever accepted red packets, their reasons for accepting the first red packet and so on. We recruited a total of 413 doctors to complete this questionnaire and conducted in-depth telephone interviews with 18 doctors from the initial group. RESULTS: Our data shows that 73 doctors claimed to have accepted red packets, accounting for 17.7% (73/413) of all respondents and 27.8% (73/263) of doctors who had been provided with red packets. 23.2% of red packets were offered after the operation and 67.1% of the doctors declared that the main reason for accepting the red packet was that they "refused the red packets more than once, but the patients/family members were sincere and it was difficult to refuse." The total amount of the red packets they received each month accounted for no more than 5% of their income. CONCLUSIONS: (1) The acceptance of red packets does exist among young doctors in China, but shows a significant decrease compared to previous studies. (2) There has been a sharp rise in the proportion of gratitude red packets. (3) Patients should also be educated regarding their behaviour in providing red packets.

Gender differences in response to medical red packets (Hongbao, monetary gifts): a questionnaire study on young doctors in China.

BACKGROUND: The acceptance of informal payments by doctors is usually viewed as unethical behavior. However, in China, such behavior is a common practice. In this study, we focus on the gender differences in accepting red packets (informal payments) by young doctors in China. METHODS: A total of 413 young doctors were selected for the study, all of whom were grouped by gender. The questionnaire was designed to include general demographic characteristics, whether they had ever been offered red packets, whether they had ever accepted red packets, the reasons for accepting red packets and so on. Wilcoxon rank-sum test, Pearson's chi-squared test, univariable and multi-variable logistic regressions were used for all analyses by Stata 17.0 SE and p-value < 0.05 was considered statistically significant. RESULTS: Compared to women, men were more likely to be offered red packets (69.5% [180/259] vs.53.9% [83/154]), and the odds ratio (OR) was statistically significant after adjusting for age, education, position and geographical areas (adjusted OR 1.81, p = 0.012). In terms of the question of whether or not they had accepted red packets, more male doctors answered "yes" compared to female doctors (33.3% [60/180] vs.15.7% [13/83], adjusted OR 2.80, p = 0.004). However, among those who had accepted red packets, we found that only 42.0% [25/60] of male doctors considered that it was normal to accept such red packets, compared to 85.0% [11/13] of women (adjusted OR 12.01, p = 0.023). CONCLUSION: The study revealed that Chinese patients and their families were more likely to offer red packets to male doctors. Secondly, among doctors who had been offered red packets, male doctors were more likely to accept red packets than female doctors. In addition, among doctors who had accepted red packets, female doctors were more likely to believe that it was not morally wrong to accept such red packets.

Glucagon-like peptide-1 receptor activation by liraglutide promotes breast cancer through NOX4/ROS/VEGF pathway.

AIMS: Scientific evidence imply the strong correlation between diabetes and breast cancer. Glucagon-like peptide-1 (GLP-1) and its analogue liraglutide, have been widely used for diabetes treatment. However, the role of GLP-1 receptor (GLP-1R) in breast cancer requires further elucidation. This study aimed to investigate the risk and the molecular mechanisms of liraglutide using in breast cancer. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction, western blot or immunohistochemistry were used to detect the expressions of GLP-1R, NADPH oxidase 4 (NOX4) and vascular endothelial growth factor (VEGF) in human triple negative breast cancer (TNBC) cells (MDA-MB-231 and MDA-MB-468) and tissues derived from BALB/cfC3H mouse bearing 4T1 cells inoculation. Cell proliferation and migration was detected using the Cell Counting Kit-8, adenosine triphosphate assay, and transwell assay, respectively. Flow cytometry was used to measure the level of reactive oxygen species (ROS). KEY FINDINGS: We found that the expression of GLP-1R increased after liraglutide treatment in breast cancer cells and the transplanted tumors. Liraglutide, at a slightly higher concentration, accelerated breast cancer progress in vitro (100 nM) and in vivo (400µg/kg) through the NOX4/ROS/VEGF signal pathway after activating GLP-1R. The GLP-1R inhibitor, Exendin (9-39), significantly inhibited the effect of liraglutide, inducing a reversed function of GLP-1R activation. SIGNIFICANCE: Our study illustrated that in an approximately toxicology context, liraglutide may promote the malignant progression of TNBC. The dosage and the phenotype of the breast cancer should be considered as important factors for the rational administration of antidiabetic drugs, especially that of liraglutide in breast cancer patients.

Blocking MCT4 SUMOylation inhibits the growth of breast cancer cells.

Monocarboxylate transporter 4 (MCT4) is highly expressed in various types of solid neoplasms including breast cancer (BC); however, the pro-tumor functions underlying its increased expression have not been explained. Here, we examined the roles of posttranslational modifications to MCT4 in BC, particularly SUMOylation. Our findings revealed that SUMOylation of MCT4 inhibited its degradation and stabilized MCT4 protein levels, while ubiquitination facilitated MCT4 degradation. The E3 ubiquitin ligases ß-TRCP and FBW7 interacted with MCT4 at the DSG-box and TPETS sequences, respectively, and Lys448 (K448) of MCT4 could be modified by SUMO chains. Our key finding was that K448 was crucial for MCT4 SUMOylation. Moreover, mutations of K448 abolished MCT4 expression, delaying the growth of BC. This study suggested that SUMOylation of K448 increased MCT4 levels, and mutations of K448 in MCT4 could have therapeutic significance in BC.

Downregulation of caveolin-1 promotes murine breast cancer cell line progression by highly glycosylated CD147.

Caveolin-1 (CAV-1) can extensively regulate lipid transportation, cell growth and cell death. In the present study, we revealed a novel function of CAV-1 in inhibiting glycosylation of other molecules in murine breast cancer cell line. After the silencing of CAV-1, we found that the mRNA and protein expressions of cluster of differentiation 147 (CD147) and its related molecules (MCT4, matrix metalloproteinase MMP2 and MMP9) increased in the breast cancer cells. Meanwhile, the migration and invasion of the breast cancer cells were significantly enhanced assessed by cell wound healing experiment and transwell assays. Further, the gelatin zymography and lactate assay in the cells also showed the strengthened enzyme activity of MMP9 and the increased extracellular lactate concentration, respectively, after the silencing of CAV-1. Notably, the glycosylation level of CD147 overtly increased after the inhibition of CAV-1 detected by Western Blot analysis, whereas upregulation of CAV-1 did the opposite. Therefore, the findings suggest that the downregulation of CAV-1 can promote breast cancer cell progression probably by highly glycosylated CD147.

The role of amplified in breast cancer 1 in breast cancer: A meta-analysis.

PURPOSE: Amplified in breast cancer 1 (AIB1) expression is known to be involved in the initiation and progression of malignant breast cancer (BC), but its prognostic role remains uncertain. This meta-analysis assessed reported studies to evaluate this relationship. METHODS: Electronic databases were systematically reviewed to collect eligible studies using pre-established criteria. Hazard ratios (HRs) or odds ratios (ORs) and 95% confidence intervals (CIs) were pooled to estimate the impact of AIB1 protein expression on overall survival (OS) and clinicopathologic properties of BC cases. RESULTS: Nine eligible studies, including 6774 patients, were finally assessed by the current clinical meta-analysis. AIB1 positivity correlated with reduced OS (pooled HR = 1.409, 95% CI 1.159-1.714, P = .001). AIB1 overexpression also impacted prognosis as shown by univariate (pooled HR = 1.420, 95% CI 1.154-1.747, P = .001) and multivariate (pooled HR = 1.446, 95% CI 1.099-1.956; P = .009) analyses. Notably, subgroup analyses also revealed that AIB1 overexpression was associated with poor OS in some subgroups, such as ER-positive group (pooled HR = 1.511, 95% CI 1.138-2.006, P = .004), ER-positive without tamoxifen administration group (pooled HR = 2.338, 95% CI 1.489-3.627, P < .001), and premenopausal women group (pooled HR = 1.715, 95% CI 1.231-2.390, P = .001). Additionally, high AIB1 protein levels were associated with HER2 positivity (pooled OR = 0.331, 95% CI 0.245-0.448; P < .001), poorly differentiated histological grade (pooled OR = 0.377, 95% CI 0.317-0.448; P < .001), high Ki67 (pooled OR = 0.501, 95% CI 0.410-0.612; P < .001), presence of lymph node metastases (pooled OR = 0.866, 95% CI 0.752-0.997; P = .045), and absence of progesterone receptor (pooled OR = 1.447, 95% CI 1.190-1.759; P < .001). CONCLUSIONS: This analysis demonstrated that AIB1 overexpression is related to aggressive phenotypes and unfavorable clinical outcomes in BC, and might involve in tamoxifen resistance. AIB1 may be a new prognostic biomarker and therapeutic target in BC.

Clinical Significance of C-X-C Motif Chemokine Receptor 4 and Integrin αvß6 Expression in Breast Cancer.

PURPOSE: C-X-C motif chemokine receptor 4 (CXCR4) and integrin αvß6 play important roles in the malignant progression of multiple cancers. However, it remains unclear whether the expression of one or both proteins in breast cancer (BC) is of clinical significance. In this study, we investigated the expression of CXCR4 and integrin αvß6 in BC tissues and their correlation with clinicopathological characteristics, including survival. METHODS: CXCR4 and αvß6 expression in 111 BC tissues was examined by immunocytochemistry. Correlations between the expression of the 2 proteins and patient clinicopathological characteristic were investigated using the Kaplan-Meier method and the Cox proportional hazards model. RESULTS: CXCR4 and αvß6 were overexpressed in BC tissue compared with normal breast tissue. Overexpression of both molecules was related to lymph node status (p = 0.013 and p = 0.022, respectively). αvß6 overexpression was also associated with tumor size (p = 0.044). A positive correlation was detected between the expression of CXCR4 and αvß6 (r = 0.649, p = 0.001), and co-overexpression of both molecules was associated with tumor size (p = 0.018) and lymph node metastasis (p = 0.015). Kaplan-Meier analysis revealed that overexpression of CXCR4, αvß6, or both molecules was associated with short overall survival (OS; p < 0.001, p < 0.001, and p = 0.009, respectively) and disease-free survival (DFS; p < 0.001, p = 0.005, and p = 0.019, respectively). Multivariate analysis indicated that lymph node metastasis was an independent prognostic factor for unfavorable OS and DFS (p = 0.002 and p = 0.005, respectively), whereas co-overexpression of CXCR4 and αvß6 was an independent prognostic factor only for OS (p = 0.043). CONCLUSION: CXCR4 and αvß6 may play synergistic roles in the progression of BC, and co-targeting of CXCR4 and αvß6 could be a potential strategy for the prevention and treatment of BC.

Significance of Vesicle-Associated Membrane Protein 8 Expression in Predicting Survival in Breast Cancer.

PURPOSE: Vesicle-associated membrane protein 8 (VAMP8) is a soluble N-ethylmaleimide-sensitive factor receptor protein that participates in autophagy by directly regulating autophagosome membrane fusion and has been reported to be involved in tumor progression. Nevertheless, the expression and prognostic value of VAMP8 in breast cancer (BC) remain unknown. This study aimed to evaluate the clinical significance and biological function of VAMP8 in BC. METHODS: A total of 112 BC samples and 30 normal mammary gland samples were collected. The expression of VAMP8 was assessed in both BC tissues and normal mammary gland tissues via a two-step immunohistochemical detection method. RESULTS: The expression of VAMP8 in BC tissues was significantly higher than that in normal breast tissues. Furthermore, increased VAMP8 expression was significantly correlated with tumor size (p=0.007), lymph node metastasis (p=0.024) and recurrence (p=0.001). Patients with high VAMP8 expression had significantly lower cumulative recurrence-free survival and overall survival (p<0.001 for both) than patients with low VAMP8 expression. In multivariate logistic regression and Cox regression analyses, lymph node metastasis and VAMP8 expression were independent prognostic factors for BC. CONCLUSION: VAMP8 is significantly upregulated in human BC tissues and can thus be a practical and potentially effective surrogate marker for survival in BC patients.