Analysis of the function, mechanism and clinical application prospect of TRPS1, a new marker for breast cancer.

It has been discovered that Trichorhinophalangeal Syndrome-1 (TRPS1), a novel member of the GATA transcription factor family, participates in both normal physiological processes and the development of numerous diseases. Recently, TRPS1 has been identified as a new biomarker to aid in cancer diagnosis and is very common in breast cancer (BC), especially in triple-negative breast cancer (TNBC). In this review, we discussed the structure and function of TRPS1 in various normal cells, focused on its role in tumorigenesis and tumor development, and summarize the research status of TRPS1 in the occurrence and development of BC. We also analyzed the potential use of TRPS1 in guiding clinically personalized precision treatment and the development of targeted drugs.

The Antimicrobial Peptide Merecidin Inhibit the Metastasis of Triple-Negative Breast Cancer by Obstructing EMT via miR-30d-5p/Vimentin.

Purpose: To investigate the inhibitory effect of antimicrobial peptide merecidin on triple-negative breast cancer (TNBC) and the mechanism of inhibiting epithelial-mesenchymal transformation (EMT) by regulating miR-30d-5p/vimentin. Methods: TNBC cell lines (MDA-MB-231, MDA-MB-468) were treated with merecidin to assess proliferation, migration, invasion ability, and EMT. Confocal laser localization was used to examine the role of merecidin and TNBC cells. The relationship between merecidin and miR-30d-5p was determined through RT-qPCR and dual-luciferase reporter gene, and the relationship between merecidin and vimentin was verified through pulling down the experiment. The effects of miR-30d-5p on the migration and invasion ability of TNBC cells were confirmed through scratch and transwell experiments. Vimentin levels, tumor volume, shape, size, and weight were observed in the MDA-MB-231 subcutaneous tumor model in nude mice. Results: merecidin inhibited the proliferation, migration, invasion, and EMT of TNBC cells. merecidin was primarily located in the cytoplasm of TNBC cells, and the expression of miR-30d-5p was low in TNBC cells. merecidin significantly up-regulated the expression of miR-30d-5p. miR-30d-5p negatively regulated vimentin. merecidin could bind to vimentin in vitro. miR-30d-5p inhibited the migration and invasion ability of TNBC cells, while vimentin promoted their migration and invasion ability. Down-regulation of miR-30d-5p or overexpression of vimentin partially counteracted the inhibitory effects of merecidin on TNBC cell migration, invasion ability, and EMT. In nude mouse tumor models, merecidin significantly suppressed tumor growth. Conclusion: Merecidin effectively blocks the EMT process and inhibits the migration and invasion of TNBC cells by regulating miR-30d-5p/vimentin.

Integrated machine learning algorithms identify KIF15 as a potential prognostic biomarker and correlated with stemness in triple-negative breast cancer.

Cancer stem cells (CSCs) have the potential to self-renew and induce cancer, which may contribute to a poor prognosis by enabling metastasis, recurrence, and therapy resistance. Hence, this study was performed to identify the association between CSC-related genes and triple-negative breast cancer (TNBC) development. Stemness gene sets were downloaded from StemChecker. Based on the online databases, a consensus clustering algorithm was conducted for unsupervised classification of TNBC samples. The variations between subtypes were assessed with regard to prognosis, tumor immune microenvironment (TIME), and chemotherapeutic sensitivity. The stemness-related gene signature was established and random survival forest analysis was employed to identify the core gene for validation experiments and tumor sphere formation assays. 499 patients with TNBC were classified into three subgroups and the Cluster 1 had a better OS than others. After that, WGCNA study was performed to identify genes important for Cluster 1 subtype. Out of all 8 modules, the subtype of Cluster 1 and the yellow module with 103 genes demonstrated the largest positive association. After that, a four-gene stemness-related signature was established. Based on the yellow module, the 39 potential pivotal genes were subjected to the random forest survival analysis to find out the gene that was relatively important for OS. KIF15 was confirmed as the targeted gene by LASSO and random survival forest analyses. In vitro experiments, the downregulation of KIF15 promoted the stemness of TNBC cells. The expression levels of stem cell markers Nanog, SOX2, and OCT4 were found to be elevated in TNBC cell lines after KIF15 inhibition. A stemness-associated risk model was constructed to forecast the clinical outcomes of TNBC patients. The downregulation of KIF15 expression in a subpopulation of TNBC stem cells may promote stemness and possibly TNBC progression.

Tumour immune characterisation of primary triple-negative breast cancer using automated image quantification of immunohistochemistry-stained immune cells.

The tumour immune microenvironment (TIME) in breast cancer is acknowledged with an increasing role in treatment response and prognosis. With a growing number of immune markers analysed, digital image analysis may facilitate broader TIME understanding, even in single-plex IHC data. To facilitate analyses of the latter an open-source image analysis pipeline, Tissue microarray MArker Quantification (TMArQ), was developed and applied to single-plex stainings for p53, CD3, CD4, CD8, CD20, CD68, FOXP3, and PD-L1 (SP142 antibody) in a 218-patient triple negative breast cancer (TNBC) cohort with complementary pathology scorings, clinicopathological, whole genome sequencing, and RNA-sequencing data. TMArQ's cell counts for analysed immune markers were on par with results from alternative methods and consistent with both estimates from human pathology review, different quantifications and classifications derived from RNA-sequencing as well as known prognostic patterns of immune response in TNBC. The digital cell counts demonstrated how immune markers are coexpressed in the TIME when considering TNBC molecular subtypes and DNA repair deficiency, and how combination of immune status with DNA repair deficiency status can improve the prognostic stratification in chemotherapy treated patients. These results underscore the value and potential of integrating TIME and specific tumour intrinsic alterations/phenotypes for the molecular understanding of TNBC.

Combined knockdown of CD151 and MMP9 may inhibit the malignant biological behaviours of triple-negative breast cancer through the GSK-3ß/ß-catenin-related pathway.

Triple-negative breast cancer (TNBC) represents a significant health concern for women worldwide, and the overproduction of MMP9 and CD151 is associated with various cancers, influencing tumour growth and progression. This study aimed to investigate how CD151 and MMP9 affect TNBC cell migration, apoptosis, proliferation, and invasion. Immunohistochemical experiments revealed that CD151 and MMP9 were positively expressed in triple-negative breast cancer, and lymph node metastasis, the histological grade, and CD151 and MMP9 expression were found to be independent prognostic factors for the survival of patients with triple-negative breast cancer. Cytological experiments indicated that the knockdown of CD151 or MMP9 slowed triple-negative breast cancer cell growth, migration, and invasion and increased the apoptosis rate. Compared with CD151 knockdown, double MMP9 and CD151 knockdown further promoted cell death and inhibited TNBC cell proliferation, migration, and invasion. Moreover, ß-catenin and p-GSK-3ß were significantly downregulated. In summary, simultaneously silencing CD151 and MMP9 further suppressed the proliferation, migration and invasion of TNBC cells and promoted their apoptosis. One possible strategy for inducing this effect is to block the GSK-3ß/ß-catenin pathway.

USP1-mediated deubiquitination of KDM1A promotes the malignant progression of triple-negative breast cancer.

Previous research has indicated the highly expressed lysine-specific histone demethylase 1A (KDM1A) in several human malignancies, including triple-negative breast cancer (TNBC). However, its detailed mechanisms in TNBC development remain poorly understood. The mRNA levels of KDM1A and Yin Yang 1 (YY1) were determined by RT-qPCR analysis. Western blot was performed to measure KDM1A and ubiquitin-specific protease 1 (USP1) protein expression. Cell proliferation, apoptosis, invasion, migration and stemness were evaluated by MTT assay, EdU assay, flow cytometry, transwell invasion assay, wound-healing assay and sphere-formation assay, respectively. ChIP and dual-luciferase reporter assays were conducted to determine the relationship between YY1 and KDM1A. Xenograft tumor experiment and IHC were carried out to investigate the roles of USP1 and KDM1A in TNBC development in vivo. The highly expressed KDM1A was demonstrated in TNBC tissues and cells, and KDM1A knockdown significantly promoted cell apoptosis, and hampered cell proliferation, invasion, migration, and stemness in TNBC cells. USP1 could increase the stability of KDM1A via deubiquitination, and USP1 depletion restrained the progression of TNBC cells through decreasing KDM1A expression. Moreover, YY1 transcriptionally activated KDM1A expression by directly binding to its promoter in TNBC cells. Additionally, USP1 inhibition reduced KDM1A expression to suppress tumor growth in TNBC mice in vivo. In conclusion, YY1 upregulation increased KDM1A expression via transcriptional activation. USP1 stabilized KDM1A through deubiquitination to promote TNBC progression.

JARID2 activation by NFYA promotes stemness of triple-negative breast cancer cells through the PI3K/AKT pathway.

BACKGROUND: This study aimed to investigate the role of Jumonji AT Rich Interacting Domain 2 (JARID2) in regulating triple-negative breast cancer (TNBC) stemness and its mechanism. RESEARCH DESIGN AND METHODS: Bioinformatics analysis examined JARID2 expression, prognosis, and transcription factors. Quantitative polymerase chain reaction, western blot, and immunohistochemistry detected expression. Dual luciferase reporter gene and chromatin immunoprecipitation assays verified binding. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assay detected viability and proliferation. Sphere formation assay detected the sphere formation efficiency. Flow cytometry detected CD44+/CD24- -marked stem cells. A xenograft tumor model verified the effect of JARID2 in vivo. RESULTS: JARID2 and nuclear transcription factor Y subunit α (NFYA) were upregulated in TNBC tissues and positively correlated. Knockdown of JARID2 or NFYA inhibited cell stemness by inhibiting the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway. Enforced JARID2 expression rescued the suppressive effect of NFYA knockdown on the PI3K/AKT signaling pathway and cell stemness. Knockdown of JARID2 inhibited tumor growth and cell stemness in mice but was alleviated by concurrent overexpression of NFYA. CONCLUSIONS: NFYA promotes TNBC cell stemness by upregulating JARID2 expression and regulating the PI3K/AKT signaling pathway, suggesting JARID2 as a potential target for innovating drugs that target TNBC stem cells.

[Effects of normal mitochondrial transplantation on proliferation, apoptosis and stemness of triple-negative breast cancer cells].

Objectives: To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells. Methods: The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation. Results: The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group (P＜0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group (P=0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group (P=0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P＜0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group (P=0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group (P＜0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group (P=0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group (P＜0.001). Conclusion: Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.

Breast Cancer Stem Cells Upregulate IRF6 in Stromal Fibroblasts to Induce Stromagenesis.

The microenvironment of a cancer stem cell (CSC) niche is often found in coexistence with cancer-associated fibroblasts (CAFs). Here, we show the first in-depth analysis of the interaction between primary triple-negative breast cancer stem cells (BCSCs) with fibroblasts. Using 2D co-culture models with specific seeding ratios, we identified stromal fibroblast aggregation at the BCSC cluster periphery, and, on closer observation, the aggregated fibroblasts was found to encircle BCSC clusters in nematic organization. In addition, collagen type I and fibronectin accumulation were also found at the BCSC-stromal periphery. MACE-Seq analysis of BCSC-encapsulating fibroblasts displayed the transformation of stromal fibroblasts to CAFs and the upregulation of fibrosis regulating genes of which the Interferon Regulatory Factor 6 (IRF6) gene was identified. Loss of function experiments with the IRF6 gene decreased fibroblast encapsulation around BCSC clusters in 2D co-cultures. In BCSC xenografts, fibroblast IRF6 expression led to an increase in the stromal area and fibroblast density in tumors, in addition to a reduction in necrotic growth. Based on our findings, we propose that fibroblast IRF6 function is an important factor in the development of the stromal microenvironment and in sustaining the BCSC tumor niche.

Digital Whole Slide Image Analysis of Elevated Stromal Content and Extracellular Matrix Protein Expression Predicts Adverse Prognosis in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis and limited treatment options. This study evaluates the prognostic value of stromal markers in TNBC, focusing on the tumor-stroma ratio (TSR) and overall stroma ratio (OSR) in whole slide images (WSI), as well as the expression of type-I collagen, type-III collagen, and fibrillin-1 on tissue microarrays (TMAs), using both visual assessment and digital image analysis (DIA). A total of 101 female TNBC patients, primarily treated with surgery between 2005 and 2016, were included. We found that high visual OSR correlates with worse overall survival (OS), advanced pN categories, lower stromal tumor-infiltrating lymphocyte count (sTIL), lower mitotic index, and patient age (p < 0.05). TSR showed significant connections to the pN category and mitotic index (p < 0.01). High expression levels of type-I collagen (>45%), type-III collagen (>30%), and fibrillin-1 (>20%) were linked to significantly worse OS (p = 0.004, p = 0.013, and p = 0.005, respectively) and progression-free survival (PFS) (p = 0.028, p = 0.025, and p = 0.002, respectively), validated at the mRNA level. Our results highlight the importance of stromal characteristics in promoting tumor progression and metastasis and that targeting extracellular matrix (ECM) components may offer novel therapeutic strategies. Furthermore, DIA can be more accurate and objective in evaluating TSR, OSR, and immunodetected stromal markers than traditional visual examination.

Decreased Expression of CD247 and CD4 Immune Marker Predicts Poor Prognosis in Triple Negative Breast Cancer.

OBJECTIVE: Triple negative breast cancer (TNBC) is an aggressive from of breast cancer and is associated with poor prognosis. Tumor microenvironment of breast cancer consists of a wide   range of cell types, including tumor-infiltrating lymphocytes (TILs). Accumulating evidence indicate that TILs play a crucial role in cancer progression and resistance to standard chemotherapy. METHOD: We used online computational tools to evaluate the prognostic significance of CD247 and CD4 in TNBC. RESULTS: TNBC patients with lower expression of CD247 and CD4 have much shorter relapse- free survival and overall survival than the patients with higher expression of these genes. CD247 and CD4 expression show a strong positive correlation with tumor-infiltrating dendritic cells, B-cells, CD4+, CD8+, and neutrophils. CONCLUSION: We've concluded that low levels of CD247 and CD4 may stop immune cells from entering the area around the tumor, which stops cancer cells from being killed and gives the patient a bad outlook. These findings suggest that CD247 and CD4 may be useful biomarkers or as a target to understand the progression of TNBC. Our findings also suggest that CD247 and CD4 targeted therapeutics should be explored in detail, and could be a potentially used as atreatment strategy for TNBC.

Identification of TAT as a Biomarker Involved in Cell Cycle and DNA Repair in Breast Cancer.

Breast cancer (BC) is the most frequently diagnosed cancer and the primary cause of cancer-related mortality in women. Treatment of triple-negative breast cancer (TNBC) remains particularly challenging due to its resistance to chemotherapy and poor prognosis. Extensive research efforts in BC screening and therapy have improved clinical outcomes for BC patients. Therefore, identifying reliable biomarkers for TNBC is of great clinical importance. Here, we found that tyrosine aminotransferase (TAT) expression was significantly reduced in BC and strongly correlated with the poor prognosis of BC patients, which distinguished BC patients from normal individuals, indicating that TAT is a valuable biomarker for early BC diagnosis. Mechanistically, we uncovered that methylation of the TAT promoter was significantly increased by DNA methyltransferase 3 (DNMT3A/3B). In addition, reduced TAT contributes to DNA replication and cell cycle activation by regulating homologous recombination repair and mismatch repair to ensure genomic stability, which may be one of the reasons for TNBC resistance to chemotherapy. Furthermore, we demonstrated that Diazinon increases TAT expression as an inhibitor of DNMT3A/3B and inhibits the growth of BC by blocking downstream pathways. Taken together, we revealed that TAT is silenced by DNMT3A/3B in BC, especially in TNBC, which promotes the proliferation of tumor cells by supporting DNA replication, activating cell cycle, and enhancing DNA damage repair. These results provide fresh insights and a theoretical foundation for the clinical diagnosis and treatment of BC.

Comparison of Mitochondrial and Antineoplastic Effects of Amiodarone and Desethylamiodarone in MDA-MB-231 Cancer Line.

Previously, we have demonstrated that amiodarone (AM), a widely used antiarrhythmic drug, and its major metabolite desethylamiodarone (DEA) both affect several mitochondrial processes in isolated heart and liver mitochondria. Also, we have established DEA's antitumor properties in various cancer cell lines and in a rodent metastasis model. In the present study, we compared AM's and DEA's mitochondrial and antineoplastic effects in a human triple-negative breast cancer (TNBC) cell line. Both compounds reduced viability in monolayer and sphere cultures and the invasive growth of the MDA-MB-231 TNBC line by inducing apoptosis. They lowered mitochondrial trans-membrane potential, increased Ca2+ influx, induced mitochondrial permeability transition, and promoted mitochondrial fragmentation. In accordance with their mitochondrial effects, both substances massively decreased overall, and even to a greater extent, mitochondrial ATP production decreased, as determined using a Seahorse live cell respirometer. In all these effects, DEA was more effective than AM, indicating that DEA may have higher potential in the therapy of TNBC than its parent compound.

Discovery and Optimization of Ergosterol Peroxide Derivatives as Novel Glutaminase 1 Inhibitors for the Treatment of Triple-Negative Breast Cancer.

In this study, novel ergosterol peroxide (EP) derivatives were synthesized and evaluated to assess their antiproliferative activity against four human cancer cell lines (A549, HepG2, MCF-7, and MDA-MB-231). Compound 3g exhibited the most potent antiproliferative activity, with an IC50 value of 3.20 µM against MDA-MB-231. This value was 5.4-fold higher than that of the parental EP. Bioassay optimization further identified 3g as a novel glutaminase 1 (GLS1) inhibitor (IC50 = 3.77 µM). In MDA-MB-231 cells, 3g reduced the cellular glutamate levels by blocking the glutamine hydrolysis pathway, which triggered reactive oxygen species production and induced caspase-dependent apoptosis. Molecular docking indicated that 3g interacts with the reaction site of the variable binding pocket by forming multiple interactions with GLS1. In a mouse model of breast cancer, 3g showed remarkable therapeutic effects at a dose of 50 mg/kg, with no apparent toxicity. Based on these results, 3g could be further evaluated as a novel GLS1 inhibitor for triple-negative breast cancer (TNBC) therapy.

Impact of RBMS 3 Progression on Expression of EMT Markers.

Epithelial-to-mesenchymal transition (EMT) is a complex cellular process that allows cells to change their phenotype from epithelial to mesenchymal-like. Type 3 EMT occurs during cancer progression. The aim of this study was to investigate the role of RNA-binding motif single-stranded interacting protein 3 (RBMS 3) in the process of EMT. To investigate the impact of RBMS 3 on EMT, we performed immunohistochemical (IHC) reactions on archived paraffin blocks of invasive ductal breast carcinoma (n = 449), allowing us to analyze the correlation in expression between RBMS 3 and common markers of EMT. The IHC results confirmed the association of RBMS 3 with EMT markers. Furthermore, we performed an in vitro study using cellular models of triple negative and HER-2-enriched breast cancer with the overexpression and silencing of RBMS 3. RT-qPCR and Western blot methods were used to detect changes at both the mRNA and protein levels. An invasion assay and confocal microscopy were used to study the migratory potential of cells depending on the RBMS 3 expression. The studies conducted suggest that RBMS 3 may potentially act as an EMT-promoting agent in the most aggressive subtype of breast cancer, triple negative breast cancer (TNBC), but as an EMT suppressor in the HER-2-enriched subtype. The results of this study indicate the complex role of RBMS 3 in regulating the EMT process and present it as a future potential target for personalized therapies and a diagnostic marker in breast cancer.

CD8-Positive T-Cells Are Key Immune Cells for Predicting the Therapeutic Effect of Neoadjuvant Chemotherapy in Triple-negative Breast Cancer.

BACKGROUND/AIM: Patients with triple-negative breast cancer (TNBC) who obtain a pathological complete response (pCR) after neoadjuvant chemotherapy have an improved prognosis. Lymphocyte-predominant breast cancer is more likely to respond to neoadjuvant chemotherapy. Here, we investigated the correlation between tumor-infiltrating lymphocytes (TILs) in pre-treatment biopsy specimens from patients with TNBC in relation to response to NAC. PATIENTS AND METHODS: The level of infiltration by immune cells expressing immune cell lineage surface markers (CD8, CD4, CD19, CD14, CD11c, and CD11b) in biopsy specimens from 52 patients with TNBC was examined using multispectral immunofluorescent labelling. RESULTS: The level of CD8-positive TILs was significantly higher in patients with a pCR (p=0.045). The Cox proportional hazard model confirmed that lymph node involvement was associated with poorer disease-free survival (p=0.008). A high level of CD8-positive TILs was related to significantly prolonged disease-free survival in patients with node-positive TNBC (p=0.018). CONCLUSION: Assessing infiltration by CD8-positive TILs in the primary tumor is a useful biomarker to predict pCR and improved outcome in patients with node-positive TNBC.

Glutathione-Disrupting Nanotherapeutics Potentiate Ferroptosis for Treating Luminal Androgen Receptor-Positive Triple-Negative Breast Cancer.

The refractory luminal androgen receptor (LAR) subtype of triple-negative breast cancer (TNBC) patients is challenged by significant resistance to neoadjuvant chemotherapy and increased immunosuppression. Regarding the distinct upregulation of glutathione (GSH) and glutathione peroxidase 4 (GPX4) in LAR TNBC tumors, we herein designed a GSH-depleting phospholipid derivative (BPP) and propose a BPP-based nanotherapeutics of RSL-3 (GDNS), aiming to deplete intracellular GSH and repress GPX4 activity, thereby potentiating ferroptosis for treating LAR-subtype TNBC. GDNS treatment drastically downregulated the expression of GSH and GPX4, resulting in a 33.88-fold enhancement of lipid peroxidation and significant relief of immunosuppression in the 4T1 TNBC model. Moreover, GDNS and its combination with antibody against programed cell death protein 1 (antiPD-1) retarded tumor growth and produced 2.83-fold prolongation of survival in the LAR-positive TNBC model. Therefore, the GSH-disrupting GDNS represents an encouraging strategy to potentiate ferroptosis for treating refractory LAR-subtype TNBC.

Dual activity of Minnelide chemosensitize basal/triple negative breast cancer stem cells and reprograms immunosuppressive tumor microenvironment.

Triple negative breast cancer (TNBC) subtype is characterized with higher EMT/stemness properties and immune suppressive tumor microenvironment (TME). Women with advanced TNBC exhibit aggressive disease and have limited treatment options. Although immune suppressive TME is implicated in driving aggressive properties of basal/TNBC subtype and therapy resistance, effectively targeting it remains a challenge. Minnelide, a prodrug of triptolide currently being tested in clinical trials, has shown anti-tumorigenic activity in multiple malignancies via targeting super enhancers, Myc and anti-apoptotic pathways such as HSP70. Distinct super-enhancer landscape drives cancer stem cells (CSC) in TNBC subtype while inducing immune suppressive TME. We show that Minnelide selectively targets CSCs in human and murine TNBC cell lines compared to cell lines of luminal subtype by targeting Myc and HSP70. Minnelide in combination with cyclophosphamide significantly reduces the tumor growth and eliminates metastasis by reprogramming the tumor microenvironment and enhancing cytotoxic T cell infiltration in 4T1 tumor-bearing mice. Resection of residual tumors following the combination treatment leads to complete eradication of disseminated tumor cells as all mice are free of local and distant recurrences. All control mice showed recurrences within 3 weeks of post-resection while single Minnelide treatment delayed recurrence and one mouse was free of tumor. We provide evidence that Minnelide targets tumor intrinsic pathways and reprograms the immune suppressive microenvironment. Our studies also suggest that Minnelide in combination with cyclophosphamide may lead to durable responses in patients with basal/TNBC subtype warranting its clinical investigation.

A SRC-slug-TGFß2 signaling axis drives poor outcomes in triple-negative breast cancers.

BACKGROUND: Treatment options for the Triple-Negative Breast Cancer (TNBC) subtype remain limited and the outcome for patients with advanced TNBC is very poor. The standard of care is chemotherapy, but approximately 50% of tumors develop resistance. METHODS: We performed gene expression profiling of 58 TNBC tumor samples by microarray, comparing chemosensitive with chemoresistant tumors, which revealed that one of the top upregulated genes was TGFß2. A connectivity mapping bioinformatics analysis predicted that the SRC inhibitor Dasatinib was a potential pharmacological inhibitor of chemoresistant TNBCs. Claudin-low TNBC cell lines were selected to represent poor-outcome, chemoresistant TNBC, for in vitro experiments and in vivo models. RESULTS: In vitro, we identified a signaling axis linking SRC, AKT and ERK2, which in turn upregulated the stability of the transcription factors, Slug and Snail. Slug was shown to repress TGFß2-antisense 1 to promote TGFß2 signaling, upregulating cell survival via apoptosis and DNA-damage responses. Additionally, an orthotopic allograft in vivo model demonstrated that the SRC inhibitor Dasatinib reduced tumor growth as a single agent, and enhanced responses to the TNBC mainstay drug, Epirubicin. CONCLUSION: Targeting the SRC-Slug-TGFß2 axis may therefore lead to better treatment options and improve patient outcomes in this highly aggressive subpopulation of TNBCs.

In our study, we focused on a particular subtype of aggressive breast cancer called Triple-Negative Breast Cancer (TNBC). We investigated a complex series of events that contribute to poor outcomes in this disease and uncovered a crucial signaling cascade driving tumor growth and progression.At the core of this signaling cascade are three key proteins: SRC, AKT, and ERK2. Together, they form a pathway that activates a transcription factor called Slug. Transcription factors act like molecular switches, controlling the expression of genes. Once Slug is activated, it strongly suppresses genes that would normally restrict cell growth and cell spread.One of the genes downregulated by Slug is TGFB2-AS1. This product of the TGFB2-AS1 gene normally controls levels of its target protein called TGF-beta2 (TGFB2), a protein which has roles in cell growth, cell migration and differentiation. Slug downregulation of TGFB2-AS1 results in higher TGFB2 levels, and this in turn contributes to the uncontrolled growth and spread of cancer cells. TGFB2, and other proteins in this pathway (SRC, AKT, ERK2, and a Slug interactor called LSD1) all maintain the stability of Slug, meaning that Slug levels remain high and drive the aggressive features of this subtype of breast cancer.Overall, our research sheds light on the intricate molecular mechanisms driving aggressive TNBC. It also identifies potential targets for future therapies, aimed at disrupting this harmful signaling pathway and potentially improving patient outcomes for this disease.

Transient splicing inhibition causes persistent DNA damage and chemotherapy vulnerability in triple-negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive type of breast cancer. While most TNBCs are initially sensitive to chemotherapy, a substantial fraction acquires resistance to treatments and progresses to more advanced stages. Here, we identify the spliceosome U2 small nuclear ribonucleoprotein particle (snRNP) complex as a modulator of chemotherapy efficacy in TNBC. Transient U2 snRNP inhibition induces persistent DNA damage in TNBC cells and organoids, regardless of their homologous recombination proficiency. U2 snRNP inhibition pervasively deregulates genes involved in the DNA damage response (DDR), an effect relying on their genomic structure characterized by a high number of small exons. Furthermore, a pulse of splicing inhibition elicits long-lasting repression of DDR proteins and enhances the cytotoxic effect of platinum-based drugs and poly(ADP-ribose) polymerase inhibitors (PARPis) in multiple TNBC models. These findings identify the U2 snRNP as an actionable target that can be exploited to enhance chemotherapy efficacy in TNBCs.