Pathogenesis Of Triple-negative Breast Cancer Research Articles

A Prognostic Risk Signature of Two Autophagy-Related Genes for Predicting Triple-Negative Breast Cancer Outcomes.

Triple-negative breast cancer (TNBC) is recognized as the most aggressive molecular subtype of breast cancer. Recent studies have highlighted the complex role of autophagy in the pathogenesis of TNBC. In this study, we evaluated 18,330 genes, including 1111 autophagy-related genes, (ARGs), across 579 TNBC samples from online databases. Differentially expressed ARGs in TNBC were identified using high-throughput RNA-seq data from the Cancer Genome Atlas (TCGA). Prognostic factors were examined through Cox regression and multivariate Cox analyses, with predictive efficacy assessed using receiver operating characteristic (ROC) curves. A nomogram integrating the risk signature with clinicopathological factors, such as TNM stage, was developed. Immunohistochemical analysis of clinical samples was also conducted. EIF4EBP1 and NPAS3 were significantly correlated with prognostic outcomes in patients with TNBC. Multivariate Cox regression analysis demonstrated that the expression levels of these two genes were accurate predictors of disease progression in TNBC samples from TCGA and the GSE31519 dataset. The efficacy of this predictive model was validated using ROC curve analysis and calibration plots, confirming its ability to accurately estimate the 1-, 2-, and 3-year survival rates for individuals with TNBC. Additionally, EIF4EBP1 and NPAS3 expression influenced drug sensitivity in TNBC cell lines, with notably lower NPAS3 expression in TNBC tissues, particularly in Stage III cases. This study is the first to report NPAS3 expression in patients with TNBC. The autophagy-related genes EIF4EBP1 and NPAS3 may serve as independent prognostic factors for individuals with TNBC.

Transcriptional regulation of DLGAP5 by AR suppresses p53 signaling and inhibits CD8+T cell infiltration in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a challenging subtype with unclear biological mechanisms. Recently, the transcription factor androgen receptor (AR) and its regulation of the DLGAP5 gene have gained attention in TNBC pathogenesis. In this study, we found a positive correlation between high AR expression and TNBC cell proliferation and growth. Furthermore, we confirmed DLGAP5 as a critical downstream regulator of AR with high expression in TNBC tissues. Knockdown of DLGAP5 significantly inhibited TNBC cell proliferation, migration, and invasion. AR was observed to directly bind to the DLGAP5 promoter, enhancing its transcriptional activity and suppressing the activation of the p53 signaling pathway. In vivo experiments further validated that downregulation of AR or DLGAP5 inhibited tumor growth and enhanced CD8+T cell infiltration. This study highlights the crucial roles of AR and DLGAP5 in TNBC growth and immune cell infiltration. Taken together, AR inhibits the p53 signaling pathway by promoting DLGAP5 expression, thereby impacting CD8+T cell infiltration in TNBC.

Restoration of TFPI2 by LSD1 inhibition suppresses tumor progression and potentiates antitumor immunity in breast cancer

Histone lysine-specific demethylase 1 (LSD1) is frequently overexpressed in triple negative breast cancer (TNBC), which is associated with worse clinical outcome in TNBC patients. However, the underlying mechanisms by which LSD1 promotes TNBC progression remain to be identified. We recently established a genetically engineered murine model by crossing mammary gland conditional LSD1 knockout mice with Brca1-deficient mice to explore the role of LSD1 in TNBC pathogenesis. Cre-mediated Brca1 loss led to higher incidence of tumor formation in mouse mammary glands, which was hindered by concurrent depletion of LSD1, indicating a critical role of LSD1 in promoting Brca1-deficient tumors. We also demonstrated that the silencing of a tumor suppressor gene, Tissue Factor Pathway Inhibitor 2 (TFPI2), is functionally associated with LSD1-mediated TNBC progression. Mouse Brca1-deficient tumors exhibited elevated LSD1 expression and decreased TFPI2 level compared to normal mammary tissues. Analysis of TCGA database revealed that TFPI2 expression is significantly lower in aggressive ER-negative or basal-like BC. Restoration of TFPI2 through LSD1 inhibition increased H3K4me2 enrichment at the TFPI2 promoter, suppressed tumor progression, and enhanced antitumor efficacy of chemotherapeutic agent. Induction of TFPI2 by LSD1 ablation downregulates activity of matrix metalloproteinases (MMPs) that in turn increases the level of cytotoxic T lymphocyte attracting chemokines in tumor environment, leading to enhanced tumor infiltration of CD8+ T cells. Moreover, induction of TFPI2 potentiates antitumor effect of LSD1 inhibitor and immune checkpoint blockade in poorly immunogenic TNBC. Together, our study identifies previously unrecognized roles of TFPI2 in LSD1-mediated TNBC progression, therapeutic response, and immunogenic effects.

Impact of microRNA variants on PI3K/AKT signaling in triple-negative breast cancer: comprehensive review.

Breast cancer (BC) is a significant cause of cancer-related mortality, and triple-negative breast cancer (TNBC) is a particularly aggressive subtype associated with high mortality rates, especially among younger females. TNBC poses a considerable clinical challenge due to its aggressive tumor behavior and limited therapeutic options. Aberrations within the PI3K/AKT pathway are prevalent in TNBC and correlate with increased therapeutic intervention resistance and poor outcomes. MicroRNAs (miRs) have emerged as crucial PI3K/AKT pathway regulators influencing various cellular processes involved in TNBC pathogenesis. The levels of miRs, including miR-193, miR-4649-5p, and miR-449a, undergo notable changes in TNBC tumor tissues, emphasizing their significance in cancer biology. This review explored the intricate interplay between miR variants and PI3K/AKT signaling in TNBC. The review focused on the molecular mechanisms underlying miR-mediated dysregulation of this pathway and highlighted specific miRs and their targets. In addition, we explore the clinical implications of miR dysregulation in TNBC, particularly its correlation with TNBC prognosis and therapeutic resistance. Elucidating the roles of miRs in modulating the PI3K/AKT signaling pathway will enhance our understanding of TNBC biology and unveil potential therapeutic targets. This comprehensive review aims to discuss current knowledge and open promising avenues for future research, ultimately facilitating the development of precise and effective treatments for patients with TNBC.

Long noncoding RNA profiling unveils LINC00960 as unfavorable prognostic biomarker promoting triple negative breast cancer progression

Long noncoding RNAs (lncRNAs) play a critical role in breast cancer pathogenesis, including Triple-Negative Breast Cancer (TNBC) subtype. Identifying the lncRNA expression patterns across different breast cancer subtypes could provide valuable insights into their potential utilization as disease biomarkers and therapeutic targets. In this study, we profiled lncRNA expression in 96 breast cancer cases, revealing significant differences compared to normal breast tissue. Variations across breast cancer subtypes, including Hormone Receptor-positive (HR + ), HER2-positive (HER2 + ), HER2 + HR + , and TNBC, as well as in relation to tumor grade and patients’ age at diagnosis were observed. TNBC and HER2+ subtypes showed distinct clustering, while HER2 + HR+ tumors clustered closer to HR+ tumors based on their lncRNA profiles. Our data identified numerous enriched lncRNAs in TNBC, notably the elevated expression of LINC00960, which was subsequently validated in two additional datasets. Analysis of LINC00960 expression in an independent TNBC cohort (n = 360) revealed elevated expression of LINC00960 to correlate with cell movement, invasion, proliferation, and migration functional categories. Depletion of LINC00960 significantly reduced TNBC cell viability, colony formation, migration, and three-dimensional growth, while increasing cell death. Mechanistically, transcriptomic profiling of LINC00960-depleted cells confirmed its tumor-promoting role, likely through sponging of hsa-miR-34a-5p, hsa-miR-16-5p, and hsa-miR-183-5p, leading to the upregulation of cancer-promoting genes including BMI1, KRAS, and AKT3. Our findings highlight the distinct lncRNA expression patterns in breast cancer subtypes and underscore the crucial role for LINC00960 in promoting TNBC pathogenesis, suggesting its potential utilization as a prognostic marker and therapeutic target.

Abstract PR011: Inhibiting eIF4E phosphorylation sensitizes triple-negative breast cancer to CDK4/6 inhibition

Abstract Despite significant progress in breast cancer treatment, Triple-Negative Breast Cancer (TNBC) presents a unique challenge due to the absence of three conventional drug targets found in other breast cancer subtypes, highlighting an urgent need for novel therapeutic strategies. A critical element of TNBC pathogenesis is the dysregulation of mRNA translation, a critical process required by rapidly dividing cancer cells to synthesize proteins as building blocks. This hallmark positions mRNA translation as a promising target for therapeutic intervention. At the forefront of regulating mRNA translation, is the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E). While its role in general mRNA translation initiation is crucial, eIF4E phosphorylation by the MNK1/2 [MAPK (Mitogen-Activated Protein Kinase)-Interacting Kinase1/2] has been implicated in tumorigenesis. Specifically, phosphorylated eIF4E (p-4E) selectively enhances the translation of mRNAs encoding proteins critical for cancer cell survival and metastasis, such as myeloid cell leukemia-1 (MCL1) and matrix metalloproteinase-3 (MMP3). Yet, inhibiting eIF4E phosphorylation markedly impairs TNBC metastasis without affecting the primary tumor’s growth rates in vivo, emphasizing its specific role in metastatic progression. To dissect the mechanism of p-4E in TNBC, we performed an in vitro shRNA synthetic lethal screen in TNBC cells to identify genes whose knockdown synergizes with p-4E inhibition to limit cell growth. A small-molecule inhibitor of MNK1/2, eFT508, was utilized to mitigate p-4E level during the screen. This screen uncovered that knockdown of cyclin-dependent kinase 4 (CDK4), known primarily for its role in cell cycle regulation, synergizes with p-4E inhibition. Through validation using various cell proliferation assays and three different FDA-approved CDK4/6 inhibitors (palbociclib, ribociclib, and abemaciclib), we demonstrated a strong synergy between CDK4/6 inhibition and eFT508 in reducing cell growth. The synergy between CDK4/6 and p-4E inhibition not only impaired TNBC cell proliferation, but also significantly reduced eIF4E activity and mRNA translation. Additionally, our findings reveal a novel role for CDK4/6 inhibitors in modulating mRNA translation via mTORC1 signaling pathways, extending beyond their canonical role in cell cycle regulation. This discovery suggests a potential combinatorial therapeutic approach for TNBC treatment involving MNK1/2 and CDK4/6 inhibitors. Such a strategy could exploit the vulnerabilities in TNBC’s dysregulated mRNA translation machinery, offering a promising direction in treating this aggressive breast cancer subtype. Citation Format: Qiyun Deng, Mehdi Amiri, Anastasija Ana Piric, Yasaman Bagherian, Zilan Li, Sidong Huang, Michael Pollak, Nahum Sonenberg. Inhibiting eIF4E phosphorylation sensitizes triple-negative breast cancer to CDK4/6 inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR011.

In silico analysis of the wild-type and mutant-type of BRCA2 gene

BackgroundThe aim of this study was to conduct an in silico analysis of a novel compound heterozygous variant in breast cancer susceptibility gene 2 (BRCA2) to clarify its structure–function relationship and elucidate the molecular mechanisms underlying triple-negative breast cancer (TNBC).MethodsA tumor biopsy sample was obtained from a 42-year-old Chinese woman during surgery, and a maxBRCA™ test was conducted using the patient’s whole blood. We obtained an experimentally determined 3D structure (1mje.pdb) of the BRCA2 protein from the Protein Data Bank (PDB) as a relatively reliable reference. Subsequently, the wild-type and mutant structures were predicted using SWISS-MODEL and AlphaFold, and the accuracy of these predictions was assessed through the SAVES online server. Furthermore, we utilized a high ambiguity-driven protein–protein docking (HADDOCK) algorithm and protein–ligand interaction profiler (PLIP) to predict the pathogenicity of the mutations and elucidate pathogenic mechanisms that potentially underlies TNBC.ResultsHistological examination revealed that the tumor biopsy sample exhibited classical pathological characteristics of TNBC. Furthermore, the maxBRCA™ test revealed two compound heterozygous BRCA2 gene mutations (c.7670 C > T.pA2557V and c.8356G > A.pA2786T). Through performing in silico structural analyses and constructing of 3D models of the mutants, we established that the mutant amino acids valine and threonine were located in the helical domain and oligonucleotide binding 1 (OB1), regions that interact with DSS1.ConclusionOur analysis revealed that substituting valine and threonine in the helical domain region alters the structure and function of BRCA2 proteins. This mutation potentially affects the binding of proteins and DNA fragments and disrupts interactions between the helical domain region and OB1 with DSS1, potentially leading to the development of TNBC. Our findings suggest that the identified compound heterozygous mutation contributes to the clinical presentation of TNBC, providing new insights into the pathogenesis of TNBC and the influence of compound heterozygous mutations in BRCA2.

The Transformative Role of 3D Culture Models in Triple-Negative Breast Cancer Research.

Advancements in cell culturing techniques have allowed the development of three-dimensional (3D) cell culture models sourced directly from patients' tissues and tumors, faithfully replicating the native tissue environment. These models provide a more clinically relevant platform for studying disease progression and treatment responses compared to traditional two-dimensional (2D) models. Patient-derived organoids (PDOs) and patient-derived xenograft organoids (PDXOs) emerge as innovative 3D cancer models capable of accurately mimicking the tumor's unique features, enhancing our understanding of tumor complexities, and predicting clinical outcomes. Triple-negative breast cancer (TNBC) poses significant clinical challenges due to its aggressive nature, propensity for early metastasis, and limited treatment options. TNBC PDOs and PDXOs have significantly contributed to the comprehension of TNBC, providing novel insights into its underlying mechanism and identifying potential therapeutic targets. This review explores the transformative role of various 3D cancer models in elucidating TNBC pathogenesis and guiding novel therapeutic strategies. It also provides an overview of diverse 3D cell culture models, derived from cell lines and tumors, highlighting their advantages and culturing challenges. Finally, it delves into live-cell imaging techniques, endpoint assays, and alternative cell culture media and methodologies, such as scaffold-free and scaffold-based systems, essential for advancing 3D cancer model research and development.

PYCR3 modulates mtDNA copy number to drive proliferation and doxorubicin resistance in triple-negative breast cancer

Triple-negative breast cancer (TNBC) poses significant challenges in treatment due to its aggressive nature and limited therapeutic targets. Understanding the underlying molecular mechanisms driving TNBC progression and chemotherapy resistance is imperative for developing effective therapeutic strategies. Thus, in this study, we aimed to elucidate the role of pyrroline-5-carboxylate reductase 3 (PYCR3) in TNBC pathogenesis and therapeutic response. We observed that PYCR3 is significantly upregulated in TNBC specimens compared to normal breast tissues, correlating with a poorer prognosis in TNBC patients. Knockdown of PYCR3 not only suppresses TNBC cell proliferation but also reverses acquired resistance of TNBC cells to doxorubicin, a commonly used chemotherapeutic agent. Mechanistically, we identified the mitochondrial localization of PYCR3 in TNBC cells and demonstrated its impact on TNBC cell proliferation and sensitivity to doxorubicin through the regulation of mtDNA copy number and mitochondrial respiration. Importantly, Selective reduction of mtDNA copy number using the mtDNA replication inhibitor 2′, 3′-dideoxycytidine effectively recapitulates the phenotypic effects observed in PYCR3 knockout, resulting in decreased TNBC cell proliferation and the reversal of doxorubicin resistance through apoptosis induction. Thus, our study underscores the clinical relevance of PYCR3 and highlight its potential as a therapeutic target in TNBC management. By elucidating the functional significance of PYCR3 in TNBC, our findings contribute to a deeper understanding of TNBC biology and provide a foundation for developing novel therapeutic strategies aimed at improving patient outcomes.

Abstract 6990: RHBDF1 is a potential modulator of EGFR activation and oncogenic biomarker in triple-negative breast cancer

Abstract Recently, our proteomics study on breast cancer (BC) reported a possible association of RHBDF1 with triple-negative breast cancer (TNBC). The overall survival of TNBC patients on METABRIC data set implicated that the RHBDF1 high-expression group has a shorter life expectancy than the RHBDF1 low-expression group. Although many TNBC patients have high expression levels of RHBDF1 and poor prognosis, the mechanism of RHBDF1 in the pathogenesis of TNBC remained unclear. Rhomboid 5 homolog 1 (RHBDF1) is a catalytically inactive pseudoprotease on the Golgi and endoplasmic reticulum membrane. Despite its inactive status of peptidase, it regulates the activity of ADAM17 protease that cleaves the extracellular portion of EGFR ligands such as TGF alpha and EGF. Eventually, RHBDF1 and ADAM17 complex activate the EGFR signaling pathway, thereby increasing cell proliferation, migration, and tumorigenesis. First of all, we recruited the clinical data of the overall survival (OS) and disease-free survival (DFS) in the hospital of Seoul National University (SNUH cohort, n= 203) and compared IHC score 0 or 1 with survival rate. To investigate the underlying biological mechanism of RHBDF1 in TNBC, the effect of knockdown or overexpression of RHBDF1 was observed in TNBC cell lines (MDA-MB-468, HCC1143 and BT20) using siRNA (small interfering RNA) and lentiviral systems. Cell proliferation assay was performed to clarify whether RHBDF1 affects cell growth. Then, we estimated the ability of migration, invasion, and sphere formation to confirm the characteristics of cancer cells including cancer stemness and metastasis. SNUH cohort with TNBC patients showed that the RHBDF1 high-expression group has a poorer disease-free survival (DFS) than the low-expression group (p<0.05). In vitro assay, RHBDF1 knockdown inhibited proliferation in MDA-MB-468 and HCC1143. RHBDF1 downregulation also decreased the ability of migration and invasion. We found that RHBDF1 activates specific cell growth signaling pathways and increases cell mobility in TNBC. The sphere formation assay was performed to measure cancer stemness characteristics. As a result, RHBDF1 knockdown TNBC cell lines couldn’t form spheres relative to RHBDF1 overexpressed TNBC cell lines. In conclusion, we demonstrated that RHBDF1, a potential triple-negative breast cancer biomarker, is highly amplified in TNBC patients and TNBC cell lines. Its functions of accelerating cell proliferation, metastasis and cancer stemness were unveiled. But we need to study ultimately how RHBDF1 regulates ADAM17 activity at Golgi and endoplasmic reticulum membrane and which transcription factor activates RHBDF1 expression. Citation Format: Hayeon Kim, Soo Young Park, Da Sol Kim, Cheng Hyun Lee, Min Ji Song, Han Suk Ryu. RHBDF1 is a potential modulator of EGFR activation and oncogenic biomarker in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6990.

20(R)-Panaxatriol enhances METTL3-mediated m6A modification of STUB1 to inhibit autophagy and exert antitumor effects in Triple-Negative Breast Cancer cells

BackgroundAberrant activation of autophagy in triple-negative breast cancer (TNBC) has led researchers to investigate potential therapeutic strategies targeting this process. The regulation of autophagy is significantly influenced by METTL3. Our previous research has shown that the Panax ginseng-derived compound, 20(R)-panaxatriol (PT), has potential as an anti-tumor agent.However, it remains unclear whether PT can modulate autophagy through METTL3 to exert its anti-tumor effects. ObjectiveOur objective is to investigate whether PT can regulate autophagy in TNBC cells and elucidate the molecular mechanisms. Study DesignFor in vitro experiments, we employed SUM-159-PT and MDA-MB-231 cells. While in vivo experiments involved BALB/c nude mice and NOD/SCID mice. MethodsIn vitro, TNBC cells were treated with PT, and cell lines with varying expression levels of METTL3 were established. We assessed the impact on tumor cell activity and autophagy by analyzing autophagic flux, Western Blot (WB), and methylation levels. In vivo, subcutaneous transplantation models were established in BALB/c nude and NOD/SCID mice to observe the effect of PT on TNBC growth. HE staining and immunofluorescence were employed to analyze histopathological changes in tumor tissues. MeRIP-seq and dual-luciferase reporter gene assays were used to identify key downstream targets. Additionally, the silencing of STIP1 Homology And U-Box Containing Protein 1 (STUB1) explored PT's effects. The mechanism of PT's action on STUB1 via METTL3 was elucidated through mRNA stability assays, mRNA alternative splicing analysis, and nuclear-cytoplasmic mRNA separation. ResultsIn both in vivo and in vitro experiments, it was discovered that PT significantly upregulates the expression of METTL3, leading to autophagy inhibition and therapeutic effects in TNBC. Simultaneously, through MeRIP-seq analysis and dual-luciferase reporter gene assays, we have demonstrated that PT modulates STUB1 via METTL3, influencing autophagy in TNBC cells. Furthermore, intriguingly, PT extends the half-life of STUB1 mRNA by enhancing its methylation modification, thereby enhancing its stability. ConclusionIn summary, our research reveals that PT increases STUB1 m6A modification through a METTL3-mediated mechanism in TNBC cells, inhibiting autophagy and further accentuating its anti-tumor properties. Our study provides novel mechanistic insights into TNBC pathogenesis and potential drug targets for TNBC.

Characterization of MYBL1 Gene in Triple-Negative Breast Cancers and the Genes' Relationship to Alterations Identified at the Chromosome 8q Loci.

The MYBL1 gene is a strong transcriptional activator involved in events associated with cancer progression. Previous data show MYBL1 overexpressed in triple-negative breast cancer (TNBC). There are two parts to this study related to further characterizing the MYBL1 gene. We start by characterizing MYBL1 reference sequence variants and isoforms. The results of this study will help in future experiments in the event there is a need to characterize functional variants and isoforms of the gene. In part two, we identify and validate expression and gene-related alterations of MYBL1, VCIP1, MYC and BOP1 genes in TNBC cell lines and patient samples selected from the Breast Invasive Carcinoma TCGA 2015 dataset available at cBioPortal.org. The four genes are located at chromosomal regions 8q13.1 to 8q.24.3 loci, regions previously identified as demonstrating a high percentage of alterations in breast cancer. We identify alterations, including changes in expression, deletions, amplifications and fusions in MYBL1, VCPIP1, BOP1 and MYC genes in many of the same patients, suggesting the panel of genes is involved in coordinated activity in patients. We propose that MYBL1, VCPIP1, MYC and BOP1 collectively be considered as genes associated with the chromosome 8q loci that potentially play a role in TNBC pathogenesis.

Identification of Subtypes in Triple-negative Breast Cancer Based on Shared Genes Between Immunity and Cancer Stemness.

The correlation between triple-negative breast cancer (TNBC) and genes related to immunity and cancer stemness, particularly shared genes, remains unclear. This study aimed to investigate the correlation of immunity and cancer stemness with the molecular subtyping and survival rates in TNBC using bioinformatics approaches. Differential gene analysis was conducted to identify TNBC-associated differentially expressed genes (DEGs). Cancer stem cell (CSC)-related genes were obtained using weighted gene coexpression network analysis. Immune-related gene sets were retrieved from the literature. Venn analysis was performed to identify the shared DEGs between immunity and cancer stemness in TNBC. Cluster analysis and survival analysis based on the expression of these genes were conducted to identify TNBC subtypes with significant survival differences. A total of 5259 TNBC-associated DEGs, 2214 CSC-related genes, 1793 immune-related genes, and 44 shared DEGs between immunity and cancer stemness were obtained. Among them, 3 shared DEGs were closely associated with TNBC survival rates ( P <0.05). Cluster and survival analyses revealed that among 3 subtypes, cluster2 exhibited the best survival rate, and cluster3 showed the worst survival rate ( P <0.05). Dendritic cells were highly infiltrated in cluster2, while plasma cells and resting mast cells were highly infiltrated in cluster3 ( P <0.05). Genes shared by immunity and cancer stemness were capable of classifying TNBC samples. TNBC patients of different subtypes exhibited significant differences in immune profiles, genetic mutations, and drug sensitivity. These findings could provide new insights into the pathogenesis of TNBC, the immune microenvironment, and the selection of therapeutic targets for drug treatment.

Formononetin triggers ferroptosis in triple-negative breast cancer cells by regulating the mTORC1/SREBP1/SCD1 pathway.

Triple-negative breast cancer (TNBC) is the most malignant type of breast cancer, and its prognosis is still the worst. It is necessary to constantly explore the pathogenesis and effective therapeutic targets of TNBC. Formononetin is an active ingredient with anti-tumor effects that we screened earlier. The main purpose of this study is to elucidate mechanism of the inhibitory effect of Formononetin on TNBC. We conducted experiments through both in vivo and in vitro methodologies. The in vivo experiments utilized a nude mice xenotransplantation model, while the in vitro investigations employed two breast cancer cell lines, MDA-MB-231 and MDA-MB-468. Concurrently, ferroptosis associated proteins, lipid peroxide levels, and proteins related to the rapamycin complex 1 were analyzed in both experimental settings. In our study, Formononetin exhibits significant inhibitory effects on the proliferation of triple TNBC, both in vivo and in vitro. Moreover, it elicits an increase in lipid peroxide levels, downregulates the expression of ferroptosis-associated proteins GPX4 and xCT, and induces ferroptosis in breast cancer cells. Concurrently, Formononetin impedes the formation of the mammalian target of rapamycin complex 1 (mTORC1) and suppresses the expression of downstream Sterol regulatory element-binding protein 1(SREBP1). The utilization of breast cancer cells with SREBP1 overexpression or knockout demonstrates that Formononetin induces ferroptosis by modulating the mTORC1-SREBP1 signaling axis. In conclusion, this study provides evidence that Formononetin exerts an anti-proliferative effect on triple-negative breast cancer by inducing ferroptosis. Moreover, the mTORC1-SREBP1 signal axis is identified as the primary mechanism through which formononetin exerts its therapeutic effects. These findings suggest that formononetin holds promise as a potential targeted drug for clinical treatment of TNBC.

Identification of new microtubule small-molecule inhibitors and microtubule-associated genes against triple negative breast cancer.

Breast cancer represents the leading cancer type and leading cause of cancer-related death among women in the world. Triple-negative breast cancer (TNBC) is a subset of breast cancer with the poorest prognosis and still lacking of effective therapeutic options. We recently screened a natural product library and identified 3 new hit compounds with selective and prominent anti-TNBC activities on different subtype of TNBC cell lines. Interestingly, all of these 3 hit compounds belong to "cytoskeletal drugs" that target tubulin and microtubule function. Our data also showed that these hit compounds showed consistently effective on TNBC cells which are resistant to those currently used antimicrotubule agents such as Paclitaxel. RNA-Sequencing analyses revealed the anti-TNBC mechanisms of these hit compounds and identified a subset of new cellular factors commonly affected by hit compounds in different subtypes of TNBC cells. Among them, we demonstrated AHCYL1 and SPG21 as new microtubule-associated proteins, which were required for TNBC cell survival with clinical implication through tissue array analysis. Our studies provide new insights into the mechanisms of TNBC pathogenesis and offer promising therapeutic directions for this aggressive breast cancer.

Comparative genomic hybridisation and transcriptome microarray analysis in triple negative breast cancer: An INDIAN study.

Triple-negative breast cancer (TNBC), which accounts for approximately 15–20% of all breast cancers, defined as lack of expression of estrogen receptor, progesterone receptor and Her-2 neu receptors. TNBC has two subtypes basal like and non-basal like, the former characterised by aggressive biology with limited therapeutic options. This study explored molecular markers involved in pathogenesis of TNBC and investigated novel potential diagnostic and therapeutic targets by CGH array and transcriptome array. aCGH analysis in TNBC demonstrated genes amplified were 3888, number of pathway hits was 1554 and major pathways amplified was found to be WNT signalling pathway and Cadherin signalling pathway. Among all metastatic sites and remission, activation of WNT signalling pathway is commonly observed. TNBC exhibited 1486 copy number variations (CNVR) which is approximately 250 times higher than controls. More than 20 CNVR was observed in all chromosomes and more than 80 CNVR was observed in Chr 1 to Chr 4, Chr 7, Chr 11 and Chr X. Common CNVR associated with amplified regions in Chr 22, Chr 14, Chr 8 and Chr 2 was observed in TNBC and CNVR associated with Chr 22q11.22–23, Chr 6p21.32–33, Chr11q12.2, Chr14q32.22–23, Chr 8p11.22–23, was observed in metastatic disease. In transcriptome array analysis a total of 11,359 differentially expressed genes with fold change 2.0 were in observed in TNBC comprise of 7639 upregulated genes and 3720 downregulated genes. Further, with fold change 10, 1526 upregulated genes and 839 down regulated genes were identified. Panther pathway analysis identified the main pathways of upregulated genes were Wnt signalling pathway, Integrin signalling pathway and Cadherin signalling pathway. The main pathways of down regulated genes were Inflammation mediated by chemokine and cytokine signalling pathways. PPI network shows that COL12A1, COL6A3, FN1, MMP3, WNT5A were key upregulated genes and ITGB7, PTPRC, ITGA4, LCK and CD247 were key down regulated genes. Cytoscape analysis followed by multiple list comparator tool identified top 5 significant hub genes were FN1, MMP3, COLL11A1, COL12A1 and COL3A1. The significant pathway genes obtained by CGH array and transcriptome array when compared, exhibited 5 common genes COL4A1, FN1, COL6A3, COL5A2 and PCDH7. These genes were not overexpressed in Controls and therefore involved in pathogenesis of TNBC. Expressions of these genes were validated by studying protein expression by immunohistochemistry. FN1 and COL6A3 protein over expression predicted worse DFS in TNBC and can be considered as therapeutic targets at diagnosis to reduce the disease metastases. These findings provide new insights into the pathogenesis of TNBC and guide for selection of targets related to diagnosis, prognosis and prediction of treatment in TNBC.

A systems biology approach and in vitro experiment indicated Rapamycin targets key cancer and cell cycle-related genes and miRNAs in triple-negative breast cancer cells.

An anticancer drug known as Rapamycin acts by inhibiting the mammalian target of the Rapamycin pathway. This agent has recently been investigated for its potential therapeutic benefits in sensitizing drug-resistant breast cancer (BC) treatment. The molecular mechanism underlying these effects, however, is still a mystery. Using a systems biology method and in vitro experiment, this study sought to discover essential genes and microRNAs (miRNAs) targeted by Rapamycin in triple-negative BC (TNBC) cells to aid prospective new medications with less adverse effects in BC treatment. We developed the transcription factor-miRNA-gene and protein-protein interaction networks using the freely accessible microarray data sets. FANMOD and MCODE were utilized to identify critical regulatory motifs, clusters, and seeds. Then, functional enrichment analyses were conducted. Using topological analysis and motif detection, the most important genes and miRNAs were discovered. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the effect of Rapamycin on the expression of the selected genes and miRNAs to verify our findings. We performed flow cytometry to investigate Rapamycin's impact on cell cycle and apoptosis. Furthermore, wound healing and migration assays were done. Three downregulated (PTGS2, EGFR, VEGFA) and three upregulated (c-MYC, MAPK1, PIK3R1) genes were chosen as candidates for additional experimental verification. There were also three upregulated miRNAs (miR-92a, miR-16, miR-20a) and three downregulated miRNAs (miR-146a, miR-145, miR-27a) among the six selected miRNAs. The qRT-PCR findings in MDA-MB-231 cells indicated that c-MYC, MAPK1, PIK3R1, miR-92a, miR-16, and miR-20a expression levels were considerably elevated following Rapamycin treatment, whereas PTGS2, EGFR, VEGFA, miR-146a, and miR-145 expression levels were dramatically lowered (p < 0.05). These genes are engaged in cancer pathways, transcriptional dysregulation in cancer, and cell cycle, according to the top pathway enrichment findings. Migration and wound healing abilities of the cells declined after Rapamycin treatment, and the number of apoptotic cells increased. We demonstrated that Rapamycin suppresses cell migration and metastasis in the TNBC cell line. In addition, our data indicated that Rapamycin induces apoptosis in this cell line. The discovered vital genes and miRNAs affected by Rapamycin are anticipated to have crucial roles in the pathogenesis of TNBC and its therapeutic resistance.

Opioids and immune checkpoint inhibitors differentially regulate a common immune network in triple-negative breast cancer.

Opioids are the primary analgesics for cancer pain. Recent clinical evidence suggests opioids may counteract the effect of immune checkpoint inhibition (ICI) immunotherapy, but the mechanism for this interaction is unknown. The following experiments study how opioids and immunotherapy modulate a common RNA expression pathway in triple negative breast cancer (TNBC), a cancer subtype in which immunotherapy is increasingly used. This study identifies a mechanism by which opioids may decrease ICI efficacy, and compares ketamine, a non-opioid analgesic with emerging use in cancer pain, for potential ICI interaction. Tumor RNA expression and clinicopathologic data from a large cohort with TNBC (N=286) was used to identify RNA expression signatures of disease. Various drug-induced RNA expression profiles were extracted from multimodal RNA expression datasets and analyzed to estimate the RNA expression effects of ICI, opioids, and ketamine on TNBC. We identified a RNA expression network in CD8+ T-cells that was relevant to TNBC pathogenesis and prognosis. Both opioids and anti-PD-L1 ICI regulated RNA expression in this network, suggesting a nexus for opioid-ICI interaction. Morphine and anti-PD-L1 therapy regulated RNA expression in opposing directions. By contrast, there was little overlap between the effect of ketamine and anti-PD-L1 therapy on RNA expression. Opioids and ICI may target a common immune network in TNBC and regulate gene expression in opposing fashion. No available evidence supports a similar interaction between ketamine and ICI.

Natural compound So-2 suppresses triple-negative breast cancer through inducing ferroptosis via downregulating transcription factor E2F7

Triple-negative breast cancer (TNBC), accounting for about 15∼18% of all breast cancers, is notorious for its poor prognosis, high rate of relapse and short overall survival. Because of lacking effective therapeutic targets or drugs, treatment of TNBC in clinical encounters great obstacle. Siegesbeckiaorientalis L. have been used as a traditional Chinese medicine “Xi-Xian-Cao” for centuries with multiple medicinal benefits including cancerous treatment. We have reported the isolation of twenty-seven germacranolides including So-2 from the aerial parts of S. orientalis with potent cytotoxicity against breast cancer cells. The studyaims to verified the anti-TNBC function of the natural compound So-2 both in vitro and vivo and uncover the underlying mechanism. The results showed that So-2 caused cell cycle arrest and suppress TNBC cell proliferation and migration. Also, So-2 was first identified to be a bona fide ferroptosis inducer in TNBC cells. So-2 effectively suppressed tumor growth of TNBC by using an orthotopic transplantation tumor model. We also characterized the oncogenic role of the transcription factor E2F7 in TNBC. E2F7 was demonstrated to be involved in the ferroptosis-inducing and tumor suppression effect of So-2. Altogether, So-2 exhibits inhibitory effect on TNBC both in vitro and vivo by inducing TNBC ferroptosis via downregulating the expression of E2F7. These findings provide valuable insight into the pathogenesis of TNBC. The natural compound So-2, isolated from Chinese traditional medicine, might be a prospective drug candidate in TNBC therapy.

Update on Classic and Novel Approaches in Metastatic Triple-Negative Breast Cancer Treatment: A Comprehensive Review.

Triple-negative breast cancer (TNBC) accounts for almost 15% of all diagnosed breast cancers and often presents high rates of relapses and metastases, with generally poor prognosis despite multiple lines of treatment. Immunotherapy has radically changed the approach of clinicians towards TNBC in the last two to three years, even if targeted and specific therapeutic options are still missing; this unmet need is further justified by the extreme molecular and clinical heterogeneity of this subtype of breast cancer and by the weak response to both single-agent and combined therapies. In March 2023, the National Comprehensive Cancer Network (NCCN), the main association of cancer centers in the United States, released the last clinical practice guidelines, with an update on classic and novel approaches in the field of breast cancer. The purpose of this comprehensive review is to summarize the latest findings in the setting of metastatic TNBC treatment, focusing on each category of drugs approved by the Food and Drug Administration (FDA) and included in the NCCN guidelines. We also introduce part of the latest published studies, which have reported new and promising molecules able to specifically target some of the biomarkers involved in TNBC pathogenesis. We searched the PubMed and Scopus databases for free full texts reported in the literature of the last 5 years, using the words "triple-negative breast cancer" or "TNBC" or "basal-like". The articles were analyzed by the authors independently and double-blindly, and a total of 114 articles were included in the review.