MAL/SPP1 axis drives tumor proliferation and immune evasion via JAK2/STAT3 and MHC class Ib in breast cancer.

Triple-negative breast cancer (TNBC) is a particularly aggressive subtype of breast cancer, known for its high malignancy, elevated risk of recurrence and metastasis, and limited therapeutic options, resulting in the poorest prognosis among breast cancer types. This study explores the anticancer effects of VALD-3, a Schiff base ligand derivative, on breast cancer cells. While VALD-3 exhibited cytotoxic effects on both triple-negative breast cancer (TNBC) and estrogen receptor-positive (ER+) MCF-7 cells, it more potently inhibited TNBC cell viability. More importantly, VALD-3 induced characteristic pyroptotic features selectively in TNBC cells, including cell swelling, balloon-like protrusions, and the release of inflammatory cytokines due to pore formation in the plasma membrane, ultimately inhibiting tumor growth. Mechanistically, VALD-3 increased reactive oxygen species (ROS) levels and JNK phosphorylation, leading to the recruitment of Bax to the mitochondria and the formation of a Bax-Bcl-2 heterodimer, which facilitated cytochrome c release into the cytoplasm. This cascade activated caspase-3 and triggered gasdermin E(GSDME)- dependent pyroptosis in TNBC cells. Thus, VALD-3 treatment initiated the ROS/JNK/Bax-mitochondrial apoptosis pathway, leading to caspase-3 activation and GSDME cleavage, thereby executing pyroptosis. These findings suggest that GSDME-dependent pyroptosis is a novel mechanism by which VALD-3 eradicates cancer cells and offer new insights into potential clinical applications for anticancer therapies.

Ubiquitin carboxyl-terminal hydrolase L3 (UCHL3), a key deubiquitinating enzyme of the UCH family, has been implicated in DNA repair in breast cancer; however, its role in regulating cell migration and invasion in triple-negative breast cancer (TNBC) remains poorly understood. The expression pattern of UCHL3 across breast cancer subtypes was analyzed using TCGA data. In TNBC cells, UCHL3 mRNA levels were quantified by qRT-PCR, while its protein expression and epithelial-mesenchymal transition (EMT) markers (Vimentin, N-cadherin, SNAIL, E-cadherin, α-SMA, TWIST) were assessed by Western blot. Functional assays included: MTT and EdU incorporation assays for proliferation; wound healing and Transwell migration assays for migratory capacity. Transcriptomic changes induced by UCHL3 knockdown were profiled via RNA sequencing. Additionally, an in vivo mice model was used to evaluate the pro-invasive effects of UCHL3 overexpression in TNBC. UCHL3 was significantly overexpressed in TNBC and correlated with poor patient prognosis. Functional studies revealed that UCHL3 enhances the proliferative capacity of TNBC cells. Furthermore, UCHL3 was demonstrated to promote TNBC cell migration and invasion through modulation of epithelial-mesenchymal transition (EMT) markers. Mechanistically, knockdown of UCHL3 attenuated activation of invasion- and migration-related signaling pathways in TNBC cells. Pharmacological inhibition of UCHL3 effectively suppressed TNBC cell migration and invasion in vitro. In vivo studies further confirmed that UCHL3 overexpression drives metastatic progression of TNBC in mice. UCHL3 is significantly upregulated in TNBC and drives tumor progression by promoting cell proliferation and activating EMT-mediated migration and invasion. These findings identify UCHL3 as a potential therapeutic target for TNBC treatment.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and currently lacks defined therapeutic targets. Although miR-152-3p functions as a tumor suppressor in various cancers, its specific mechanism and regulatory network in TNBC remain poorly understood. To investigate the expression and tumor-suppressive function of miR-152-3p in TNBC cells and to elucidate its mechanism of targeting STAT3, RELA and ADCY6. miR-152-3p expression was compared between MDA-MB-231 and MCF-10A cells using qRT-PCR. MDA-MB-231 cells were transfected with miR-152-3p mimics or inhibitors and cell proliferation, apoptosis and invasion were assessed by MTT assay, flow cytometry and Transwell assay, respectively. Direct target interactions were validated by a dual-luciferase reporter assay, and protein levels of STAT3, RELA, and ADCY6 were examined by Western blot. Key findings were further validated in Hs 578T cells. miR-152-3p expression was significantly downregulated in TNBC cells. Overexpression of miR-152-3p markedly inhibited proliferation and invasion while promoting apoptosis in MDA-MB-231 cells. Dual-luciferase reporter assays confirmed that miR-152-3p directly binds to the 3'untranslated regions of STAT3, RELA and ADCY6. Overexpression of miR-152-3p significantly reduced STAT3 and RELA protein levels while upregulating ADCY6 expression. Rescue experiments demonstrated that restoration of STAT3 expression partially reversed the tumor-suppressive effects of miR-152-3p. These findings were recapitulated in Hs 578T cells, suggesting generalizability across TNBC subtypes. miR-152-3p suppresses TNBC progression by downregulating STAT3/RELA and upregulating ADCY6, thereby activating cAMP signaling. These findings provide a foundation for further investigation into the potential of miR-152-3p as a multi-target therapeutic strategy for TNBC.

Silver-selenium hybrid nanocomposite with combined cytotoxic and metabolic reprogramming effects in triple-negative breast cancer.

The processes regulating cellular dormancy in tumor cells remain inadequately characterized. Further, there are very few models available that can recapitulate a dormancy-like phenotype invitro. In this study, we investigated the role of microenvironmental cues in inducing a dormancy-like phenotype in estrogen receptor-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cell lines. We characterized an invitro model in distinct conditions defined hierarchically by the following: (a) normoxia or true hypoxia, (b) dishes coated with fibronectin or laminin or none, and (c) presence or absence of 10 ng/mL basic fibroblast growth factor (FGF-2) in the culture medium, added on day 0 of culture. Cells were cultured at clonogenic densities for 9 days (day -1, day 0, up to day +7). Cells were characterized for dormancy- like behavior using increased p-p38 and decreased p-ERK expression (dormant, p-p38High and p-ERKLow expression), reduced Ki67 expression, elevated p21 and p27 levels by immunofluorescence, absence of senescence using β-galactosidase staining and resistance to doxorubicin. Fibronectin or laminin were sufficient for the induction of a dormancy-like phenotype in MCF-7 cells under hypoxic conditions, whereas the addition of FGF-2 to fibronectin (but not laminin) could induce a dormancy-like phenotype under normoxic conditions. FGF-2 was required with laminin to induce dormancy in MDA-MB-231 cells under hypoxic conditions (FGF-2 plus fibronectin were unable to induce dormancy), whereas laminin or fibronectin alone were sufficient under normoxic conditions. Overall, we established culture conditions that recapitulate an invitro breast cancer dormancy-like phenotype and reveal subtype-specific differences in dormancy regulation.

NOS2 and COX2 impact the spatial landscape of CD8+ T cells in ER-breast cancer, providing novel mechanistic insight that drives tumor progression and poor survival.

The identification of novel therapeutic targets and agents to overcome chemoresistance remains a central challenge in triple-negative breast cancer (TNBC). Here, we report three key innovations: the discovery of a novel oncogenic circRNA, circPARPBP, as a driver of chemoresistance; the elucidation of its mechanism through the recruitment of the SRCAP complex to activate CCL20 transcription and cancer stemness; and the demonstration that the natural compound isoliquiritigenin (ISL) effectively suppresses this axis to overcome chemoresistance. TNBC is the most aggressive subtype of breast cancer with poor prognosis and limited treatment options. In this study, we identified circPARPBP, a circRNA (hsa_circ_0000432) derived from PARPBP gene, which was aberrantly upregulated in TNBC tissues and cells. Clinically, the upregulation of circPARPBP was notably associated with TNBC chemoresistance. In vitro and in vivo experiments demonstrated that circPARPBP promoted TNBC progression and chemoresistance. Mechanistically, circPARPBP recruited the SNF2-related CBP activator protein (SRCAP) complex to activate C-C motif chemokine ligand 20 (CCL20) transcription and thus contributed to cancer stemness and chemoresistance. ISL, a key bioactive substance extracted from licorice root, effectively suppressed the circPARPBP-SRCAP-CCL20 signaling pathway. Notably, patient-derived xenograft models demonstrated that ISL treatment effectively overcame TNBC chemoresistance, with a superior benefit when in combination with conventional chemotherapy. Collectively, our study identifies circPARPBP as a novel regulator of TNBC progression and chemoresistance via SRCAP-mediated CCL20 activation, and provides a preclinical rationale for the clinical development of ISL as a potential therapy targeting this axis.

PMEPA1 promotes mTOR inhibitor resistance in triple-negative breast cancer: Targeting the TGF-β/PMEPA1 axis as a therapeutic strategy to overcome resistance.

Dual-targeting of mitochondrial and c-Myc G-quadruplexes by a photoactive coumarin-benzothiazolium conjugate inhibits triple-negative breast cancer cell growth.

Urea-based lysophosphatidic acid receptor 1 antagonists as potential migrastatics for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype lacking targeted therapies due to the absence of hormone receptors and HER2 expression, resulting in poor clinical outcomes and limited treatment options. Identifying novel vulnerabilities is therefore critical to advancing TNBC therapeutics. Mitochondrial metabolism has emerged as a key regulator of cancer cell survival and proliferation, with serine hydroxymethyltransferase 2 (SHMT2) playing a central role in mitochondrial one-carbon metabolism by supplying one-carbon units for nucleotide biosynthesis and maintaining redox homeostasis. Despite its established importance in cancer metabolism, the functional role and therapeutic potential of SHMT2 in TNBC remain underexplored. Here, we demonstrate that gambogic acid (GA), a natural product with reported anticancer properties, exerts potent and selective cytotoxicity against TNBC cells by covalently targeting SHMT2. GA binds specifically to the critical cysteine residue Cys241, inhibiting SHMT2 enzymatic activity and disrupting mitochondrial function. This leads to bioenergetic collapse, activation of the Nrf2/HO-1 axis, iron overload, and induction of ferroptosis, a non-apoptotic form of cell death increasingly recognized for its therapeutic potential. Our integrative chemoproteomic and mechanistic studies reveal a novel SHMT2-mitochondria-Nrf2/HO-1-ferroptosis axis driving GA's anti-TNBC activity. Moreover, SHMT2 overexpression in TNBC correlates with tumor aggressiveness and poor prognosis, underscoring its role as a metabolic oncogene and promising drug target. These findings establish GA as a novel covalent SHMT2 inhibitor and provide a new framework for exploiting metabolic vulnerabilities to overcome TNBC treatment resistance.

Oxytocin is a nine-amino-acid peptide hormone renowned for its roles in reproduction and childbirth. Beyond these classical functions, it has attracted increasing research interest for its broader biological activities. Additionally, clinical studies have proposed oxytocin as a potential therapeutic agent for breast cancer, particularly triple-negative breast cancer, due to its ability to modulate cell proliferation and migration. Despite its wide recognition across various biological systems, the full scope of oxytocin's activity remains incompletely understood. Early studies have shown that metal ions can affect oxytocin's function. Building on this, our group previously demonstrated that oxytocin's redox state, regulated by its two cysteine residues, also modulates oxytocin-dependent signaling through Cu(II) and Zn(II) binding in HEK293T cells expressing the oxytocin receptor. In this study, we investigated the effects of metal-oxytocin preparations on triple-negative breast cancer cells (MDA-MB-231), focusing on cell migration and invasion. Our findings show that samples containing both oxytocin and copper (CuOT) differentially influence cellular behavior in a manner that depends on the redox state of oxytocin. Preparations combining Cu(II) with oxidized oxytocin (CuoxOT) promote cell invasion, while preparations combining Cu(II) with reduced oxytocin (CurOT) enhance migration. LC-MS analysis revealed that the cellular environment promotes partial reduction of oxOT and distinct structural rearrangements among CuOT species, suggesting dynamic redox modulation of OT in a tumor microenvironment. To assess the signaling mechanisms underlying these effects, we found that CuoxOT significantly downregulated PI3K and β-arrestin 2 expressions. These changes may support the distinct cellular responses observed with CuoxOT, particularly in relation to migration and invasion. This study highlights the potential of redox- and metal state-dependent oxytocin species as modulators of distinct signaling pathways in triple-negative breast cancer, offering new perspectives for targeted therapeutic strategies.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype with few available therapeutic strategies. Berberine Hydrochloride (BBR) has shown anticancer potential but its mechanisms in TNBC remain unclear. We used cell viability assays, immunohistochemistry, western blotting, and metabolic assays to investigate the effects of BBR on TNBC cells. BBR markedly reduced the viability of TNBC cell lines and increased apoptosis compared with untreated controls. Relative to adjacent non-tumor tissues, TNBC tumor samples showed higher levels of total p65, p65 K310 acetylation, and p65 S536 phosphorylation; the same pattern appeared in TNBC cell lines compared with normal breast epithelial cells. In TNBC cells, both BBR and NAM increased p65 K310 acetylation while reducing S536 phosphorylation relative to vehicle treatment. SIRT2 expression was elevated in TNBC tumors and cell lines compared with their controls, whereas BBR decreased SIRT2 levels. BBR also induced stronger oxidative stress than controls and caused mitochondrial dysfunction, including membrane potential loss and reduced oxygen consumption and acidification rates. BBR induces apoptosis in TNBC cells and is associated with p65 acetylation/phosphorylation changes, SIRT2 downregulation, and mitochondrial dysfunction. This study provides a novel mechanistic basis for BBR as a potential therapeutic agent for TNBC.

ONC201 is a first-in-class, FDA-approved small molecule activator of the mitochondrial ATP-dependent caseinolytic peptidase P (ClpP). This and other related small molecules referred to as ClpP agonists, exert antiproliferative effects in several cancer cell types. We report that ONC201 and highly potent second generation ClpP agonists (TR-57, TR-107), promote induction of senescence in triple-negative breast cancer (TNBC) cell lines. Senescence was determined by increased β-galactosidase (β-gal) activity, downregulation of phosphorylated Rb, c-Myc (Myc), and lamin B1, upregulation of senescent-associated secretory phenotype (SASP), and extended cell proliferation assays. These responses were not observed in ClpP knockout cell lines, demonstrating ClpP-dependence. Proteomics analyses identified multiple events related to the development of senescence including cell cycle arrest and mitochondrial dysfunction. Flow cytometry confirmed an S-phase arrest and DNA damage was detected by Comet assay, 53BP1, phospho-S*Q, and γH2A.X immunostaining. In parallel with this, activation of the ATM pathway and phosphorylation of Chk2 was observed. We determined that ClpP agonist-induced senescence was irreversible in both in vitro and in vivo studies. Following TR-57 treatment and drug washout, cells remained growth arrested which coincided with loss of mitochondrial membrane potential and ability to produce ATP by oxidative phosphorylation. β-gal staining after TR-57 treatment and drug washout demonstrated a sustained increase in β-gal activity, indicating cells are senescent after drug washout. This response was reproduced in vivo wherein senescent 4T1-Luc cells did not develop tumors following injection into mice. Finally, the combination of a ClpP agonist with a known senolytic (venetoclax), synergistically increased the amount of cell death observed. In summary, we show that ClpP agonists stably induce an irreversible senescence in a ClpP-dependent manner that synergizes with venetoclax in TNBC cells.

Tamoxifen (TMX), a selective estrogen receptor modulator, is widely used to treat hormone-dependent breast cancer (BC). Nevertheless, low bioavailability, and emerging deleterious effects considerably restrict TMX clinical application. This study aimed to develop and comprehensively characterize TMX-loaded solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs), to comparatively assess their physicochemical performance and to evaluate their therapeutic potential against triple-negative BC (TNBC). After production, those lipid nanoparticles (LNPs) were characterized in terms of particle size, polydispersity index, zeta potential, morphological investigation through transmission electron microscopy analysis, identifications of interactions via FT-IR and differential scanning calorimetry, entrapment efficiency, drug loading, drug release study, long-term stability for 7 months, cell viability and flow cytometry analysis to evaluate antitumor efficacy of drug carriers. TMX loaded LNPs (15mg) were produced with small size (< 200nm), homogeneous dispersion (< 0.3) and high entrapment efficiency (> 90%). FT-IR spectra and differential scanning calorimetry profiles confirmed TMX conjugation into LNPs. TMX showed higher release from NLCs, whereas SLNs showed retarded release. Particle concentration was reduced 10-30 times after TMX loading. Moreover, TMX-loaded LNPs effectively inhibited cell viability on TNBC (MDA-MB-231), mainly based on apoptosis. These findings suggest that TMX-loaded LNPs represent a promising preclinical strategy for enhancing TMX efficacy against TNBC cells.

A Ruthenium–BODIPY Photosensitizer for Light-Triggered Apoptosis in Triple-Negative Breast Cancer Cells

Triple-negative breast cancer (TNBC) is aggressive and lacks targeted therapeutic options. Mixed lineage kinase 3 (MLK3) plays a key role in TNBC progression. To enhance the therapeutic impact of MLK3 inhibition in TNBC, a novel MLK3 inhibitor scaffold was optimized to incorporate inhibition of NAMPT, often upregulated in TNBC to meet the increased demand for NAD+. Cell-free and cell-based assays for MLK3 and NAMPT inhibition drove the design and optimization of GR2-128, a dual inhibitor with matched nanomolar inhibition for MLK3 and NAMPT, antiproliferative in TNBC cell lines, with acceptable metabolic stability and bioavailability. GR2-128 attenuated tumorigenesis in a syngeneic mouse breast cancer model, increasing apoptosis via inhibition of MLK3 and NAMPT, attenuating macrophage and neutrophil infiltration and increasing tumor T-cell markers without change in B-cell count. These data support the dual inhibition of MLK3/NAMPT as a therapeutic approach to TNBC, with the potential for tumor immuno-sensitization.

Triple-negative breast cancer (TNBC) is highly invasive and has limited effective treatment options. Here, we developed natural extracellular nanovesicles derived from Taxus leaves (TLENs), which share the same plant origin as that of paclitaxel (PTX), to explore their potential as a targeted therapy carrier for TNBC. Through nontargeted metabolomics, it was found that it contains a large amount of flavonoids, with naringenin being the most abundant compound, as confirmed by LC-MS/MS analysis. Experiments have shown that the nanovesicles have significant anti-TNBC effects and, more importantly, can improve sensitivity to paclitaxel. At the cellular level, TLENs are internalized by TNBC cells via macropinocytosis, triggering an intracellular surge of reactive oxygen species that activates the JNK/p38 MAPK signaling cascade, thereby driving apoptosis while inhibiting cell proliferation, epithelial-mesenchymal transition, and migration. Beyond direct cytotoxicity, the oral coadministration of TLENs and PTX fundamentally remodeled the tumor microenvironment. The treatment effectively reversed immunosuppression by promoting dendritic cell maturation and enhancing cytotoxic CD8+ T cell infiltration while concurrently depleting regulatory T cells (Tregs) and myeloid-derived suppressor cells. This work innovatively proposes a "homologous combination" strategy that combines TLENs derived from the same plant with PTX. This combination exhibits enhanced anti-TNBC effects that are greater than those of either agent alone, providing a novel combination therapy for the treatment of TNBC.

The pronounced therapeutic recalcitrance of triple-negative breast cancer (TNBC) fundamentally stems from its molecular target paucity and aggressive pathophysiology. Although paclitaxel and quercetin (PTX/Q) have been explored as complementary agents, their concurrent use is limited by poor solubility, rapid systemic clearance, and dose-related toxicities. To address these constraints, we developed a C-peptide-functionalized solid lipid nanoparticle (SLN) system designed to enhance the codelivery of PTX and Q to αvβ3-expressing TNBC cells. The optimized SLN-PTX-Q-pep formulation exhibited a spherical morphology, a mean hydrodynamic diameter of approximately 415 nm, and high encapsulation efficiencies for both PTX (96.9 ± 0.6%) and Q (91.8 ± 1.5%). Thermal analysis and fluorescence spectroscopy confirmed that the drugs were molecularly dispersed within the lipid matrix. The formulation demonstrated excellent blood compatibility, with less than 1.5% hemolysis, and exhibited pH-responsive drug release, with accelerated liberation under mildly acidic, tumor-relevant conditions (pH 5.8). In vitro studies on 4T1 TNBC cells revealed that the targeted nanoformulation achieved a lower PTX IC50 value compared to nontargeted controls. Functional assays confirmed enhanced apoptosis (Annexin V staining), reduced cell migration, and suppression of intracellular reactive oxygen species (ROS). Competitive binding assays verified that the peptide-functionalized SLNs specifically interact with the αvβ3 integrin receptor. In vivo, SPECT imaging demonstrated superior tumor accumulation of the targeted nanoparticles. Correspondingly, mice treated with SLN-PTX-Q-pep showed a significant reduction in tumor growth and lower final tumor burdens, with no signs of systemic toxicity as confirmed by stable body weight, serum biochemistry, and histological analysis of major organs. Collectively, these findings establish that C-peptide-functionalized SLNs are a robust platform for the coordinated delivery of PTX and Q, enhancing tumor-targeted accumulation and therapeutic efficacy in a preclinical TNBC model. This work provides a strong basis for the continued development of dual-drug nanocarriers for difficult-to-treat breast cancers.