Triple-negative Breast Cancer Cells Research Articles

Synthesis, anticancer activity and mechanism of action of Fe(III) complexes.

To inhibit the growth and metastasis of triple-negative breast cancer (TNBC), two Fe(III) thiosemicarbazone complexes (Fe1 and Fe2) were designed and synthesized. The structures of the Fe(III) complexes were characterized by single crystal X-ray diffraction. The antiproliferative activity of Fe1 and Fe2 against four cancer lines (MDA-MB-231, T98G, HepG2, 143B) and human renal proximal tubular epithelial cell line (HK-2) was evaluated by MTT assay. Among all cells, Fe2 showed significant cytotoxicity to TNBC cells (MDA-MB-231), with an IC50 value of 12.38 μM. Furthermore, Fe2 showed less toxicity to HK-2 cells. The two Fe(III) complexes can produce excess of reactive oxygen species, decrease of mitochondrial membrane potential, and induce DNA damage, then lead to apoptosis of MDA-MB-231 cells. In addition, Fe1 and Fe2 can also inhibit migration and invasion of MDA-MB-231 cells. This study provides guidance for the development of metal complexes that inhibit the growth and metastasis of TNBC.

Significance of LIF/LIFR Signaling in the Progression of Obesity-Driven Triple-Negative Breast Cancer

American women with obesity have an increased incidence of triple-negative breast cancer (TNBC). The impact of obesity conditions on the tumor microenvironment is suspected to accelerate TNBC progression; however, the specific mechanism(s) remains elusive. This study explores the hypothesis that obesity upregulates leukemia inhibitory factor receptor (LIFR) oncogenic signaling in TNBC and assesses the efficacy of LIFR inhibition with EC359 in blocking TNBC progression. TNBC cell lines were co-cultured with human primary adipocytes, or adipocyte-conditioned medium, and treated with EC359. The effects of adiposity were measured using cell viability, colony formation, and invasion assays. Mechanistic studies utilized RNA-Seq, Western blotting, RT-qPCR, and reporter gene assays. The therapeutic potential of EC359 was tested using xenograft and patient-derived organoid (PDO) models. The results showed that adipose conditions increased TNBC cell proliferation and invasion, and these effects correlated with enhanced LIFR signaling. Accordingly, EC359 treatment reduced cell viability, colony formation, and invasion under adipose conditions and blocked adipose-mediated organoid growth and TNBC xenograft tumor growth. RNA-Seq analysis identified critical pathways modulated by LIF/LIFR signaling in diet-induced obesity mouse models. These findings suggest that adiposity contributes to TNBC progression via the activation of the LIF/LIFR pathway, and LIFR inhibition with EC359 represents a promising therapeutic approach for obesity-associated TNBC.

Cationized extracellular vesicles for gene delivery

Last decade, extracellular vesicles (EVs) attracted a lot of attention as potent versatile drug delivery vehicles. We reported earlier the development of EV-based delivery systems for therapeutic proteins and small molecule chemotherapeutics. In this work, we first time engineered EVs with multivalent cationic lipids for the delivery of nucleic acids. Stable, small size cationized EVs were loaded with plasmid DNA (pDNA), or mRNA, or siRNA. Nucleic acid loaded EVs were efficiently taken up by target cells as demonstrated by confocal microscopy and delivered their cargo to the nuclei in triple negative breast cancer (TNBC) cells and macrophages. Efficient transfection was achieved by engineered cationized EVs formulations of pDNA- and mRNA in vitro. Furthermore, siRNA loaded into cationized EVs showed significant knockdown of the reporter gene in Luc-expressing cells. Overall, multivalent cationized EVs represent a promising strategy for gene delivery.

The molecular anti-metastatic potential of CBD and THC from Lebanese Cannabis via apoptosis induction and alterations in autophagy

The medicinal plant Cannabis sativa L. (C. sativa) is currently being extensively studied to determine the full extent of its therapeutic pharmacological potential. Δ9-tetrahydocannabinol (THC) and cannabidiol (CBD) are the most thoroughly investigated compounds. We aimed to explore the anticancer activity of cannabinoids mixture isolated from the Lebanese C. sativa plant in ratios comparable to the local medicinal plant, to elucidate its mechanism of action in breast cancer cells in vitro. Cells were subjected to cytotoxicity assay, cell cycle analysis, Annexin V/PI dual staining, cell death ELISA, immunofluorescence, in addition to western blot analysis of apoptotic and autophagy markers. We further evaluated the anti-metastatic effect of cannabinoids on MDA-MB-231 using the scratch wound-healing, trans-well migration and invasion assays. Our results revealed the promising therapeutic benefits of CBD/THC on inhibiting the growth of breast cancer cells by promoting cellular fragmentation, phosphatidylserine translocation to the outer membrane leaflet and DNA fragmentation in both cell lines while inhibiting the motility of the triple negative breast cancer cells. In our study, CBD/THC mixture was found to exhibit a pro-apoptotic activity via the activation of the mitochondrial apoptotic pathway, independent from ROS production while also suggesting the activation of a caspase-dependent apoptotic pathway. Even though autophagy was altered upon exposure to the cannabinoid mixture, our data suggested that it is not the mechanism responsible of inducing cell death. In conclusion, our study demonstrates the promising therapeutic benefits of CBD and THC isolated from the Lebanese C. sativa plant on breast cancer cells in vitro.

Synergistic Anti-Tumor Effects of Newcastle Disease Virus and Doxorubicin: Evidence from A Murine Breast Cancer Model

BackgroundDespite the recent advances in the diagnosis and treatment of breast cancer, triple-negative breast cancer (TNBC) remains a clinical challenge due to its aggressive nature and resistance to conventional therapies. Virotherapy has emerged as a promising cancer treatment strategy, leveraging the ability of viruses to specifically target and replicate in cancerous cells. This study evaluated the oncolytic potential of a combined therapeutic strategy, utilizing Newcastle disease virus (NDV) and Doxorubicin hydrochloride (Dox) both in vitro and in vivo. MethodsThe in vitro experiments involved exposing human and mouse TNBC cell lines (MDA-MB-231 and 4T1, respectively) to NDV and Dox, individually or in combination. Cell viability assays and flow cytometry analyses were conducted to assess the synergistic effects of NDV and Dox on regulating breast cancer cell behavior in vitro. Furthermore, the immune-stimulating potential of NDV was investigated by examining its effects on dendritic cell (DC) maturation using flow cytometry and T cell proliferation. The in vitro anti-tumor effects of NDV were examined in both parental and tamoxifen-resistant cancer cells to assess its efficacy against chemoresistance. Animal models of breast cancer were treated with NDV in combination with Dox. The body weight changes, tumor volume, and survival rates of the mice were monitored throughout the study. Histopathological analyses were conducted to evaluate the potential toxic effects of the treatments. ResultsBased on the MTT results, NDV at optimal concentrations synergized the effect of Dox to reduce the viability of both MDA-MB231 and 4T1 cell lines (Isobologram combination index of less than 1). Additionally, individual treatment with NDV was able to significantly reduce the viability of patient-derived breast cancer cells, compared to the untreated control (P < 0.05) without affecting the cells of normal adjacent tissue. Furthermore, a combination of NDV and Dox significantly enhanced the percentage of early and late apoptotic cells in MDA-MB-231 (P < 0.0001) and late apoptotic cells in 4T1 (P < 0.0001), in comparison with individual treatment with these agents. Flow cytometry results showed that, compared to wild type MDA-MB-231 cells, NDV-infected MDA-MB-231 cells were better inducers of T cell proliferation and DC maturation as evidenced by increased proliferation index (P < 0.05) and elevated expression of CD1a, CD83, and CD86 (P < 0.0001), respectively. Moreover, co-treatment of both wild-type and (tamoxifen) TAM-resistant MCF-7/TAMR-1 cells with TAM and NDV significantly reduce the viability of the cancer cells (P < 0.0001). In tumor-bearing mice locally engrafted with 4T1 cells, combined treatment of NDV and Dox exhibited a marked reduction in median tumor volume compared to the control group, validating our in vitro findings on their synergistic anti-tumor effects. These findings suggest that combining NDV with Dox can effectively inhibit tumor progression and has the potential to reduce the dose, and consequently the toxic side-effects, of Dox in breast cancer therapy.

DLAT promotes triple-negative breast cancer progression via YAP1 activation

ABSTRACT Background Breast cancer (BC) is the most prevalent malignant tumor in women globally. Triple-negative breast cancer (TNBC) represents the most malignant and invasive subtype of BC. New therapeutic targets are urgently needed for TNBC owing to its receptor expression characteristics, which render it insensitive to traditional targeted and endocrine therapies for BC. The role and mechanisms of dihydrolipoamide S-acetyltransferase (DLAT) as a crucial molecule in glycometabolism and cuproptosis-related biological processes in tumors remain to be explored. Methods DLAT expression was investigated using bioinformatics methods and quantitative real-time polymerase chain reaction. Subsequently, the MTT assay, colony formation assay, and migration-invasion assay were performed to validate the effect of DLAT on TNBC cell viability, proliferation, and migration. Cytoplasmic-nuclear separation experiments, western blot analysis, and co-immunoprecipitation assays were performed to elucidate the underlying molecular mechanisms. Results This study revealed a robust correlation between elevated DLAT expression in BC and unfavorable prognosis in patients, with higher expression of DLAT compared to other subtypes in TNBC. Functional cytology experiments indicated that DLAT plays a tumor-promoting role in TNBC. Mechanistic studies showed that DLAT directly interacts with YAP1, leading to the dephosphorylation and activation of YAP1 and its increased nuclear translocation, thereby transcriptionally activating and regulating downstream oncogenes, promoting the malignant phenotype of TNBC. Rescue experiments indicated that DLAT promotes the malignant behavior of TNBC through a YAP1-dependent pathway. Conclusions Our research unveiled the significant involvement of DLAT in TNBC, along with the potential for modulating DLAT/YAP1 activity as a targeted treatment strategy for TNBC.

FOS-Mediated PLCB1 Induces Radioresistance and Weakens the Antitumor Effects of CD8+ T Cells in Triple-Negative Breast Cancer.

Radioresistance and immune evasion are interactive and crucial events leading to treatment failure and progression of human malignancies. This research studies the role of phospholipase C beta 1 (PLCB1) in these events in triple-negative breast cancer (TNBC) and the regulatory mechanism. PLCB1 was bioinformatically predicted as a dysregulated gene potentially linked to radioresistance in TNBC. Parental TNBC cell lines were exposed to fractionated radiation for 6 weeks. PLCB1 expression was decreased in the first 2 weeks but gradually increased from Week 3. PLCB1 knockdown increased the radiosensitivity of the cells, as manifested by a decreased half-inhibitory dose of irradiation, reduced cell proliferation, apoptosis resistance, mobility, and tumorigenesis in mice. The FOS transcription factor promoted PLCB1 transcription and activated the PI3K/AKT signaling. Knockdown of FOS similarly reduced radioresistance and T cells-mediated immune evasion. However, the radiosensitivity of TNBC cells and the antitumor effects of CD8+ T cells could be affected by a PI3K/AKT activator or by the PLCB1 upregulation. The PLCB1 or FOS knockdown also suppressed radioresistance and tumorigenesis of the TNBC cells in mice. In conclusion, FOS-mediated PLCB1 induces radioresistance and weakens the antitumor effects of CD8+ T cells in TNBC by activating the PI3K/AKT signaling pathway.

Chaperone-Derived Copper(I)-Binding Peptide Nanofibers Disrupt Copper Homeostasis in Cancer Cells.

Copper (Cu) is a transition metal that plays crucial roles in cellular metabolism. Cu+ homeostasis is upregulated in many cancers and contributes to tumorigenesis. However, therapeutic strategies to target Cu+ homeostasis in cancer cells are rarely explored because small molecule Cu+ chelators have poor binding affinity in comparison to the intracellular Cu+ chaperones, enzymes, or ligands. To address this challenge, we introduce a Cu+ chaperone-inspired supramolecular approach to disrupt Cu+ homeostasis in cancer cells that induces programmed cell death. The Nap-FFMTCGGCR peptide self-assembles into nanofibers inside cancer cells with high binding affinity and selectivity for Cu+ due to the presence of the unique MT/CGGC motif, which is conserved in intracellular Cu+ chaperones. Nap-FFMTCGGCR exhibits cytotoxicity towards triple negative breast cancer cells (MDA-MB-231), impairs the activity of Cu+ dependent co-chaperone super oxide dismutase1 (SOD1), and induces oxidative stress. In contrast, Nap-FFMTCGGCR has minimal impact on normal HEK 293T cells. Control peptides show that the self-assembly and Cu+ binding must work in synergy to successfully disrupt Cu+ homeostasis. We show that assembly-enhanced affinity for metal ions opens new therapeutic strategies to address disease-relevant metal ion homeostasis.

In vitro and computational investigation of antioxidant and anticancer properties of Streptomyces coeruleofuscus SCJ extract on MDA-MB-468 triple-negative breast cancer cells

This study aimed to explore the antioxidant potential of the ethyl acetate extract of Streptomyces coeruleofuscus SCJ strain, along with its inhibitory effects on the triple-negative human breast carcinoma cell line (MDA-MB-468). The ethyl acetate extract’s total phenolic and flavonoid contents were quantified, and its antioxidant activity was investigated using DPPH (1,1-Diphenyl-2-picrylhydrazyl), ABTS (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), and FRAP (Ferric Reducing Antioxidant Power) assays. Furthermore, the cytotoxic effect of the organic extract from Streptomyces coeruleofuscus SCJ on MDA-MB-468 cancer cells was assessed via the crystal violet assay. In tandem, a thorough computational investigation was conducted to explore the pharmacokinetic properties of the identified components of the extract, utilizing the SwissADME and pKCSM web servers. Additionally, the molecular interactions between these components and Estrogen Receptor Beta, identified as a potential target, were probed through molecular docking studies. The results revealed that ethyl acetate extract of SCJ strain exhibited remarkable antioxidant activity, with 39.899 ± 1.56% and 35.798 ± 0.082% scavenging activities against DPPH and ABTS, respectively, at 1 mg/mL. The extract also displayed significant ferric reducing power, with a concentration of 1.087 ± 0.026 mg ascorbic acid equivalents per mg of dry extract. Furthermore, a strong positive correlation (p < 0.0001) between the antioxidant activity, the polyphenol and the flavonoid contents. Regarding anticancer activity, the SCJ strain extract demonstrated significant anticancer activity against TNBC MDA-MB-468 cancer cells, with an inhibition percentage of 62.76 ± 0.62%, 62.67 ± 0.93%, and 58.07 ± 4.82% at 25, 50, and 100 µg/mL of the extract, respectively. The HPLC-UV/vis analysis revealed nine phenolic compounds: gallic acid, sinapic acid, p-coumaric acid, cinnamic acid, trans-fereulic acid, syringic acid, chloroqenic acid, ellagic acid, epicatechin. Streptomyces coeruleofuscus SCJ showed promise for drug discovery, exhibiting antioxidant and anticancer effects.

Loss of miR-634 contributes to the formation FOXA1-positive triple negative breast cancer subtype

BackgroundTriple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, lacks targeted therapies, posing a substantial challenge for treatment. Therefore, investigating its pathogenesis is a crucial research focus. FOXA1 and miR-634 are involved in tumorigenesis. However, the molecular mechanisms underlying the aberrant upregulation of FOXA1 expression in TNBC remain unclear. Therefore, we explore the role of miR-634 in the FOXA1-positive TNBC subtype.MethodsQuantitative reverse transcription polymerase chain reaction was used to detect miR-634 expression in breast cancer tissues and cell lines. Aberrantly activated signaling pathways and related genes in TNBC were analyzed using The Cancer Genome Atlas. The potential target of miR-634 was predicted by TargetScan, the TNBC cell proliferation rate was detected using an MTT assay, the in vitro metastatic capacity was determined by transwell assay and the cell cycle distribution was tested using flow cytometry. Western blotting was performed to measure the expression of proteins involved in the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway.ResultsThe expression of miR-634 was significantly down-regulated in both TNBC tissues and cells, compared with adjacent non-cancerous tissues and MCF10A, respectively. Ectopic expression of miR-634 inhibits breast cancer cell proliferation and in vitro metastasis. The TCGA-based expression profile analysis of TNBC revealed that aberrantly activated PI3K/AKT signaling may contribute to its malignant phenotype. FOXA1, the top hit of aberrantly upregulated genes in TNBC, was a direct target of miR-643. Moreover, forced expression of miR-643 drastically suppressed FOXA1 expression by the inactivation PI3K/AKT signaling.ConclusionMiR-634 suppresses FOXA1 to inhibit the proliferation and metastasis of TNBC cells by inactivating the PI3K/AKT signaling pathway.

TRPC3-mediated NFATc1 calcium signaling promotes triple negative breast cancer migration through regulating glypican-6 and focal adhesion.

Canonical transient receptor potential isoform 3 (TRPC3), a calcium-permeable non-selective cation channel, has been reported to be upregulated in breast cancers and a modulator of cell migration. Calcium-sensitive transcription factor NFATc1, which is important for cell migration, was shown to be frequently activated in triple negative breast cancer (TNBC) biopsy tissues. However, whether TRPC3-mediated calcium influx would activate NFATc1 and affect the migration of TNBC cells, and, if yes, the underlying mechanisms involved, remain to be investigated. By immunostaining followed by confocal microscopy, TNBC lines MDA-MB-231 and BT-549 were both found to express TRPC3 on their plasma membrane while ER+ line MCF-7 and HER2+ line SK-BR3 do not. Blockade of TRPC3 by pharmacological inhibitor Pyr3 or stable knockdown of TRPC3 by lentiviral vector both inhibited cell migration as measured by wound healing assay. Importantly, blocking TRPC3 by Pyr3 or knockdown of TRPC3 both caused the translocation of NFATc1 from the nucleus to the cytosol as revealed by confocal microscopy. Interestingly, NFATc1 was found to bind to the promoter of glypican 6 (GPC6) as determined by chromatin immunoprecipitation assay. Consistently, knockdown of TRPC3 decreased the expression of GPC6 as revealed by western blotting. Moreover, long-term knockdown of GPC6 by lentiviral vector also consistently decreased the migration of TNBC cells. Intriguingly, GPC6 proteins physically interact with vinculin in MDA-MB-231 as determined by co-immunoprecipitation. Blockade of TRPC3, knockdown of TRPC3 or knockdown of GPC6 all induced larger, stabilized actin-bound peripheral focal adhesion (FA) formations in TNBC cells as determined by co-staining of actin and vinculin followed by confocal microscopy. These large, stabilized actin-bound peripheral FAs indicated a defective FA turnover, and were reported to be responsible for impairing directed cell migration. Our results suggest that, in TNBC cells, calcium influx through TRPC3 channel positively regulates NFATc1 nuclear translocation and GPC6 expression, which maintains the dynamics of FA turnover and optimal cell migration. Our study reveals a novel TRPC3-NFATc1-GPC6-vinculin signaling cascade in maintaining the migration of TNBC cells.

Blestriarene C exerts an inhibitory effect on triple-negative breast cancer through multiple signaling pathways

IntroductionBreast cancer is the most common cancer worldwide, the leading cause of cancer death in women, and the fifth leading cause of cancer death. Triple negative breast cancer (TNBC), with high metastasis and mortality rates, is the most challenging subtype in breast cancer treatment. There is an urgent need to develop anti-TNBC drugs with significant efficacy, low side effects and good availability. In early drug screening, blestriarene C was found to have inhibitory effects on TNBC cells. In this article, we further explore the mechanisms associated with blestriarene C for breast cancer.MethodsIn this article, we take the approach of network pharmacology combined with in vivo and in vitro experiments. Network pharmacology analysis was used to predict the active components in Baiji, and to investigate the hub targets and related mechanisms of BC in TNBC treatment. The mechanism of anti-TNBC in vitro was evaluated by CCK-8 assay, cell apoptosis and cell cycle assays, wound healing assay, WB assay, and molecular docking analysis. The inhibition effect in vivo was test in subcutaneous tumor models established in mice.ResultsThrough network pharmacology analysis and experiments, we screened out BC as the main active ingredient, and found that BC could inhibit the Ras/ERK/c-Fos signaling pathway while downregulating the expression of HSP90AA1 and upregulating the expression of PTGS2, thereby promoting apoptosis, causing S-phase cycle arrest, and inhibiting the proliferation and migration of BT549 cells. The in vivo results illustrated that BC inhibited the growth of TNBC tumors and has a high safety profile. By integrating network pharmacology with in vitro and in vivo experiments, this study demonstrated that BC inhibited the proliferation and migration of TNBC cells by inhibiting the Ras/ERK/c-Fos signaling pathway, promoting apoptosis, and causing S-phase cycle arrest.DiscussionThis study provides new evidence for the use of BC as a novel drug for TNBC treatment.

A BPTF Inhibitor That Interferes with the Multidrug Resistance Pump to Sensitize Murine Triple-Negative Breast Cancer Cells to Chemotherapy

Triple-negative breast cancer (TNBC) is associated with a generally poor prognosis due to its highly aggressive and metastatic nature, lack of targetable receptors, as well as the frequent development of resistance to chemotherapy. We previously reported that AU1, a small molecule developed as an inhibitor of BPTF (bromodomain PHD finger-containing transcription factor), was capable of sensitizing preclinical models of TNBC to chemotherapy in part via the promotion of autophagy. In studies reported here, we identify an additional property of this compound, specifically that sensitization is associated with the inhibition of the P-glycoprotein (P-gp) efflux pump. In silico molecular docking studies indicate that AU1 binds to active regions of the efflux pump in a manner consistent with the inhibition of the pump function. This work identifies a novel chemical structure that can influence multidrug efflux, an established mechanism of drug resistance in TNBC, that has not yet been successfully addressed by clinical efforts.

High prevalence of chromosome 17 in breast cancer micronuclei: a means to get rid of tumor suppressors?

Micronuclei (MN), defined as small extra-nuclear chromatin bodies enclosed by a nuclear envelope, serve as noticeable markers of chromosomal instability (CIN). The MN have been used for breast cancer (BC) screening, diagnosis, and prognosis. However, more recently they have gained attention as seats for active chromosomal rearrangements. BC subtypes exhibit differential CIN levels and aggressiveness. This study aimed to investigate MN chromosomal contents across BC subtypes, exploring its potential role in aggressiveness and pathogenesis. Immunostaining of BC cells was performed with anti-centromeric antibody followed by confocal microscopy. Further, fluorescence in situ hybridization (FISH) was done to check the presence of specific chromosomes in the MN. The real time PCR was also done from the RNA isolated from MN to check the expression of TP53 gene. BC cell lines (CLs) showed the presence of both centromere-positive ( +) and -negative ( -) MN, with significant variation in frequency among hormone and human epidermal growth factor receptor positive and triple-negative (TN) BC cells. FISH targeting chromosomes 1, 3, 8, 11, and 17 detected centromeric signals for all the above chromosomes in MN with a relatively higher prevalence of chromosome 17 in all the CLs. Out of all the CLs, TNBC cells demonstrated the highest frequency of centromere + and chromosome 17 + MN. TP53 expression could also be demonstrated inside the MN by FISH and real time PCR. Patient sample imprints also confirmed the presence of chromosome 17 in MN with polysomy of the same in corresponding nuclei. The high prevalence of chromosome 17 in BC MN may connote the importance of its rearrangements in the pathogenesis of BC. Further, the higher prevalence of chromosome 17 and 1 signals in TNBC MN point towards the significance of pathogenetic events involving the genes located in these chromosomes in evolution of this more aggressive phenotype.

Breast cancer secretes anti-ferroptotic MUFAs and depends on selenoprotein synthesis for metastasis.

The limited availability of therapeutic options for patients with triple-negative breast cancer (TNBC) contributes to the high rate of metastatic recurrence and poor prognosis. Ferroptosis is a type of cell death caused by iron-dependent lipid peroxidation and counteracted by the antioxidant activity of the selenoprotein GPX4. Here, we show that TNBC cells secrete an anti-ferroptotic factor in the extracellular environment when cultured at high cell densities but are primed to ferroptosis when forming colonies at low density. We found that secretion of the anti-ferroptotic factors, identified as monounsaturated fatty acid (MUFA) containing lipids, and the vulnerability to ferroptosis of single cells depends on the low expression of stearyl-CoA desaturase (SCD) that is proportional to cell density. Finally, we show that the inhibition of Sec-tRNAsec biosynthesis, an essential step for selenoprotein production, causes ferroptosis and impairs the lung seeding of circulating TNBC cells that are no longer protected by the MUFA-rich environment of the primary tumour.

WNT1/ROR2 pathway enhances the Triple-Negative breast cancer invasion, migration, and Epithelial-Mesenchymal transition.

It has been evidenced that ROR2 influences the growth of many tumors, including non-small cell lung cancer, osteosarcoma, and breast cancer. This research examined the effect of the WNT1/ROR2 signaling pathway on the progression of triple-negative breast cancer (TNBC). Bioinformatics analysis results demonstrated that ROR2 had a higher messenger RNA (mRNA) expression level in TNBC tissues and was positively correlated with poor patient prognosis. Western blot analysis (WB) and quantitative reverse transcription polymerase chain reaction demonstrated that TNBC cells had relatively higher ROR2 mRNA and protein levels than normal cell lines. Transwell and Cell Counting Kit-8 (CCK-8) assay further proved that downregulating ROR2 expression dramatically slowed the MDA-MB-231 cell progression. WB detection of epithelial-mesenchymal transition (EMT)-related proteins suggested that knocking down ROR2 could alleviate the EMT tendency of cancer cells. The WNT1/ROR2 signaling pathway could be inhibited by the WNT inhibitor pyrvinium pamoate (PP). Experiments on in vitro cell functional recovery have demonstrated that PP could restore malignant phenotypes caused by ROR2 overexpression. Finally, the mouse model experiments further validated the anticancer effect of PP on TNBC. Generally speaking, the malignant progression of TNBC could be stimulated by the WNT1/ROR2 signaling pathway which can be inhibited by PP, suggesting the potential value of PP in controlling TNBC.

Activation of the γ-secretase/NICD-PXR/Notch pathway induces Taxol resistance in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is currently the only subtype lacking efficient targeted therapies. Taxol is the primary chemotherapeutic agent for TNBC. However, Taxol resistance often develops in the treatment of TNBC patients, which importantly contributes to high mortality and poor prognosis in TNBC patients. Recent preclinical studies have shown that the inhibition of Notch pathway by γ-secretase inhibitors can slow down the progression of TNBC. Our studies in bioinformatic analysis of breast cancer patients and TNBC/Taxol cells in vitro showed that there was high correlation between the activation of Notch pathway and Taxol resistance in TNBC. Increased γ-secretase activity (by the overexpression of catalytic core PSEN-1) significantly reduced Taxol sensitivity of TNBC cells, and enhanced biological characteristics of malignancy in vitro, and tumour growth in vivo. Mechanistically, increased γ-secretase activity led to the accumulation of NICD in the nucleus, promoting the interaction between NICD and PXR to activate PXR, which triggered the transcription of PXR downstream associated drug resistance genes. Furthermore, we showed that pharmacological inhibition of γ-secretase with γ-secretase inhibitors (Nirogacestat and DAPT) can reverse Taxol resistance in vivo and in vitro. Our results for the first time demonstrate that the activation of γ −secretase/NCD-PXR/Notch pathway is one of important mechanisms to cause Taxol resistance in TNBC, and the blockades of this pathway may represent a new therapeutic strategy for overcoming Taxol resistance in TNBC.

A novel copper(II) complex with a salicylidene carbohydrazide ligand that promotes oxidative stress and apoptosis in triple negative breast cancer cells.

We report the synthesis, characterization, anti-cancer activity and mechanism of action of a novel water-soluble Cu(II) complex with salicylidene carbohydrazide as the ligand and o-phenanthroline as the co-ligand. The synthesized complex (1) was characterized by FT-IR, EPR, and electronic spectroscopy, as well as single crystal X-ray diffraction. This compound was found to be paramagnetic from EPR spectra and X-ray crystallography revealed that the molecule crystallized in an orthorhombic crystal system. The crystal lattice was asymmetric containing two distinct binuclear copper complexes containing the Schiff base as the major ligand, o-phenanthroline as a co-ligand, two nitrate anions, and two water molecules. The Cu(II) in the first site coordinated with the enolised ligand comprising enolate O-, phenolate O-, and the imine N and N,N from o-phen. The major part of this complex exists as Cu(II) coordinated with two H2O molecules at the second site with nitrate acting as the counter anion. However, a smaller portion of the complex exists where Cu(II) is coordinated with NO3- and H2O, and the remaining water molecule acts as lattice water. It was tested for DNA binding and cleavage properties which revealed that it binds in an intercalative mode to CT-DNA with Kb value of 1.25 × 104 M-1. Furthermore, cleavage studies reveal that the complex has potential for efficient DNA cleavage under both oxidative and hydrolytic conditions. It was able to enhance the rate of cleavage by 2.8 × 108 times. The complex shows good cytotoxicity to breast cancer monolayer (2D) as well as spheroid (3D) systems. The IC50 values for MDA-MB-231 and MCF-7 monolayer culture was calculated as 1.86 ± 0.17 μM and 2.22 ± 0.08 μM, respectively, and in (3D) spheroids of MDA-MB-231 cells, the IC50 value was calculated to be 1.51 ± 0.29 μM. It was observed that the complex outperformed cisplatin in both breast cancer cell lines. The cells treated with complex 1 underwent severe DNA damage, increased oxidative stress and cell cycle arrest which finally led to programmed cell death or apoptosis in triple negative breast cancer cells through an intrinsic pathway.

Amine-Terminated Silver Nanoparticles Exhibit Potential for Selective Targeting of Triple-Negative Breast Cancer

Silver nanoparticles (AgNPs) demonstrate potential in treating aggressive cancers such as triple-negative breast cancer (TNBC) in preclinical models. To further the development of AgNP-based therapeutics for clinical use, it is essential to clearly define the specific physicochemical characteristics of the nanoparticles and connect these properties to biological outcomes. This study addresses this knowledge gap through detailed investigations into the structural and surface functional relationships, exploring the mechanisms, safety, and efficacy of AgNPs in targeting TNBC. The surface functionality of nanoparticles is crucial not only for their internalization into cancer cells but also for enhancing their toxicity toward tumor cells. Although the nanoparticles internalized into cancer cells, they failed to exhibit their full toxicity against the cancer. Herein we report a solvent-assisted synthesis amine, mercaptohexanol and bifunctional silver nanoparticles and performing comparative study to understand their selectivity and toxicity toward TNBC cells. The nanoparticles are fully characterized by UV–visible absorption spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and dynamic light scattering measurement (DLS). The synthesis method achieves an extremely high yield and surface coating ratio of synthesized colloidal AgNPs. Our findings reveal that the amine-capped AgNPs exhibit significant selective toxicity against TNBC cell lines MCF7 and MDA-MB-231 at a concentration of 40 µg/mL without affecting normal breast cell lines MCF10A. This study underscores the potential of functionalized AgNPs in developing safe and targeted therapeutic approaches for treating aggressive cancers like TNBC, laying the groundwork for future clinical advancements.

Abstract B022: Tumoral GLI1 controls the resident innate and adaptive tumor immune microenvironment in triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer (BC) cases but due to its aggressive nature and the lack of targeted treatment options, represents a disproportionately higher number of BC-related deaths. Compared to other BC subtypes, TNBCs are more inflamed with M2-like tumor-associated macrophages (TAMs) and cytotoxic T-lymphocytes (CTLs) which are associated with poor and good prognosis, respectively. Clinical trials have proven that immunogenic cancers can be effectively treated through immune checkpoint blockade (ICB), a treatment strategy that prevents cancer cells from silencing CTL-mediated attack. However, CTLs can be suppressed by M2-like TAMs which can limit ICB efficacy and support tumor growth. The hedgehog (HH) signaling pathway is highly activated in TNBC and higher expression of GLI1 is associated with worse overall survival. Recent pan-cancer analyses highlight a significant correlation between activated HH signaling and characteristics of immune evasion. We aim to clarify how activated HH signaling regulates resident innate and adaptive immune cells in the tumor microenvironment (TME) in TNBC. Methods: Primary K14-cre; Brca1 fl/fl ; P53 fl/fl (KBP) murine TNBC cells were transfected with CRISPR/Cas9 + sgRNA targeting GLI1. After isolating single cell clones and confirming GLI1 knockout (KO), KBP and KBP-GLI1-KO cells were orthotopically injected into the mammary fat pad of syngeneic immune-competent female mice and allowed to grow for 6-8 weeks. Tumor volume was measured once tumors were palpable, and tumors were harvested once the largest tumor reached ethical endpoint. Tumors were weighed at harvest and fluorescence activated cell sorting (FACS) was performed to determine if there was an effect on the composition of immune populations. Results: At harvest, KBP-GLI1-KO tumors were delayed in growth and significantly smaller than KBP tumors. FACS analysis showed that GLI1-KO tumors had increased CD4+ and CD8+ T cells and a reduced amount of Foxp3+ CD4+ Tregs. Furthermore, GLI1-KO tumors had significantly fewer F4/80+ CD11b+ TAMs within the TME. Amongst TAM populations, there was reduced CD206+ M2-like TAMs and an increase in CD80+ M1-like TAMs upon loss of GLI1. Similar trends were observed when KBP allografts were treated with the GLI1 inhibitor, GANT61. Immunohistochemical and RNAseq analysis are ongoing. Similar experiments with other KBP-GLI1-KO clones are currently being done. Conclusion: Here, we show that tumoral GLI1 not only promotes tumor growth, but also supports an immunosuppressive TME in TNBC. Abolishing GLI1 converted tumors to a pro-inflammatory phenotype marked by an increase in CD4+ and CD8+ T cells and a reduction in CD206+ M2-like TAMs. Future experiments are aimed at revealing the mechanisms by which GLI1 regulates immune cell recruitment to the TME and testing the effect of combination immunotherapy. Overall, these preliminary findings identify GLI1 as a potential therapeutic target to limit tumor growth and promote anti-tumor immunity. Citation Format: Aidan J Gray, Wanda Marini, Kiichi Murakami, Michael Reedijk. Tumoral GLI1 controls the resident innate and adaptive tumor immune microenvironment in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B022.