BT474 Breast Cancer Cells Research Articles

G protein selectivity profile of GPR56/ADGRG1 and its effect on downstream effectors.

GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor's constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-a-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56's extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1-385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of b-arrestin-2 but GPR56 internalization was β-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells.

G protein selectivity profile of GPR56/ADGRG1 and its effect on downstream effectors

GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor’s constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-α-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56’s extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1-385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of β-arrestin-2 but GPR56 internalization was β-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells.

Exosomal Delivery Enhances the Antiproliferative Effects of Acid-Hydrolyzed Apiaceae Spice Extracts in Breast Cancer Cells.

Breast cancer remains a leading cause of death worldwide. The Apiaceae plant family includes many culinary spices that have been shown to have medicinal properties. Many phytochemicals exhibit potent bioactivities but often suffer from poor uptake and oral bioavailability. Bovine milk and colostrum exosomes are a compelling drug delivery platform that could address this issue; these natural nanoparticles can be loaded with hydrophilic and lipophilic small molecules and biologics, resulting in lower doses needed to inhibit cancer growth. Ethanolic extracts of eight Apiaceae spices were examined for phytochemical content and antiproliferative potential. Acid hydrolysis (AH) was employed to remove glycosides, asses its impacts on extract efficacy, and evaluate its effects on exosome loading and subsequent formulation efficacy. Antiproliferative activity was assessed through MTT assays on T-47D, MDA-MB-231, and BT-474 breast cancer cells; all extracts exhibited broad antiproliferative activity. AH enhanced the bioactivity of cumin, caraway, and fennel in T-47D cells. Celery, cumin, anise, and ajwain showed the highest activity and were assayed in exosomal formulations, which resulted in reduced doses required to inhibit cellular proliferation for all extracts except AH-cumin. Apiaceae spice extracts demonstrated antiproliferative activities that can be improved with AH and further enhanced with exosomal delivery.

Revealing the Molecular Signatures of miR-185-5p on Breast Cancer Cells Using Proteomic Analysis.

Breast cancer is a heterogeneous type of disease in which genetic and environmental factors play a crucial role. There are several types of treatment for breast cancer (BC) patients. However, the biggest problem in the treatment of breast cancer is the resistance that occurs during the treatment with chemotherapeutic agents. Usnic acid, a secondary metabolite of lichen, has been identified as a drug candidate molecule in cancer treatment. The determination of miRNA target proteins is essential for the understanding of molecular mechanisms of miRNA-related tumorigenesis. We determined that mir-185-5p has therapeutic potential at the miRNA level by applying usnic acid to BT-474 breast cancer cells in a previous study. Herein, we aimed to investigate the molecular mechanisms of miR-185-5p on BT-474 breast cancer cells using a proteomics approach. We explored the changes in the protein expression level of BT-474 breast cancer cells in response to the up-regulation of miR-185-5p after applying usnic acid as a novel candidate anti-- cancer drug molecule. We performed quantitative proteome analysis based on an LC-MS/MS assay, which was validated by western blotting. The differentially expressed proteins were analyzed using the latest data available in bioinformatics tools. The up-regulated expression of YWHAE, Cathepsin D, and the down-regulated levels of PAK-1 were demonstrated by western blot assay. According to the results, 86 proteins showing >2-fold change were identified as differentially expressed between breast cancer and normal breast epithelial cells. The apoptosis pathway was the main clade containing most of the proteins regulated by miR-185-5p. The results indicate that miR-185-5p modulates apoptosis signaling pathways in BT-474 breast cancer cells. Breast cancer inhibition due to increased expression of YWHAE, Cathepsin D, and decreased expression of PAK-1 is likely to be mediated by inducing miR-185-5p mediated apoptosis. In this study, the identification of miR-185-5p protein targets demonstrated the potential for the development of targeted therapy and the development of miRNA-based therapeutics and presented it as a biomarker for breast cancer diagnosis, prognosis, and treatment response. In this regard, proteome analyses provided an understanding of the molecular mechanism underlying the effect of miR-185-5p on breast cancer.

MiRNA-105–5p regulates the histone deacetylase HDAC2 through FOXG1 to affect the malignant biological behavior of triple-negative breast cancer cells

BackgroundTriple-negative breast cancer (TNBC) is a specific subtype of breast cancer (BC). Some potential molecular targets have been identified, and miR-105–5p was found to be abnormally expressed in TNBC tissues. ObjectiveThe objective of this study was to probe the effect of miR-105–5p on TNBC via FOXG1/HDAC2-mediated acetylation. MethodsAn animal model of TNBC was established by injecting BC cells into the axillary area of nude mice. The levels of miR-105–5p, FOXG1, HDAC2, Bcl-2, Bax, and Ki67 were detected via RT‒qPCR, Western blotting and immunohistochemistry. Flow cytometry, CCK-8, Transwell and colony formation assays were used to measure apoptosis, proliferation and migration, respectively. Total histone acetylation levels were measured by ELISA. The binding of FOXG1 to HDAC2 was detected by co-immunoprecipitation. The binding relationship between miR-105–5p and FOXG1 was verified using a dual-luciferase reporter gene assay. ResultsIn this study, miR-105–5p and HDAC2 were highly expressed in the MDA-MB-231 and BT-549 BC cell lines, whereas FOXG1 was expressed at low levels. The inhibition of miR-105–5p inhibited the proliferation and migration of MDA-MB-231 and BT-549 cells and promoted their apoptosis. Bioinformatics analysis revealed that miR-105–5p and FOXG1 had a negative targeting regulatory relationship. FOXG1 overexpression had a similar effect on cancer cells as the inhibition of miR-105–5p. Moreover, experiments revealed that FOXG1 and HDAC2 could bind to each other and that HDAC2 overexpression or treatment with the histone acetyltransferase inhibitor Garcinol weakened the effect of FOXG1 overexpression. In addition, FOXG1 knockdown inhibited the effect of the miR-105–5p inhibitor, while Garcinol treatment further enhanced the effect of FOXG1 knockdown, inhibited histone acetylation, promoted the proliferation and migration of cancer cells, and inhibited apoptosis. Moreover, the in vivo results confirmed the in vitro results. ConclusionmiR-105–5p promotes HDAC2 expression by reducing FOXG1, inhibits histone acetylation, and aggravates the malignant biological behavior of TNBC cells.

β2-microglobulin induced apoptosis of tumor cells via the ERK signaling pathway by directly interacting with HFE in HER2-overexpressing breast cancer

BackgroundOur previous study demonstrated that β2-microglobulin (β2M) promoted ER+/HER2– breast cancer survival via the SGK1/Bcl-2 signaling pathway. However, the role of β2M has not been investigated in ER–/HER2+ breast cancer. Here, we aimed to determine the role of β2M in ER–/HER2+ breast cancer.MethodsThe interaction between β2M and HFE was confirmed by co-immunoprecipitation, mass spectrometry, yeast two-hybrid screening, and His pull-down. The knockdown and overexpression of β2M or HFE were performed in MDA-MB-453 cells, and ERK signaling pathway was subsequently analyzed via western blotting. Apoptotic cells were detected using flow cytometer. β2M, HFE, and p-ERK1/2 were examined in tumor and paired adjacent tissues via immunohistochemistry.ResultsHFE was found to be an interacting protein of β2M in ER–/HER2+ breast cancer cells MDA-MB-453 by co-immunoprecipitation and mass spectrometry. A yeast two-hybrid system and His-pull down experiments verified that β2M directly interacted with HFE. β2M and HFE as a complex were mainly located in the cytoplasm, with some on the cytomembrane of MDA-MB-453 cells. In addition to breast cancer cells BT474, endogenous β2M directly interacted with HFE in breast cancer cells MDA-MB-453, MDA-MB-231, and MCF-7. β2M activated the ERK signaling pathway by interacting with HFE and induced apoptosis of MDA-MB-453 cells. The expression of HFE and p-ERK1/2 showed significantly high levels in HER2-overexpressing breast cancer tumor tissue compared with adjacent normal tissue, consistent with the results obtained from the cell experiments.Conclusionsβ2M induced apoptosis of tumor cells via activation of the ERK signal pathway by directly interacting with HFE in HER2-overexpressing breast cancer.

Ruxolitinib-loaded poly-ɛ-caprolactone (PCL) nanoparticles inhibit JAK2/STAT5 signaling in BT474 breast cancer cells by downregulating Bcl-2 and Mcl-1.

JAK/STAT signaling plays an important role in regulating cell proliferation. Reducing proliferation and inducing cell death with gene-specific inhibitors such as ruxolitinib, Receptor tyrosine kinases (RTK) inhibitor targeting JAK1/2, are therapeutic approaches. The use of nanoparticles can reduce the toxicity and side effects of drugs, as they act directly on cancer cells and can selectively increase drug accumulation in tumor cells. Poly-ɛ-caprolactone (PCL) is a polymer that is frequently used in drug development. In this study, Rux-PCL-NPs were synthesized to increase the effectiveness of ruxolitinib. In addition, this study aimed to determine the effect of Rux-PCL-NPs on JAK/STAT signaling and apoptotic cell death. Rux-PCL-NPs were synthesized by nanoprecipitation. The Rux-PCL-NPs had a spherical and mean particle size of 219 ± 88.66nm and a zeta potential of 0.471 ± 0.453 mV. In vitro cytotoxicity and antiproliferative effects were determined by MTT and soft agar colony formation assays, respectively. The effects of ruxolitinib, PCL-NPs, and Rux-PCL-NPs on apoptosis and the JAK/STAT pathway in cells were examined by western blot analysis. PCL-NPs did not have a toxic effect on the cells. The IC50 value of Rux-PCL-NPs was decreased 50-fold compared to that of ruxolitinib. Rux-PCL-NPs promoted cell death by downregulating JAK2 and STAT5, thereby inhibiting the JAK/STAT pathway. Our results revealed that Rux-PCL-NPs, which increased the efficacy of ruxolitinib, regulated apoptosis and the JAK2/STAT5 pathway.

A novel PIK3CA hot-spot mutation in breast cancer patients detected by HRM-COLD-PCR analysis.

The PI3K protein is involved in the PI3K/AKT/mTOR pathway. Deregulation of this pathway through PIK3CA mutation is common in various tumors. The aim of this work is to identify hotspot mutation at exons 9 and 20 in Tunisian patients with sporadic or hereditary breast cancer. Hotspot mutations in exon 9 and exon 20 of the PIK3CA gene were identified by QPCR-High Resolution Melting followed by COLD-PCR and sequencing in 63 (42 sporadic cases and 21 hereditary cases) tumor tissues collected from Tunisian patient with breast cancer. MCF7, and BT20 breast cancer cell lines harboring the PIK3CA hotspot mutations E545K and H1047R in exon 9 and exon 20 respectively, were used as controls in HRM experiments. PIK3CA hotspot mutations were detected in 66.7% (28 out of 42) of sporadic BC cases, and in 14.3% (3 out of 21) of hereditary BC. The E545K and the H1048Y were the most prevalent mutations identified in patients with sporadic and hereditary BC, whereas the H1047R hotspot mutation was not found in our patients. Statistical analysis showed that PIK3CA mutation associated with an aggressive behavior in patients with sporadic BC, while it's correlated with age, tumor stage and tumor size in the group patients with hereditary breast cancer. Our results showed a novel PIK3CA hotspot mutation in Tunisian breast cancer patients detected by HRM-COLD-PCR. Moreover, the absence of PIK3CA hotspot mutation associated with good prognosis.

Structural Basis for Substrate Binding, Catalysis and Inhibition of Breast Cancer Target Mitochondrial Creatine Kinase by Covalent Inhibitor via Cryo-EM.

Mitochondrial creatine kinases are key players in maintaining energy homeostasis in cells by working in conjunction with cytosolic creatine kinases for energy transport from mitochondria to cytoplasm. High levels of MtCK observed in Her2+ breast cancer and inhibition of breast cancer cell growth by substrate analog, cyclocreatine, indicate dependence of cancer cells on the 'energy shuttle' for cell growth and survival. Hence, understanding the key mechanistic features of creatine kinases and their inhibition plays an important role in the development of cancer therapeutics. Herein, we present the mutational and structural investigation on understudied ubiquitous mitochondrial creatine kinase (uMtCK). Our cryo-EM structures and biochemical data on uMtCK showed closure of the loop comprising residue His61 is specific to and relies on creatine binding and the reaction mechanism of phosphoryl transfer depends on electrostatics in the active site. In addition, the previously identified covalent inhibitor CKi showed inhibition in breast cancer BT474 cells, however our biochemical and structural data indicated that CKi is not a potent inhibitor for breast cancer due to strong dependency on the covalent link formation and inability to induce conformational changes upon binding.

Role of mesoporous silica functionalized with boronic acid derivative in targeted delivery of doxorubicin and co-delivery of doxorubicin and resveratrol

Phenylboronic acid derivatives have gained interest due to their ability to reversibly bind to 1,2-diols, such as sialic acid receptors overexpressed by breast cancer cells. In this study, two types of mesoporous silica, MCM-41 and SBA-15, were functionalized with 4-carboxyphenylboronic acid (CPBA) through the amidation reaction, and the resulting materials MCM-CPBA and SBA-CPBA were used as carriers for doxorubicin (Dox) or for co-delivery of doxorubicin and resveratrol. In the case of MCM-CPBA material, all amine groups were involved in the condensation reaction with boronic acid derivative, while in the case of SBA-CPBA, free amine groups remained on the silica surface. Dox release profiles, performed in phosphate buffer solution pH 5.5, showed a faster release kinetics of Dox and a higher cumulative drug release for co-delivery system. Larger pores of SBA-15-type carrier influenced the Dox release profile as the diffusion of drug molecules was favored, a higher cumulative drug release being obtained in the case of SBA-CPBA than for MCM-CPBA. Biological assessment of the developed drug delivery systems demonstrated lower cytotoxicity on BJ fibroblasts than on BT474 breast cancer cells. Evaluation of drug delivery systems by hyperspectral microscopy evidenced a higher internalization rate of Dox when was loaded on functionalized silica carriers compared to the free drug into BT474 cells. The internalization rate of doxorubicin-loaded carrier depended on the type of carrier; a better internalization was observed for cancer cells when were treated with Dox-loaded MCM-CPBA nanoparticles than for Dox-loaded SBA-CPBA that might be attributed to smaller size of MCM-CPBA nanoparticles.

Bispecific aptamer-decorated and light-triggered nanoparticles targeting tumor and stromal cells in breast cancer derived organoids: implications for precision phototherapies

BackgroundBased on the established role of cancer-stroma cross-talk in tumor growth, progression and chemoresistance, targeting interactions between tumor cells and their stroma provides new therapeutic approaches. Dual-targeted nanotherapeutics selectively acting on both tumor and stromal cells may overcome the limits of tumor cell-targeting single-ligand nanomedicine due to the complexity of the tumor microenvironment.MethodsGold-core/silica-shell nanoparticles embedding a water-soluble iridium(III) complex as photosensitizer and luminescent probe (Iren-AuSiO2_COOH) were efficiently decorated with amino-terminated EGFR (CL4) and PDGFRβ (Gint4.T) aptamers (Iren-AuSiO2_Aptamer). The targeting specificity, and the synergistic photodynamic and photothermal effects of either single- and dual-aptamer-decorated nanoparticles have been assessed by confocal microscopy and cell viability assays, respectively, on different human cell types including mesenchymal subtype triple-negative breast cancer (MES-TNBC) MDA-MB-231 and BT-549 cell lines (both EGFR and PDGFRβ positive), luminal/HER2-positive breast cancer BT-474 and epidermoid carcinoma A431 cells (only EGFR positive) and adipose-derived mesenchymal stromal/stem cells (MSCs) (only PDGFRβ positive). Cells lacking expression of both receptors were used as negative controls. To take into account the tumor-stroma interplay, fluorescence imaging and cytotoxicity were evaluated in preclinical three-dimensional (3D) stroma-rich breast cancer models.ResultsWe show efficient capability of Iren-AuSiO2_Aptamer nanoplatforms to selectively enter into target cells, and kill them, through EGFR and/or PDGFRβ recognition. Importantly, by targeting EGFR+ tumor/PDGFRβ+ stromal cells in the entire tumor bulk, the dual-aptamer-engineered nanoparticles resulted more effective than unconjugated or single-aptamer-conjugated nanoparticles in either 3D spheroids cocultures of tumor cells and MSCs, and in breast cancer organoids derived from pathologically and molecularly well-characterized tumors.ConclusionsOur study proposes smart, novel and safe multifunctional nanoplatforms simultaneously addressing cancer-stroma within the tumor microenvironment, which are: (i) actively delivered to the targeted cells through highly specific aptamers; (ii) localized by means of their luminescence, and (iii) activated via minimally invasive light, launching efficient tumor death, thus providing innovative precision therapeutics. Given the unique features, the proposed dual targeted nanoformulations may open a new door to precision cancer treatment.

Abstract 3999: Augmentation of antitumor effects by CAR-T cells secreting anti-PD-1 single-chain antibody fused with IL-21

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy has shown remarkable responses in hematologic malignancies. However, many patients suffer from limited response and tumor relapse due to lack of persisting CAR-T cells and immune escape. Delivery of immunomodulatory cytokines is one of the strategies to improve immune function; however, the systemic effect of cytokines can be detrimental. Methods: To prevent the toxic effects of cytokines, we proposed the concept of targeting cytokines to tumor-specific T cells in vivo. To this end, we developed CAR-T cells secreting a fusion protein (PD-1Ab21) composed of anti-PD-1 single chain antibody and IL-21, which target IL-21 to CAR-T cells to promote their proliferation and differentiation into memory cells while blocking the PD-1 signal of CAR-T cells. The secretion and functions of PD-1Ab21 were identified. The proliferation and differentiation of CAR-T cells secreting PD-1Ab21 (PD-1Ab21-CAR-T) were compared in vitro with that of the conventional CAR-T cells. The therapeutic effects of CAR-T cells secreting PD-1Ab21 were studied in immunodeficient mice engrafted with human Burkitt lymphoma Daudi, multiple myeloma cell U266 and Her2/neu+ breast cancer cell BT474. Results: The results showed that the culture supernatant of PD-1Ab21-CAR-T cells, like anti-PD-1 antibody, blocked the binding of PD-1 to PD-L1 and stimulated STAT3 phosphorylation in human T lymphocytic leukemia cell Hut78 with the same activity as IL-21. In the conventional culture milieu with high dose of IL-2, the proliferation of the PD-1Ab21-CAR-T cells was not different from that of conventional CAR-T cells. While cultured with low dose of IL-2 that could not support the proliferation of conventional CAR-T cells, the PD-1Ab21-CAR-T cells still showed significant proliferation. A major fraction of the PD-1Ab21-CAR-T products was shown to co-express CCR7, CD62L and CD45RA, and express high level of TCF-1, indicating a memory stem-like phenotype. While the most of conventional CAR-T cells showed effector phenotype defined as CD45RA+CCR7-. Furthermore, adoptive transfer of PD-1Ab21-CAR-T cells exhibited significantly improved CAR-T cell expansion and resulted in superior tumor eradication in the xenograft models of human B-cell malignancy, multiple myeloma and Her2/neu+ breast cancer. The enhanced cellular function by secreting PD-1Ab21 was mediated through an autocrine and/or paracrine manners. Conclusion: We developed an enhanced CAR-T cells. Our data provide preclinical evidence to support the translation of this design for an improved CAR-T cell-mediated anti-tumor response. Citation Format: Ying Li, Jitao Zhao, Xiangrui Cheng, Hang Li, Ran Tian, Qianqian He, Shengdian Wang. Augmentation of antitumor effects by CAR-T cells secreting anti-PD-1 single-chain antibody fused with IL-21 [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 3999.

Abstract 2672: GD3 synthase (ST8SIA1) is a key immunomodulator in GD2+ breast cancer cells

Abstract Background: Gangliosides are acidic glycosphingolipids involved in cell adhesion, proliferation, and modulation of signal transduction pathways. It has been reported that tumor-shed gangliosides influence the activity of immune cells, including macrophages, NK cells, and T cells. GD3 synthase (GD3S) is the key enzyme that regulates the biosynthesis of the b and c-series gangliosides, particularly GD3 and GD2, and studies have shown that GD3S is upregulated in most tumors and plays a role in tumor progression. Similarly, we have found GD3S to be significantly upregulated in GD2+ breast cancer stem cells (BCSC) compared to GD2− cells, and the knockdown of this gene prevented tumor formation in mice. Here, we hypothesize that GD3 synthase has an immunomodulatory function in breast cancer (BC) cells through the upregulation of GD2. Methods: We stably overexpressed GD3S in BT549 and MCF-7 breast cancer cell lines by lentiviral transfection and performed CRISPR-Cas9 knockout of GD3S in BT549 and SUM 159 cell lines. We examined the effect of GD3 synthase on macrophage-mediated phagocytosis, NK cell-mediated killing, and T-cell cytotoxicity of BC cells using the live cell imaging system, IncuCyte®. Naxitamab is a US FDA-approved fully humanized anti-GD2 monoclonal antibody for the treatment of neuroblastoma. We evaluated the effects of naxitamab in GD2+ BC cell lines (HCC1395, Hs578T) using the Incucyte, and also examined its effect on in vivo tumor growth using TNBC PDX models. Results: We observed a highly significant decrease in macrophage-mediated phagocytosis (p<0.005; p<0.0001) and NK-mediated killing (p<0.0001; p<0.0001) in BT549 and MCF-7 cell overexpressing GD3S compared to the control cells. Interestingly, we observed a significant increase in phagocytosis in GD3S knockout BT549 and SUM 159 cells (p<0.05; p<0.005) compared to their wild-type counterparts. Moreover, the knockout of GD3S enhanced NK cell-mediated apoptosis in BT549 cells (p<0.001) and T cell killing in BT549 and SUM 159 cells (p<0.0001; p<0.0001). Finally, we found that naxitamab dose-dependently enhances macrophage-mediated phagocytosis and NK cell-mediated apoptosis in HCC1395 and Hs578T BC cells, with 20µg/ml being the most effective concentration in comparison to IgG control (p<0.0001; p<0.0001). In addition, we observed a reduction in tumor volume in naxitamab-treated mice compared to IgG control (p<0.05) Conclusion: GD3 synthase regulates macrophage-mediated phagocytosis, NK cell-induced apoptosis, and T-cell cytotoxicity in BC cells. Naxitamab enhances immune cell killing of breast cancer cells with high GD2 expression. Furthermore, naxitamab inhibited tumor growth in GD2+ TNBC PDX models. Citation Format: Bolutyfe Oderinde, Vivek Anand, Maryam Siddiqui, Anudishi Tyagi, Borgman Jenny, Venkata Lokesh Battula. GD3 synthase (ST8SIA1) is a key immunomodulator in GD2+ breast cancer cells [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 2672.

Abstract 3071: Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt inhibitors

Abstract Advances in research have shown that cancer cells have distinctive bioenergetic alterations to meet their energy requirements. It has been established that certain cancers are significantly reliant on mitochondrial respiration, suggesting that modulation of mitochondrial function may have therapeutic value. The Wnt signaling pathway is a potential therapeutic target of cancer, including breast and colorectal cancers. Recent findings suggest that bi-directional crosstalk between Wnt signaling and mitochondrial function plays a crucial role in tumorigenesis, as well as differentiation and self-renewal of stem cells. In this study, 71 agents known to alter Wnt signaling were screened for effects on mitochondrial function in BT474 and Hs578T breast cancer cell lines, which have highly oxidative and highly glycolytic bioenergetic phenotypes, respectively. This screen was performed using the Agilent Seahorse XF Mito Tox assay, which enables sensitive detection and characterization of mitochondrial modulators via a standardized, quantitative parameter, the Mito Tox Index (MTI). The MTI facilitates the comparison of drug efficacy by detecting modes of mitochondrial toxicity, inhibition, and uncoupling. The MTI-based screening of the Wnt modulators identified one inhibitor and two uncouplers as potent agents inducing mitochondrial dysfunction for both BT474 and Hs578T. Despite their distinctive bioenergetic phenotypes, MTI-based dose-response assays revealed no significant differences in the efficacy of these modulators between BT474 and Hs578T cell types. In contrast, significant differential effects on ATP production rates were detected, showing cellular energy depletion induced by these agents. Using the Agilent Seahorse XF Real-Time ATP Rate assay, BT474 cells showed significant metabolic energy crisis in the presence of all 3 compounds, whereas Hs578T cells maintained stable total ATP production. This differential impact on ATP production rate corresponds directly to differences in cell viability, with BT474 showing higher susceptibility to these compounds. These results suggest that the anti-cancer efficacy of mitochondrial modulators can be different depending on the metabolic phenotype of the target cell and should be considered in therapeutic design. Citation Format: Yoonseok Kam, Lisa Winer, George W. Rogers, Natalia Romero. Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt inhibitors [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 3071.

CLEC3B inhibits proliferation and radioresistance and induces apoptosis in BT549 breast cancer cells

Among all breast cancer subtypes, triple-negative breast cancer (TNBC) has the worst prognosis due to its resistance to treatment, including radiotherapy. The purpose of this study was to screen and validate potential targets related to radioresistance in TNBC. First, a radiation-resistant TNBC cell model, was generated using BT549 cells through fractionated radiation (30 Gy in total, 10 fractions). Then, mRNA sequencing was used to screen potential target genes related to radioresistance. Furthermore, bioinformatics, proteomics and genomics methods were used to screen and validate the key target genes. The results showed that BT549 cells that had undergone fractionated radiation, namely, BT549R cells, had greater proliferation and radioresistance and a lower apoptosis rate than parental BT549 cells. C-type lectin domain family 3 member B (CLEC3B) was identified as a potential key target gene related to radioresistance. The expression of CLEC3B was closely related to the radioresistance of breast cancer and the survival of breast cancer patients. Downregulation of CLEC3B increased cell viability and radioresistance and reduced apoptosis in BT549R cells, which could be reversed by dihydroartemisinin (DHA). The overexpression of CLEC3B with or without DHA treatment inhibited the activity of the PI3K/AKT signaling pathway. DHA increased the expression of CLEC3B in a concentration-dependent manner. In summary, our study illustrated that the downregulation of CLEC3B increased radioresistance in BT549 breast cancer cells and that this effect could be reversed by DHA.

The Transcriptome of BT-20 Breast Cancer Cells Exposed to Curcumin Analog NC2603 Reveals a Relationship between EGR3 Gene Modulation and Cell Migration Inhibition.

Breast cancer represents a critical global health issue, accounting for a substantial portion of cancer-related deaths worldwide. Metastasis, the spread of cancer cells to distant organs, is the primary cause of approximately 90% of breast cancer-related fatalities. Despite advances in cancer treatment, conventional chemotherapeutic drugs often encounter resistance and demonstrate limited efficacy against metastasis. Natural products have emerged as promising sources for innovative cancer therapies, with curcumin being one such example. However, despite its therapeutic potential, curcumin exhibits several limitations. Analogous compounds possessing enhanced bioavailability, potency, or specificity offer a promising avenue for overcoming these challenges and demonstrate potent anti-tumor activities. Our study investigates the antimetastatic potential of the curcumin analog NC2603 in breast cancer cells, utilizing BT-20 cells known for their migratory properties. Cell viability assessments were performed using the MTT reduction method, while migration inhibition was evaluated through scratch and Transwell migration assays. Transcriptome analysis via next-generation sequencing was employed to elucidate gene modulation and compound mechanisms, with subsequent validation using RT-qPCR. The IC50 of NC2603 was determined to be 3.5 μM, indicating potent inhibition of cell viability, and it exhibited greater specificity for BT-20 cells compared with non-cancerous HaCaT cells, surpassing the efficacy of doxorubicin. Notably, NC2603 demonstrated superior inhibition of cell migration in both scratch and Transwell assays compared with curcumin. Transcriptome analysis identified 10,620 modulated genes. We validated the expression of six: EGR3, ATF3, EMP1, SOCS3, ZFP36, and GADD45B, due to their association with migration inhibition properties. We hypothesize that the curcumin analog induces EGR3 expression, which subsequently triggers the expression of ATF3, EMP1, SOCS3, ZFP36, and GADD45B. In summary, this study significantly advances our comprehension of the intricate molecular pathways involved in cancer metastasis, while also examining the mechanisms of analog NC2603 and underscoring its considerable potential as a promising candidate for adjuvant therapy.

Synthesis and characterization of bis-amide SSE1917 as a microtubule-stabilizing anticancer agent

Microtubule dynamics are critical for spindle assembly and chromosome segregation during cell division. Pharmacological inhibition of microtubule dynamics in cells causes prolonged mitotic arrest, resulting in apoptosis, an approach extensively employed in treating different types of cancers. The present study reports the synthesis of thirty-two novel bis-amides (SSE1901-SSE1932) and the evaluation of their antiproliferative activities. N-(1-oxo-3-phenyl-1-(phenylamino)propan-2-yl)benzamide (SSE1917) exhibited the most potent activity with GI50 values of 0.331 ± 0.01 µM in HCT116 colorectal and 0.48 ± 0.27 µM in BT-549 breast cancer cells. SSE1917 stabilized microtubules in biochemical and cellular assays, bound to taxol site in docking studies, and caused aberrant mitosis and G2/M arrest in cells. Prolonged treatment of cells with the compound increased p53 expression and triggered apoptotic cell death. Furthermore, SSE1917 suppressed the growth of both mouse and patient-derived human colon cancer organoids, highlighting its potential therapeutic value as an anticancer agent.

Progesterone receptor-Grb2 interaction is associated with better outcomes in breast cancer

In addition to mediating nuclear transcription, PR mediates extranuclear functions mainly through the PR polyproline domain (PPD) interaction with the SH3 domain of cytoplasmic signaling molecules. PR-PPD-SH3 interaction inhibits EGF-mediated signaling and decreases lung cancer cell proliferation. Grb2 is an essential adaptor molecule with an SH2 domain flanked by two SH3 domains. In this study, we examined whether PR, through interaction between PR-PPD and Grb2-SH3, can interact with Grb2 in cells and breast cancer tissues. Our previous study shows that interaction between PR-PPD and Grb2 could interfere with cytoplasmic signaling and lead to inhibition of EGF-mediated signaling. GST-pulldown analysis shows that PR-PPD specifically interacts with the SH3 domains of Grb2. Immunofluorescence staining shows colocalization of PR and Grb2 in both the nucleus and cytoplasm in BT-474 breast cancer cells. Using Bimolecular Fluorescence Complementation (BiFC) analysis, we show that PR and Grb2 interact in breast cancer cells through the Grb2-SH3 domain. Proximity Ligation Assay (PLA) analysis of 43 breast cancer specimens shows that PR-Grb2 interaction is associated with low histological stage and negatively correlates with lymph node invasion and metastasis in breast cancer. These results, together with our previous findings, suggest that PR-PPD interaction with Grb2 plays an essential role in PR-mediated growth factor signaling inhibition and could contribute significantly to better prognosis in PR- and Grb2-positive breast cancer. Our finding provides a basis for additional studies to explore a novel therapeutic strategy for cancer treatment.

In Vitro Evaluation of Cytotoxic and Antitumor Activities of The Tamoxifen and Doxorubicin Combination on MCF-7 and BT-474 Breast Cancer Cell Lines

The combination therapy of breast cancer has preferred for the patients to minimize possible side effects, drug resistance, recurrence and toxic effects. In this study, we aim to investigate the cytotoxic and antitumor activities the tamoxifen and doxorubicin combination in breast cancer cell lines, MCF-7 and BT-474. Tamoxifen (Tam) and doxorubicin (Dox) and their combination with different concentrations (0.625–20 μM Tam; 0.0625–2 μM Dox and 5 μM Tam+ 0.5/1.0/1.5 μM Dox combination were applied to MCF-7 and BT-474 cells for 48 hours. Afterthat, their cytotoxic activities were analyzed with MTT assay. Bcl-2, Mapt and Mrp1 are genes that induce cell proliferation, inhibit apoptosis and play role in drug resistance in cancer cells. To evaluate the antitumor activities of these genes in combination treatment, mRNA levels were analyzed by quantitative PCR. According to the MTT assay, it was determined that IC50 values as 17.26 μM and 16.65 μM for tamoxifen on MCF-7 and BT-474 breast cancer cell lines. IC50 values of doxorubicin in MCF-7 and BT-474 cells were 1.65 μM and 1.57 μM, respectively. It was found that the application of the combination drugs (15 μM tamoxifen and 1.5 μM doxorubicin) in MCF-7 and BT-474 cells have the lowest combination index values as 1.09 and 1.26, respectively. Therefore, the combination of 15 μM tamoxifen and 1.5 μM doxorubicin was selected and applied to both breast cancer cell lines for gene expression analysis. It was found that while Mrp1 and Mapt genes expressions were significantly upregulated, Bcl-2 gene expression was downregulated in MCF-7 cells. However, Bcl-2, Mrp1 and Mapt genes expressions in BT-474 cells were not significantly regulated. Altogether, these findings suggest that the combination of these two drugs may have a potential antagonistic interaction according combination index values.

Targeted Hyperbranched Nanoparticles for Delivery of Doxorubicin in Breast Cancer Brain Metastasis.

Breast cancer brain metastases (BM) are associated with a dismal prognosis and very limited treatment options. Standard chemotherapy is challenging in BM patients because the high dosage required for an effective outcome causes unacceptable systemic toxicities, a consequence of poor brain penetration, and a short physiological half-life. Nanomedicines have the potential to circumvent off-target toxicities and factors limiting the efficacy of conventional chemotherapy. The HER3 receptor is commonly expressed in breast cancer BM. Here, we investigate the use of hyperbranched polymers (HBP) functionalized with a HER3 bispecific-antibody fragment for cancer cell-specific targeting and pH-responsive release of doxorubicin (DOX) to selectively deliver and treat BM. We demonstrated that DOX-release from the HBP carrier was controlled, gradual, and greater in endosomal acidic conditions (pH 5.5) relative to physiologic pH (pH 7.4). We showed that the HER3-targeted HBP with DOX payload was HER3-specific and induced cytotoxicity in BT474 breast cancer cells (IC50: 17.6 μg/mL). Therapeutic testing in a BM mouse model showed that HER3-targeted HBP with DOX payload impacted tumor proliferation, reduced tumor size, and prolonged overall survival. HER3-targeted HBP level detected in ex vivo brain samples was 14-fold more than untargeted-HBP. The HBP treatments were well tolerated, with less cardiac and oocyte toxicity compared to free DOX. Taken together, our HER3-targeted HBP nanomedicine has the potential to deliver chemotherapy to BM while reducing chemotherapy-associated toxicities.