Metastatic Breast Cancer Cell Line Research Articles

Inhibition of glycolysis and Src/Akt signaling reduces Caveolin-1-enhanced metastasis

Metastasis is the leading cause of cancer-related deaths, making the development of novel, more effective therapies imperative to alleviate patient suffering. Metabolic switching is a hallmark of cancer cells that facilitates metastasis. Cancer cells obtain most of their energy and intermediate metabolites, which are required to proliferate and metastasize, through aerobic glycolysis. Previous work from our laboratory has shown that Caveolin-1 (CAV1) expression in cancer cells promotes glycolysis and metastasis. Here, we sought to determine if limiting glycolysis reduced CAV1-enhanced metastasis and to identify the mechanism(s) involved. We evaluated the effects of the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) in metastatic melanoma and breast cancer cell lines expressing or not CAV1. Non-cytotoxic concentrations of 2-DG (1 mM) inhibited the migration of B16-F10 melanoma and MDA-MB-231 breast cancer cells. CAV1-mediated activation of Src/Akt signaling was required for CAV1-enhanced migration and was blocked in the presence of 2-DG. Moreover, inhibition of Akt reduced CAV1-enhanced lung metastasis of B16-F10 cells. Collectively, these findings highlight the importance of CAV1-induced metabolic reprogramming for metastasis and point towards possible therapeutic approaches to prevent metastatic disease by inhibiting glycolysis and Src/Akt signaling.

A NIR-Fluorochrome for Live Cell Dual Emission and Lifetime Tracking from the First Plasma Membrane Interaction to Subcellular and Extracellular Locales.

Molecular probes with the ability to differentiate between subcellular variations in acidity levels remain important for the investigation of dynamic cellular processes and functions. In this context, a series of cyclic peptide and PEG bio-conjugated dual near-infrared emissive BF2-azadipyrromethene fluorophores with maxima emissions at 720 nm (at pH > 6) and 790 nm (at pH < 5) have been developed and their aqueous solution photophysical properties determined. Their inter-converting emissions and fluorescence lifetime characteristics were exploited to track their spatial and temporal progression from first contact with the plasma membrane to subcellular locales to their release within extracellular vesicles. A pH-dependent reversible phenolate/phenol interconversion on the fluorophore controlled the dynamic changes in dual emission responses and corresponding lifetime changes. Live-cell confocal microscopy experiments in the metastatic breast cancer cell line MDA-MB-231 confirmed the usability of the dual emissive properties for imaging over prolonged periods. All three derivatives performed as probes capable of real-time continuous imaging of fundamental cellular processes such as plasma membrane interaction, tracking endocytosis, lysosomal/large acidic vesicle accumulation, and efflux within extracellular vesicles without perturbing cellular function. Furthermore, fluorescence lifetime imaging microscopy provided valuable insights regarding fluorophore progression through intracellular microenvironments over time. Overall, the unique photophysical properties of these fluorophores show excellent potential for their use as information-rich probes.

Comprehensive studies on the biological activities of human metastatic (MDA-MB-231) and non-metastatic (MCF-7) breast cancer cell lines, directly or combinedly treated using non-thermal plasma-based approaches

Progressive incidence and a pessimistic survival rate of breast cancer in women worldwide remains one of the most concerning topics. Progressing research indicates a potentially high effectiveness of use cold atmospheric plasma (CAP) systems. The undoubted advantage seems its simplicity in combination with other anti-cancer modalities. Following observed trend of studies, one inventory CAP system was applied to directly treat human breast cancer cell lines and culturing in two different Plasma Activated Media (PAM) for combined utilization. Proposed CAP treatments on MCF-10 A, MCF-7, and MDA-MB-231 cell lines were studied in terms of impact on cell viability by MTT assay. Disturbances in cell motility following direct and combined CAP application were assessed by scratch test. Finally, the induction of apoptosis and necrosis was verified with annexin V and propidium iodide staining. Reactive species generated during CAP treatment were determined based on optical emission spectrometry analysis along with colorimetric methods to qualitatively assess the NO2−, NO3−, H2O2, and total ROS with free radicals concentration. The most effective approach for CAP utilization was combined treatment, leading to significant disruption in cell viability, motility and mostly apoptosis induction in breast cancer cell lines. Determined CAP dose allows for mild outcome, showing insignificant harm for the non-cancerous MCF-10 A cell line, while the highly aggressive MDA-MB-231 cell line shows the highest sensitivity on proposed CAP treatment. Direct CAP treatment seems to drive the cells into the sensitive state in which the effectiveness of PAM is boosted. Observed anti-cancer response of CAP treatment was mostly triggered by RNS (mostly NO2− ions) and ROS along with free radicals (such as H2O2, OH•, O2-•, 1O2, HO2•). The combined application of one CAP source represent a promising alternative in the development of new and effective modalities for breast cancer treatment.

Iron oxide nanoparticles synthesized using Mentha spicata extract and evaluation of its antibacterial, cytotoxicity and antimigratory potential on highly metastatic human breast cells

Iron oxide nanoparticles (Fe2O3 NPs) were synthesized utilizing Mentha spicata sourced from Cyprus as a stabilizing agent. The study delved into assessing the antimicrobial, cytotoxic, anti-proliferative, and anti-migratory potential of Fe2O3 NPs through disc diffusion, trypan blue, and 3-[4, 5-dimethylthiazol-2-yl]−2, 5-diphenyltetrazolium bromide (MTT) assay, respectively. Characterization of the synthesized Fe2O3 NPs was conducted using Fourier-transform infrared spectroscopy (FTIR), x-ray diffraction (XRD), UV–vis spectroscopy (UV–vis), scanning electron microscopy (SEM), and energy-dispersive x-ray spectroscopy (EDX). The FTIR, XRD, and SEM-EDX spectra confirmed the successful formation of Fe2O3 NPs. The analysis of UV–vis spectra indicates an absorption peak at 302 nm, thereby confirming both the successful synthesis and remarkable stability of the nanoparticles. The nanoparticles exhibited uniform spherical morphology and contained Fe, O, and N, indicating the synthesis of Fe2O3 NPs. Additionally, the Fe2O3 NPs formed through biosynthesis demonstrated antimicrobial capabilities against Escherichia coli and Bacillus cereus. The significant anti-migratory potential on MDA-MB 231 human breast cancer cells was observed with lower concentrations of the biosynthesized Fe2O3 NPs, and higher concentrations revealed cytotoxic effects on the cells with an IC50 of 95.7 μg/ml. Stable Fe2O3 NPs were synthesized using Mentha spicata aqueous extract, and it revealed antimicrobial activity on E. coli and B. cereus, cytotoxic, anti-proliferative and anti-migratory effect on highly metastatic human breast cancer cell lines.

Abstract 1881: Screening of Rac1 and Cdc42 inhibitors as anti-metastatic compounds

Abstract Metastasis is responsible for &amp;gt;90% of cancer-related deaths and is a key cause of failure of cancer therapy. The development of effective targeted therapy to prevent and treat metastasis has been challenging. Homologous Rho GTPases Rac and Cdc42 are viable metastasis targets because they have a crucial role in the signaling pathways that regulate cell survival, proliferation, and migration, so dysregulation of these proteins leads to cancer progression and metastasis of diverse cancer types. Therefore, targeting these regulatory proteins results in anticancer and antimetastatic activity, as observed with our dual Rac/Cdc42 inhibitor, MBQ-167, in triple-negative breast cancer. MBQ-167 has an IC50 of ~100nM for Rac and Cdc42 activation inhibition and a GI50 of 130nM. MBQ-167 inhibits the binding of guanine nucleotides to Rac and Cdc42 with similar efficacy. The purpose of this study was to identify MBQ-167 derivatives with similar or greater efficacy and specificity, with a range of inhibitory mechanisms, that may be specific to the different guanine nucleotide exchange factors expressed in distinct cancer cell types. Currently, over 30 different compounds with potential Rac1 and Cdc42 inhibitory activity are being evaluated. So far, some of the evaluated compounds have shown comparable GI50 values to MBQ-167 in metastatic breast cancer cell lines. From this screening, CPV-337 was selected as a compound with a GI50 of ~55nM and ~70% Rac activation inhibition at 50nM, therefore being 2X more effective than our leading compound MBQ-167. However, evaluation of CPV-337 in the viability of the MCF-10 non-tumorigenic epithelial cell line demonstrated a GI50 of ~63nM, which is similar to that of breast cancer cells. Therefore, CPV-337 may have cytotoxic effects on breast cancer. We are continuing to screen the library of MBQ-167 derivatives in breast, pancreatic, lung, and prostate cancer cell lines. Citation Format: Jessica Colón González, María C. Santa María Fuentes, Cornelis Vlaar, Suranganie Dharmawardhane. Screening of Rac1 and Cdc42 inhibitors as anti-metastatic compounds [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 1881.

Abstract 6764: Vascularized tumor-on-chip model for evaluating chemotherapeutic-mediated damage to adjacent healthy tissue

Abstract Introduction: Systemic chemotherapy is an effective anticancer treatment for all stages of breast cancer, with intravenous (IV) infusion being the principal method of administration. In addition to targeting cancer, systemic chemotherapy can impact the viability and function of surrounding normal tissues and vasculature. Early-discovery research tools comparing novel and existing systemic treatment strategies are urgently needed to support predictive efficacy and toxicity efforts. Microfluidic systems have the potential to fill this need while simultaneously reducing animal use. The overall goal of the study is to use an advanced microfluidic tumor-on-chip system to simultaneously evaluate the therapeutic and toxic effects of IV chemotherapy on normal and cancerous tissues. Materials and Methods: Synthetic Tumor Networks, which are comprised of primary and secondary tissue sites separated by an interconnected vasculature, were developed using in vivo images and fabricated using soft lithography. Primary vascular endothelial cells were used to establish microvasculature. A normal breast cell line (MCF 10A), or a GFP-labeled metastatic human breast cancer cell line (MDA-MB-231/GFP), were cultured in the tissue sites in a three-dimensional (3D) environment using a human-derived hydrogel. The vascular networks were perfused with endothelial cell media under physiological fluid flow conditions to establish the model. An EZH2 methyltransferase inhibitor (EZH2 inhibitor III) was delivered by IV infusion to the model tissues. Real-time monitoring of cellular growth, tumor invasion and extravasation, and cellular viability were performed using fluorescence microscopy over a two-week period. Results and Discussion: Metastatic MDA-MB-231/GFP breast cancer cells proliferated rapidly in the primary tumor site. Tumor intravasation into the vascular channels was observed in models that received no anticancer treatment, as well as extravasation into the secondary tissue site and invasion of the healthy breast tissue. Differences in tumor viability and migratory behavior were observed between untreated models or models receiving IV infusions of the EZH2 inhibitor through the vascular networks. Vascular barrier integrity and normal breast tissue viability were monitored at early (24-72 hr) and late (7-14 days) timepoints after anticancer treatment to measure short- and long-term adverse drug impacts. These results provide a unique perspective on the in vivo realism of an in vitro system for monitoring both therapeutic and toxic effects of systemic chemotherapies. Conclusions: We have developed a 3D vascularized tumor-on-chip model for monitoring the impacts of systemic chemotherapeutic agents on tumor and healthy tissues. This model can be used to investigate therapeutic and toxic effects on multiple tissue types simultaneously using real-time imaging techniques. Citation Format: Deborah Ramsey, Jenna Rosano, Cristabel Gordon, Gwen Fewell. Vascularized tumor-on-chip model for evaluating chemotherapeutic-mediated damage to adjacent healthy tissue [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 6764.

O-Glycome Profiling of Breast Cancer Cell Lines to Understand Breast Cancer Brain Metastasis.

Breast cancer is the second leading cause of cancer-related death among women and a major source of brain metastases. Despite the increasing incidence of brain metastasis from breast cancer, the underlying mechanisms remain poorly understood. Altered glycosylation is known to play a role in various diseases including cancer metastasis. However, profiling studies of O-glycans and their isomers in breast cancer brain metastasis (BCBM) are scarce. This study analyzed the expression of O-glycans and their isomers in human breast cancer cell lines (MDA-MB-231, MDA-MB-361, HTB131, and HTB22), a brain cancer cell line (CRL-1620), and a brain metastatic breast cancer cell line (MDA-MB-231BR) using nanoLC-MS/MS, identifying 27 O-glycan compositions. We observed significant upregulation in the expression of HexNAc1Hex1NeuAc2 and HexNAc2Hex3, whereas the expression of HexNAc1Hex1NeuAc1 was downregulated in MDA-MB-231BR compared to other cell lines. In our isomeric analysis, we observed notable alterations in the isomeric forms of the O-glycan structure HexNAc1Hex1NeuAc1 in a comparison of different cell lines. Our analysis of O-glycans and their isomers in cancer cells demonstrated that changes in their distribution can be related to the metastatic process. We believe that our investigation will contribute to an enhanced comprehension of the significance of O-glycans and their isomers in BCBM.

Low-dose decitabine enhances the efficacy of viral cancer vaccines for immunotherapy

Cancer immunotherapy requires a specific antitumor CD8+ Tcell-driven immune response; however, upon genetic and epigenetic alterations of the antigen processing and presenting components, cancer cells escape the CD8+ Tcell recognition. As a result, poorly immunogenic tumors are refractory to conventional immunotherapy. In this context, the use of viral cancer vaccines in combination with hypomethylating agents represents a promising strategy to prevent cancer from escaping immune system recognition. In this study, we evaluated the sensitivity of melanoma (B16-expressing ovalbumin) and metastatic triple-negative breast cancer (4T1) cell lines to FDA-approved low-dose decitabine in combination with PeptiCRAd, an adenoviral anticancer vaccine. The two models showed different sensitivity to decitabine invitro and invivo when combined with PeptiCRAd. In particular, mice bearing syngeneic 4T1 cancer showed higher tumor growth control when receiving the combinatorial treatment compared to single controls in association with a higher expression of MHC class I on cancer cells and reduction in Tregs within the tumor microenvironment. Furthermore, remodeling of the CD8+ Tcell infiltration and cytotoxic activity toward cancer cells confirmed the effect of decitabine in enhancing anticancer vaccines in immunotherapy regimens.

The emerging role of breast cancer derived extracellular vesicles-mediated intercellular communication in ovarian cancer progression and metastasis.

Breast cancer is one of the most occurring cancer types in women worldwide and metastasizes to several organs such as bone, lungs, liver, brain, and ovaries. Extracellular vesicles (EVs) mediate intercellular signaling which has a profound effect on tumor development and metastasis. Recent developments in the field of EVs provide an opportunity to investigate the roles of EVs released from tumor cells in metastasis. In this study, we compared the effects of metastatic breast cancer-derived EVs on both nonluteinized granulosa HGrC1 and ovarian cancer OVCAR-3 cells in terms of proliferation, invasion, apoptosis, and gene expression levels. EVs were isolated from the culture medium of metastatic breast cancer cell line MDA-MB-231 by ultracentrifugation. Cell proliferation, apoptosis, cell cycle, invasion, and cellular uptake analysis were performed to clarify the roles of tumor-derived EVs in both cells. 6.85 × 108 nanoparticles of BCD-EVs were markedly increased cell proliferation as well as invasion capacity. Exposing the cells with BCD-EVs for 24h, resulted in an accumulation of both cells in G2/M phase as determined by flow cytometry. The apoptosis assay results were consistent with cell proliferation and cell cycle results. The uptake of the BCD-EVs was efficiently internalized by both cells. In addition, marked variations in fatty acid composition between cells were observed. BCD-EVs appeared new fatty acids in HGrC1. Besides, BCD-EVs upregulated epithelial-mesenchymal transition (EMT) and proliferation-related genes. In conclusion, an environment of tumor-derived EVs changes the cellular phenotype of cancer and noncancerous cells and may lead to tumor progression and metastasis.

Abstract B105: Antineoplastic effects and expression modulations in proliferation/apoptosis related genes by riproximin in breast cancer cells

Abstract Background: Ribosomal inactivating proteins (RIPs) target the ribosomal machinery of target cells and stop the translational machinery irreversibly. Riproximin is a plant-based RIP which was extracted and purified from the plant “Ximenia americana”. Riproximin has demonstrated its anticancer effects against various in vitro and in vivo cancer models. In this study, the focus was to evaluate the effects of riproximin on proliferation and apoptosis of breast cancer cells at functional and molecular levels. Methods: MTT dye reduction assay and DAPI nuclear staining methodologies were used to highlight the riproximin mediated effects on proliferation and apoptosis in primary (MCF-7) and metastatic (MDA-MB-231) breast cancer cell lines. Afterwards, the two cell lines were used to investigate the expression modulations in genes related to PIK3/Akt and apoptosis pathways (84 genes/pathway) via real-time PCR methodology. The expression modulations were used to build the signalling pathways via Ingenuity Pathway Analysis supported by Qiagen software system. Results: Riproximin successfully halted the proliferation of the breast cancer cells with an average 50% inhibitory concentrations of 1.2 and 2.9 ng/ml after 72 hours exposure interval in MCF-7 and MDA-MB-231 cells, respectively. The protein also induced apoptotic effects in the two cell lines as reflected by shrinkage, blebbing and fragmentation of the nucleus/DNA content in a concentration dependant format. Substantial alterations (≥2fold) were observed in PIK3/Akt pathway genes (MCF-7: 49/84, MDA-MB-231: 28/84) in response to riproximin exposure. Similarly, the protein induced de-regulation (≥2fold) of apoptosis related 44/84 and 34/84 genes in MCF-7 and MDA-MB-231 cell lines, respectively. The Ingenuity Pathway Analysis revealed involvement of these genes at distinct positions in cell survival and death mechanisms. Conclusion: Riproximin demonstrated the substantial potential to induce the cytotoxic effects in breast cancer cells. The protein also de-regulated a significant number of proliferation and apoptosis related genes in breast cancer primary and metastatic cells. Further in vitro investigations are required to understand the fine molecular tuning being operated by this natural plant protein in cancer cells. Citation Format: Asim Pervaiz, Asma Khan, Kiran Umer, Martin R. Berger. Antineoplastic effects and expression modulations in proliferation/apoptosis related genes by riproximin in breast cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B105.

Metabolic Alterations Associated with Loss of TET2 Potentiates Inflammation in Myeloid Cells Driving Breast Cancer Progression

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by the clonal expansion of hematopoietic stem cells possessing somatic mutations commonly associated with cancer, but with no detectable hematological cancer. Certain CHIP driver mutations, such as loss of function in Tet2 result in hyperinflammatory myeloid cells. The proinflammatory cytokine IL-1β promotes breast cancer progression and correlates with disease severity. Activation of the NLRP3 inflammasome in myeloid cells is the main source of IL-1β processing. Here, we demonstrate growth of the metastatic breast cancer cell line, E0771, in both germline and chimeric Tet2 +/- mice was 2-fold greater than that observed in Tet2 +/+ mice (Figure 1). The increased tumor burden in Tet2 +/- mice was shown to be NLRP3-dependent as Tet2 +/-mice receiving the NLRP3 inhibitor, OLT1177, exhibited significantly decreased tumor growth compared to Tet2 +/- mice with no treatment (Figure 1). Cellular composition of the tumor microenvironment was also assessed. Notably, we observed marked increases in tumor-infiltrating granulocytes in the Tet2 +/- mice. This observation was consistent with elevated serum cytokines associated with neutrophil recruitment and activation. Next, considering NLRP3 inflammasome activation is tightly linked to metabolic alterations, we sought to characterize the metabolic profile of Tet2 +/- myeloid cells. To start, global metabolic analysis revealed increased abundance of metabolites from glycolysis and pentose phosphate pathway in Tet2 +/- myeloid cells compared to Tet2 +/+ myeloid cells. Consistent with the metabolic demands of a hyperinflammatory phenotype, Seahorse Flux assays revealed significantly increased ECAR (glycolysis) and OCR (pentose phosphate pathway) as measured at baseline in Tet2 +/- myeloid cells compared to Tet2 +/+ myeloid cells. Lastly, to confirm whether metabolic alterations drive the hyperinflammatory phenotype in Tet2 +/- myeloid cells, we assessed cytokine production after LPS stimulation in Tet2 +/-and Tet2 +/+ myeloid cells treated with 2-DG (glycolysis inhibitor) or 6-AN (pentose phosphate pathway inhibitor). Expectedly, Tet2 +/- myeloid cells produced significantly more IL-1β, IL-6, TNFα and IL-18 compared to Tet2 +/+ myeloid cells after LPS stimulation. LPS induced IL-1β and IL-6 production was significantly decreased for both genotypes after 2-DG and 6-AN treatment. Importantly, LPS induced TNFα and IL-18 only decreased after 2-DG and 6-AN treatment in Tet2 +/- myeloid cells suggesting metabolic changes associated with loss of TET2 affect not only NLRP3 priming but TNFα secretion as well. Overall, these studies demonstrate how TET2-driven CHIP alters the tumor microenvironment in breast cancers through aberrant NLRP3 activation and characterizes the underlying metabolic alterations subsequent to loss of TET2, offering further mechanistic insight into the hyperinflammatory phenotype in myeloid cells associated with TET2-driven CHIP. Figure 1. Tet2+/- driven breast cancer progression is NLRP3 activation-dependent. Experimental designs; BM chimeras were generated by transplanting 75%WT:25%Tet2+/+ or Tet2+/- mixed BM, mice were orthotopically implanted with E0771 breast tumors and given standard or OLT1177 diet (OLT). Tumor growth curve from Tet2+/+(WT) or Tet2 +/- chimeric mice implanted with E0771 cells receiving standard or OLT1177 diet(OLT) (N=10, two independent experiments). ***p&amp;lt;0.001, **p&amp;lt;0.01, *p&amp;lt;0.05.

Immuno-Modulatory Effects of Korean Mistletoe in MDA-MB-231 Breast Cancer Cells and THP-1 Macrophages

Korean mistletoe (Viscum album var. coloratum) has been traditionally used as a remedy for cancer, diabetes, and hypertension. This study investigated the immuno-modulatory effects of Korean mistletoe water extract, specifically on MDA-MB-231 cells, a highly metastatic breast cancer cell line, when co-cultured with THP-1 human macrophage cells. When compared to MDA-MB-231 cells cultured alone, the co-culture of MDA-MB-231/THP-1 cells treated with mistletoe extract showed a significant reduction in IL-6 secretion. Additionally, these co-cultures exhibited elevated levels of IL-4, TGF-β, and IFN-y. These results suggest that water extracts from mistletoe have the potential to induce mitochondria-targeted apoptosis in MDA-MB 231 cells stimulated by THP macrophages. Regarding apoptosis, in MDA-MB-231 cells co-cultured with THP-1 macrophages, mistletoe water extract treatment triggered a significant increase in Bax/Bcl-2 ratio, caspase-3 activation, and PARP inactivation. In addition, there was a significant increase in E-cadherin and a decrease in N-cadherin. Treatment of Korean mistletoe also led to significant reductions in both MMP-2 and -9. Furthermore, inhibition of cell migration in MDA-MB-231/THP-1 co-cultured cells was observed. In summary, this study highlights the potential of Korean mistletoe as a prospective drug for the treatment of triple-negative breast cancer, particularly through its ability to regulate human immunity.

Manipulating PTPRD function with ectodomain antibodies.

Protein tyrosine phosphatases (PTPs) are critical regulators of signal transduction but have yet to be exploited fully for drug development. Receptor protein tyrosine phosphatase δ (RPTPδ/PTPRD) has been shown to elicit tumor-promoting functions, including elevating SRC activity and promoting metastasis in certain cell contexts. Dimerization has been implicated in the inhibition of receptor protein tyrosine phosphatases (RPTPs). We have generated antibodies targeting PTPRD ectodomains with the goal of manipulating their dimerization status ectopically, thereby regulating intracellular signaling. We have validated antibody binding to endogenous PTPRD in a metastatic breast cancer cell line, CAL51, and demonstrated that a monoclonal antibody, RD-43, inhibited phosphatase activity and induced the degradation of PTPRD. Similar effects were observed following chemically induced dimerization of its phosphatase domain. Mechanistically, RD-43 triggered the formation of PTPRD dimers in which the phosphatase activity was impaired. Subsequently, the mAb-PTPRD dimer complex was degraded through lysosomal and proteasomal pathways, independently of secretase cleavage. Consequently, treatment with RD-43 inhibited SRC signaling and suppressed PTPRD-dependent cell invasion. Together, these findings demonstrate that manipulating RPTP function via antibodies to the extracellular segments has therapeutic potential.

Bee Venom Induces the Interaction between Phosphorylated Histone Variant, H2AX, and the Intracellular Site of beta-Actin in Liver and Breast Cancer Cells.

Bee venom is a natural mixture and candidate anti-cancer agent with selective cytotoxic effect on some cancer cells. However, the cellular mechanisms of how bee venom selectively targets cancer cells remain elusive. The aim of this study was to reveal the genotoxic effect of bee venom in concordance with the location of β-actin protein throughout the nucleus or/and cytoplasm. For this aim, the level of H2AX phosphorylation (γH2AX) and intracellular location of β-actin were assessed by immunofluorescence in liver (HEPG2) and metastatic breast (MDA-MB-231) cancer cell lines compared to normal fibroblasts (NIH3T3) after bee venom treatment. Colocalisation profiles of γH2AX and β-actin in each cell line were also analysed. The results showed that the levels of γH2AX staining decreased in normal cells but increased in cancer cells. The majority of β-actin was localised within the cytoplasm of normal cells after bee venom treatment, but it was mostly accumulated within the nucleus in cancer cells. Colocalisation of β-actin and γH2AX both in nucleus and cytoplasm was induced in each cancer cell by different patterns. The results showed that normal and cancerous cells had different responses against bee venom, and suggested that bee venom induced a cellular response by the interaction between γH2AX and β-actin.

Cytotoxic Activity, Anti-Migration and In Silico Study of Black Ginger (Kaempferia parviflora) Extract against Breast Cancer Cell.

Metastatic breast cancer remains the leading cause of death in women worldwide. This condition necessitates extensive research to find an effective treatment, one of which is the natural medicine approach. Kaempferia parviflora (KP) is a plant believed to possess anticancer properties. Therefore, this study aims to determine KP's bioactive compound, cytotoxic, and anti-migration activity in the highly metastatic breast cancer cell line model 4T1, also in the breast cancer cell model MCF-7 and noncancerous cell line NIH-3T3. Maceration with ethanol (EEKP) and infusion with distilled water (EWKP) was used for extraction. The MTT assay was used to test for cytotoxicity, and the scratch wound healing assay was used to test for the inhibition of migration. Phytochemical profiling of EEKP was performed using UHPLC-MS, and the results were studied for in silico molecular docking. Result showed that EEKP had a better cytotoxic activity than EWKP with an IC50 value of 128.33 µg/mL (24 h) and 115.09 µg/mL (48 h) on 4T1 cell line, and 138.43 µg/mL (24 h) and 124.81 µg/mL (48 h) on MCF-7 cell line. Meanwhile, no cytotoxic activity was observed at concentrations ranging from 3-250 µg/mL in NIH-3T3. EEKP also showed anti-migration activity in a concentration of 65 µg/mL. Mass Spectrophotometer (MS) structures from EEKP are 5-Hydroxy-7,4'-dimethoxyflavanone (HDMF), 5-Hydro-7,8,2'-trimethoxyflavanone (HTMF), Retusine, and Denbinobin. The in silico docking was investigated for receptors Bcl-2, Bcl-XL, ERK2, and FAK, as well as their activities. In silico result indicates that HTMF and denbinobin are bioactive compounds responsible for EEKP's cytotoxic and anti-migration activity. These two compounds and standardized plant extract can be further studied as potential breast cancer treatment candidates.

Abstract 2657: Preclinical model to evaluate the safety and efficacy of PODO83: A monoclonal antibody against podocalyxin that targets tumor growth and metastasis

Abstract Podocalyxin (PODXL) is a highly glycosylated transmembrane sialomucin normally found on the apical free surface of mesothelial, vascular endothelial, luminal mammary epithelial cells, kidney podocytes and pluripotent and hematopoietic stem cells. We were the first to show that PODXL is upregulated on human cancers and an independent predictor of poor prognosis in breast and high-grade serous ovarian carcinoma (HGSOC). We also demonstrated that PODXL expression is crucial for primary tumor growth, local invasion, and experimental metastasis to distant organs. Subsequently, these results were extended to a wide range of other tumor types. We next developed a large series of high-affinity monoclonal antibodies (mAb) to human PODXL and evaluated these for therapeutic activity against the PODXL+ metastatic breast cancer cell line, MDA-MB-231, in xenografted NOD-SCID IL2Rγ-/- (NSG) mice. One of these, PODO83, potently attenuated primary tumor growth and formation of distal metastases. The PODO83 reactive epitope maps to the juxtamembrane stalk domain (non-glycosylated region) of the PODXL core protein. As a promising therapeutic it is imperative to perform toxicity studies and determine if PODO83 adversely affects the function of PODXL on normal tissues. Accordingly, we generated a transgenic mouse strain (C57Bl/6 congenic) replacing 4 of the 8 mouse PODXL-encoding exons with the orthologous human exons (including those encoding the PODO83 epitope). These mice were healthy and we confirmed by immunohistochemistry that they exhibit appropriate PODO83 immunoreactivity in all expected tissues (podocytes, endothelia, etc). Strikingly, administration of PODO83 in vivo at high doses (4 treatments of 5 mg/kg over 14 days) resulted in no overt signs of toxicity. To now fully assess the safety and efficacy profile of the PODO83 mAb in this preclinical humanized mouse model we have developed mouse metastatic tumor cell line expressing human PODXL and will evaluate the ability of PODO83 to inhibit tumor growth and metastasis while sparing normal tissues in vivo. The results from this study will help us advance PODO83 mAb as a safe and effective approach to treating the wide variety of cancer subtypes that express PODXL. Citation Format: Julyanne Brassard, Michael R. Hughes, Pamela Dean, Diana Canals Hernaez, Janella Schwab, Yasmine Lau, Calvin D. Roskelley, Kelly M. McNagny. Preclinical model to evaluate the safety and efficacy of PODO83: A monoclonal antibody against podocalyxin that targets tumor growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2657.

Abstract 2466: Detection of extracellular vesicle-associated DNA in mouse metastatic breast cancer cells

Abstract Background: Breast cancer is the second most common type of cancer worldwide, and while primary disease is often well controlled, metastatic breast cancer (MBC) is responsible for the majority of deaths. MBC cells can metastasize to distant sites including the liver, lung, brain and bone, and the mechanisms underlying this organotropism remain largely unknown. MBC has high genetic heterogeneity, and studies have reported mutations in oncogenic pathways such as TP53, PIK3CA and ESR1. Liquid biopsy has been used to detect extracellular vesicle-associated DNA (EV-DNA), bypassing the limits of tissue biopsies often invasive and difficult to obtain. EVs are nanoparticles of diverse sizes released in the extracellular environment by all types of cells and are known to carry cargos including EV associated proteins, lipids, RNA and DNA with the potential to interact with surrounding cells and be explored as markers of disease progression. The goal of our study was to detect EV associated oncogenes derived from MBC cell lines. EV-DNA can potentially be incorporated into cancer diagnosis and prognosis in the clinical setting. Methods: In this study, we isolated EVs from 4T1 mouse mammary tumor model with primary cells and 4T1 induced liver-metastatic variants (2776, 2792 cells). We isolated and characterized the EVs using nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) for particle size and concentration, as well as western blot (WB) for EV markers. EV-DNA was isolated using the Gentra puregene blood kit (Qiagen) and quantified using a Qubit fluorometer, and oncogenic mutations assessed using droplet digital polymerase chain reaction (ddPCR) to detect mutant PIK3CA H1047R, TP53 P31T, and ESR1 D538G. Results: EVs isolated from primary and liver metastatic MBC cell lines were positive for EV markers synthenin-1 and CD63. NTA showed higher concentrations of EVs from liver-metastatic variants cell lines (2776: 1.11x1011 particles/ml, mean size 109.2 nm; 2792: 1.37x1011 particles/ml, mean size 122.7 nm) compared to primary tumor cells (4T1: 5.83x1010 particles/ml; mean size: 120.3 nm). These results suggest an increased EV emission from metastatic variants cell lines compared to parental cells. Interestingly, significantly higher EV-DNA concentration was found in liver-metastatic cells (2776: 2.85 ng/μl; 2792: 1.48 ng/μl) than in parental cells (0.23 ng/μl). Conclusions: These findings suggest circulating oncogenic EV-DNA may be a useful biomarker to monitor MBC patients throughout disease progression. For future investigation of EV enriched material, we will assess the mutant copies of oncogenes TP53 P31T, PIK3CA H1047R and ESR1 D538G associated with the EVs of parental cells and metastatic variants. Extensive studies in vitro and in vivo are required to validate and clarify definitive roles of EVs in tumor invasion and to further our understanding of the role of EV cargo in spreading malignancies. Citation Format: Amélie Nadeau, Kyle Dickinson, Thupten Tsering, Emilie Solymoss, Sébastien Tabariès, Peter Siegel, Julia Burnier. Detection of extracellular vesicle-associated DNA in mouse metastatic breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2466.

Abstract 2051: MDN1, a novel potential early biomarker for endocrine resistant breast cancer

Abstract Midasin (MDN1) is a chaperone protein required for maturation and nuclear export of pre-ribosomal RNA. Recent investigation regarding the mutational status of MDN1 in breast cancer (BC) revealed an increased tumor mutational burden in BC patients. Interestingly, our previously published work demonstrated significantly high MDN1 protein levels in AI-resistant mammospheres. Therefore, we investigated the overall status of MDN1 in the TCGA database in breast cancer tumors and identified MDN1 expression in breast cancer cells that were sensitive or resistant to AIs. The purpose of this study was to evaluate whether there is an association between MDN1 expression or mutation status to examine if MDN1 functions as an early high-risk indicator for breast cancers resistant to AIs or endocrine therapy. Using the cBioportal for Cancer Genomics, in silico analysis was conducted and BC patients with altered MDN1 status demonstrated several clinical attributes that were significantly associated with higher mutation counts (Kruskal Wallis test, p value = 1.41 × 10-10 and q value = 4.11 × 10-9); MSI sensor score, (p value = 2.74 × 10-5 and q value = 2.27 × 10-4); and Fraction Altered Genome score (p value = 2.37 × 10-3 and q value = 0.011). Next, aneuploidy in breast cancer significantly evolved and was positively correlated with higher MDN1 expression (Spearman: 0.64, p value = 3.92 × 10-122). Compared to the other BC subtypes, MDN1 was significantly elevated in basal breast cancer (Chi square test p value &amp;lt; 10.0 × 10-10, q value = 4.11 × 10-9) and significantly higher in deceased versus living BC patients. The DepMap breast cancer cell line database analysis also revealed significantly increased MDN1 expression in both ER+/HER2- cell lines (p value = 7.93 × 10-3; 12 cell lines) and in ER-/HER2- cell lines (p value = 2.88 × 10-2; 31 cell lines). Primary and metastatic BC cell lines were compared and MDN1 expression was significantly higher in metastatic BC cells (p value = 8.04 × 10-5; 33 metastatic cell lines). Likewise, AI-resistant cell lines exhibited altered MDN1 expression compared to their sensitive counterparts. Additionally, there was a statistically significant association between higher ER copy number and increased MDN1 expression in BC cell lines (p value = 1.44 × 10-3; 61 cell lines). Further in-depth analysis of proteins that were coexpressed in MDN1-altered tumors revealed a panel of genes (MSH2, MSH6, RBM15, FOXM1,CCNE2, and CHEK1) that could represent a signature of early markers of AI-resistant BC since there was a positive correlation between MDN1 expression and associated pathways. In conclusion, increased expression of MDN1 in BC tumors or cells may serve as preliminary marker to predict resistance to endocrine therapy due to its significant association with genes such as MSH2, MSH6 and FOXM1 that are strongly established as indicators of endocrine resistance. This work was supported by NIH grant # SC1GM126617 and U54MD007582. Citation Format: Afia Ohemeng, Akash Gupta, Bipika Banjara, Mounika M. Pamukuntla, Manasa Kotina, A Michael Davidson, Syreeta L. Tilghman. MDN1, a novel potential early biomarker for endocrine resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2051.

Sodium regulates PLC and IP3 R-mediated calcium signaling in invasive breast cancer cells.

Intracellular Ca2+ signaling and Na+ homeostasis are inextricably linked via ion channels and co-transporters, with alterations in the concentration of one ion having profound effects on the other. Evidence indicates that intracellular Na+ concentration ([Na+ ]i ) is elevated in breast tumors, and that aberrant Ca2+ signaling regulates numerous key cancer hallmark processes. The present study therefore aimed to determine the effects of Na+ depletion on intracellular Ca2+ handling in metastatic breast cancer cell lines. The relationship between Na+ and Ca2+ was probed using fura-2 and SBFI fluorescence imaging and replacement of extracellular Na+ with equimolar N-methyl-D-glucamine (0Na+ /NMDG) or choline chloride (0Na+ /ChoCl). In triple-negative MDA-MB-231 and MDA-MB-468 cells and Her2+ SKBR3 cells, but not ER+ MCF-7 cells, 0Na+ /NMDG and 0Na+ /ChoCl resulted in a slow, sustained depletion in [Na+ ]i that was accompanied by a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+ ]i ). Application of La3+ in nominal Ca2+ -free conditions had no effect on this response, ruling out reverse-mode NCX activity and Ca2+ entry channels. Moreover, the Na+ -linked [Ca2+ ]i increase was independent of membrane potential hyperpolarization (NS-1619), but was inhibited by pharmacological blockade of IP3 receptors (2-APB), phospholipase C (PLC, U73122) or following depletion of endoplasmic reticulum Ca2+ stores (cyclopiazonic acid). Thus, Na+ is linked to PLC/IP3 -mediated activation of endoplasmic reticulum Ca2+ release in metastatic breast cancer cells and this may have an important role in breast tumors where [Na+ ]i is perturbed.

MiRNA let-7a inhibits invasion, migration, anchorage-independent growth by suppressing EZH2 and promotes mesenchymal to epithelial transition in MDAMB-231

Enhancer of zeste homolog 2 (EZH2) is a transcriptional repressor belonging to the polycomb group protein family (PRC) and is frequently linked with aggressive breast cancer. Micro RNAs (miRNAs) are single-stranded 19–25 nucleotide long RNAs that control gene expression at the post-transcriptional level. Previous reports indicated that epigenetic modulation through EZH2-mediated histone methylation can silence the miR-200 cluster. Therefore, we attempted to identify the miRNAs that regulate the expression of EZH2 in metastatic breast cancer. Our results with microarray studies showed that upon knock-down of EZH2 lead to enhanced expression of tumor-suppressive miRNAs including miR-203, miR-200a, miR-200b, and let-7a in MDAMB-231, a metastatic breast cancer cell line. Further studies to confirm the expression of EZH2 upon overexpression of the mentioned miRNAs showed that only let-7a could post-transcriptionally regulate EZH2 expression significantly. Luciferase-based 3′UTR (untranslated region) reporter assay of EZH2 confirmed a significant reduction in the reporter activity upon co-transfection of let-7a and reporter construct 3′UTR of EZH2. Interestingly, overexpression of let-7a leads to decreased expression of another PRC2 group of protein SUZ12 and produced a lowered expression of H3K27me3 methylation. Additionally, let-7a miRNA promoted mesenchymal to epithelial transition i.e., by downregulating the key metastatic markers including Vimentin, Zeb, N-cadherin, and Snail, thus leading to reduced migration and invasion ability in MDA-MB-231 cells. This data poses an impact on understanding and developing therapeutics for breast cancer.