MDA-MB231 Cell Lines Research Articles

The Effect of Ginger Extract on the Expression of Epidermal Growth Factor Receptor in MDA-mb231 and HT-29 Cell Lines

Background: Cancer is a neoplastic disease that continues to be a global challenge, with a reported prevalence increasing annually. Epidermal growth factor receptor (EGFR) is overexpressed in various epithelial tumors such as breast and colon cancer, and for this reason, it is used for cancer diagnosis and treatment. Because of the high mortality rate associated with cancer and the side effects of chemotherapy and radiotherapy, patients need replaced strategies for therapy. Ginger has biological effects, including antioxidant and anticancer activities. Objectives: This study aims to evaluate ginger extract's effect on the expression of EGFR in MDA-mb231 and HT-29 cell lines. Methods: Fresh ginger rhizomes were purchased from a local food market, washed, grated, and then ginger extract was prepared using ethanol. MDA-mb231 and HT-29 cell lines were treated with different concentrations of ginger (5, 10, and 20 µgr/mL) extract in RPMI-1640 plus 10 % FCS for 18 h. The gene expression of EGFR was measured by real-time PCR. Results: The level of EGFR expression in MDA-mb231 and HT-29 cell lines after treatment with different concentrations of ginger extract was not changed significantly (P > 0.05). Conclusions: The current data indicate that the ginger extract does not have a significant dose-dependent effect, in the concentration range of 5 to 20 µgr/mL, on the expression of EGFR in these cancer cell lines. It is suggested to repeat the experiments with higher concentrations of ginger and in a time-dependent manner.

The Impact of Silver Nanoparticle-Induced Photothermal Therapy and Its Augmentation of Hyperthermia on Breast Cancer Cells Harboring Intracellular Bacteria.

Breast cancer can harbor intracellular bacteria, which may have an impact on metastasis and therapeutic responses. Silver nanoparticles are FDA-approved for their antimicrobial potential, plus they have pleiotropic benefits for eradicating cancer cells. In the current work we synthesized photothermal silver nanoparticles (AgNPs) with an absorption at 800 nm for heat generation when exposed to near-infrared laser irradiation. Breast cell lines MCF 10A, MCF7, and MDA MB 231 were infected with Pseudomonas aeruginosa, and their response to AgNPs, heat, or photothermal therapy (PTT) was evaluated. The results demonstrate that the application of a brief heating of cells treated with AgNPs offers a synergistic benefit in killing both infected and non-infected cells. Using 10 µg/mL of AgNPs plus laser stimulation induced a temperature change of 12 °C, which was sufficient for reducing non-infected breast cells by 81-94%. Infected breast cells were resistant to PTT, with only a reduction of 45-68%. In the absence of laser stimulation, 10 µg/mL of AgNPs reduced breast cell populations by 10-65% with 24 h of exposure. This concentration had no impact on the survival of planktonic bacteria with or without laser stimulation, although infected breast cells had a 42-90% reduction in intracellular bacteria. Overall, this work highlights the advantages of AgNPs for the generation of heat, and to augment the benefits of heat, in breast cancer cells harboring intracellular infection.

Quercetin-loaded solid lipid nanoparticles exhibit antitumor activity and suppress the proliferation of triple-negative MDA-MB 231 breast cancer cells: implications for invasive breast cancer treatment.

Quercetin (QC) is a naturally occurring flavonoid found in abundance in fruits and vegetables. Its anti-cancer and anti-inflammatory properties have been previously demonstrated, but its low bioavailability hampers its clinical use. Triple-negative breast cancer is a subtype of breast cancer with a poor response to chemotherapy. This study investigates the anti-cancer effects of quercetin-solid lipid nanoparticles (QC-SLN) on the triple-negative breast cancer cell line MDA-MB231. MCF-7 and MDA-MB231 cells were treated with 18.9 µM of QC and QC-SLN for 48h. Cell viability, apoptosis, colony formation assay, and the anti-angiogenic effects of the treatment were evaluated. QC-SLN displayed optimal properties (particle size of 154nm, zeta potential of -27.7 mV, encapsulation efficiency of 99.6%, and drug loading of 1.81%) and exhibited sustained release of QC over 72h. Compared to the QC group, the QC-SLN group showed a significant decrease in cell viability, colony formation, angiogenesis, and a substantial increase in apoptosis through the modulation of Bax and Bcl-2 at both gene and protein levels. The augmentation in the proportion of cleaved-to-pro caspases 3 and 9, as well as poly (ADP-ribose) polymerase (PARP), under the influence of QC-SLN, was conspicuously observed in both cancer cell lines. This study showcases quercetin-solid lipid nanoparticles (QC-SLN) as a promising therapy for triple-negative breast cancer. The optimized QC-SLN formulation improved physicochemical properties and sustained quercetin release, resulting in reduced cell viability, colony formation, angiogenesis, and increased apoptosis in the MDA-MB231 cell line. These effects were driven by modulating Bax and Bcl-2 expression, activating caspases 3 and 9, and poly (ADP-ribose) polymerase (PARP). Further in vivo studies are needed to confirm QC-SLN's efficacy and safety.

Structural design, and biological assessment of Co2+, Ni2+, Cu2+and Zn2+ complexes with N-(phenyl)-2-imino-2H-chromene-3-carboxamide

A new series of complexes based on N-(phenyl)-2-imino-2H-chromene-3-carboxamide ligand (NPICC) with Co2+, Ni2+, Cu2+ and Zn2+ metal ions have been synthesized and characterized using IR, H-NMR, UV–Vis, and TGA techniques. In vitro antimicrobial activity of the synthesized compound were conducted using well diffusion method against Gram-positive and Gram-negative strains bacteria and fungi. The results showed that complexes possess good inhibition activity against Klebsiella pneumoniae (G-), staphylococcus epidermis and staphylococcus aureus (G+) as well as candida albicans fungi than ligand and comparable with commercial drugs. Furthermore, the titled compounds were tested for their anti-tumor activity against SKOV-3 and MDA-MB231 cell lines. Results indicated that NiNPICC complex exhibited best activity with IC50 values of 6.42 and 3.95 µg/mL against SKOV-3 and MDA-MB231 cell lines, respectively. To understand the mode of antimicrobial action, quantum mechanics calculation based on DFT theory and docking study were performed for all compounds. Electronic structure analysis and docking energy were investigated. All the titled compounds were docked in a good manner within the active sites of the bacterial and fungi enzymes and exhibited good binding energies. Complexes showed higher electronic activity than ligand supporting the experimental results obtained.

A Potential Antibody–Drug Conjugate Targeting Human LIV1 for the Treatment of Triple-Negative Breast Cancer

AbstractTriple-negative breast cancer (TNBC), which accounts for 15 to 20% of incidents of breast cancer, is the only breast cancer subtype that lacks targeted treatments. It was reported in the literature that LIV1 was highly expressed in TNBC and other solid tumors. This makes LIV1 a potential target for the treatment of TNBC. This study aimed to develop an anti-LIV1 antibody for the treatment of TNBC. In this study, a novel anti-LIV1 antibody Ab1120 was developed and conjugated with monomethyl auristatin E (MMAE) to obtain the antibody–drug conjugate, Ab1120-vcMMAE. The Cell Counting Kit-8 method was used to assess the killing effect of the antibody–drug conjugate on cell lines MDA-MB−231 (high LIV1 expression of breast cancer cell line), MDA-MB-468 (low LIV1 expression of breast cell line), and 293C18 (LIV1-negative human embryonic kidney cell). The antitumor effect of Ab1120-vcMMAE on an MDA-MB-231 xenograft model was determined by evaluating the tumor volume and body weight after its treatment. In vitro analysis showed that Ab1120-vcMMAE is a potent inhibitor against the proliferation of a LIV1 overexpression cell line. The in vivo results demonstrated its antitumor activity in the cell-derived xenograft breast tumor mouse model. The results of this study suggest that Ab1120-vcMMAE may be used as a new therapeutic drug for patients with LIV1 high-expression breast cancer.

Chorein-dependent microfilament organization in tumor cells

Chorein is variably expressed in different cancer cells. In various non-malignant cell types, this protein regulates cytoskeleton microstructure and signaling. However, the role of chorein in regulating the actin cytoskeleton in tumor cells remains elusive. Here, we investigated chorein expression in various breast tumor cells and the involvement of this protein in microfilament organization. We used laser scanning microscopy to analyze microfilaments architecture, Triton X-100 fractionation to quantify G and Total actin levels, and quantitative RT-PCR to assess chorein gene transcription. We show that in line with previous observations, the less differentiated MCF7 breast cancer cells exhibited the highest relative expression of chorein compared to MDA-MB231 and T47D cell lines. Contrastingly, in less differentiated ZF rhabdomyosarcoma cells expressing high chorein levels, silencing of this protein was followed by clear depolymerization of actin microfilaments as apparent from IF morphological analysis. Quantification of G- and F-actin levels by Triton X-100 fractionation that revealed a significant increase in this ratio fully supported this finding. These results disclose that chorein is highly expressed in less differentiated tumor cells. In addition, silencing of this protein induces significant structural disorganization of the actin network, providing clear evidence that chorein regulates microfilament cytoskeleton architecture in tumor cells of higher malignant potential.

Synergistic effects of trastuzumab and gambogic acid on MDA-MB-453 breast cancer cell line

Trastuzumab is one of the most commonly used monoclonal antibodies in the treatment of breast cancer. It is effective because it binds to the HER2 receptor and inhibits the signaling pathways for proliferation and metastasis. However, intrinsic or acquired resistance resulting from the various signaling pathways of heterogeneous breast cancer reduces its efficacy. To increase its efficacy, it is combined with different agents in the treatment. In this study, the synergistic effect of the combination of trastuzumab and gambogic acid on the breast cell line MDA-MB -453 was investigated in vitro. At the end of 12-, 24-, 48-, and 72-hour incubation, their antiproliferative effects were determined by XTT method. Combination Index (CI) values of the study were calculated using CompuSyn.exe software. The highest synergistic effect was obtained with 12.5 µg/mL trastuzumab + 1.25 µM gambogic acid (CI-4) at the 72nd hour. The activity of the antioxidant enzyme catalase (CAT) was also measured for the applications. We observed that combination of trastuzumab and gambogic acid decreased CAT activity at 24, 48 and 72nd hours. It was concluded that gambogic acid may be a good candidate for combination with other agents in targeted drug design.

Novel Iron Chelator SK4 Drives Cytotoxicity through Inhibiting Mitochondrial Metabolism in Ovarian and Triple Negative Breast Cancer Cell Lines.

Anti-cancer therapy by iron chelation has been shown to inhibit many cellular processes including DNA replication, mitochondrial metabolism and oncogenic signalling pathways (e.g., EGFR). Iron chelator SK4 represents a double pronged approach towards treating cancer. SK4 enters through LAT1, a commonly overexpressed amino acid transporter in tumours, thus targeting iron addiction and LAT1 overexpression. The aim of this study was to characterise the mode of action of SK4 through proteomics, metabolomics, lipidomics and seahorse real-time analysis in ovarian cell line SKOV3 and triple negative breast cancer cell line MDA MB 231. Pathway enrichment of proteomics data showed an overrepresentation of metabolism related pathways. Metabolic change after SK4 exposure have been confirmed in investigations of changes in basal and maximal mitochondrial respiration using seahorse real-time analysis of mitochondrial metabolism. Metabolomics also showed an increase in AMP and glucose-1-phosphate. Interestingly, our lipidomics data show a decrease in phospholipid synthesis in the SKOV3 cells which is in contrast with previous data which showed an upregulation of ceramide driven apoptosis. In summary, our data highlight impairment of energy metabolism as a mechanism of action underlying SK4 apoptosis, but also suggest a potential role of ceramide induction in the phenotypic outcome of the cell model.

Insights into the mechanisms governing P01 scorpion toxin effect against U87 glioblastoma cells oncogenesis.

The emerging concept of small conductance Ca2+-activated potassium channels (SKCa) as pharmacological target for cancer treatment has significantly increased in recent years. In this study, we isolated the P01 toxin from Androctonus australis (Aa) scorpion venom and investigated its effect on biological properties of glioblastoma U87, breast MDA-MB231 and colon adenocarcinoma LS174 cancer cell lines. Our results showed that P01 was active only on U87 glioblastoma cells. It inhibited their proliferation, adhesion and migration with IC50 values in the micromolar range. We have also shown that P01 reduced the amplitude of the currents recorded in HEK293 cells expressing SK2 channels with an IC50 value of 3pM, while it had no effect on those expressing SK3 channels. The investigation of the SKCa channels expression pattern showed that SK2 transcripts were expressed differently in the three cancer cell lines. Particularly, we highlighted the presence of SK2 isoforms in U87 cells, which could explain and rely on the specific activity of P01 on this cell line. These experimental data highlighted the usefulness of scorpion peptides to decipher the role of SKCa channels in the tumorigenesis process, and develop potential therapeutic molecules targeting glioblastoma with high selectivity.

Insights into the therapeutic outcomes of trimetazidine/doxorubicin combination in Ehrlich solid-phase carcinoma mouse tumor model

One of the key features of cancer is metabolic reprogramming that can be exploited to sensitize cancer cells to chemotherapy. Trimetazidine (TMZ) is a metabolic anti-ischemic drug that blocks the activity of long-chain 3-ketoacyl CoA thiolase leading to the inhibition of fatty acid oxidation. AimsThe objective of the current investigation was to evaluate the idea that TMZ could synergize the antitumor activity of doxorubicin (DOX). Main methodsThe hypothesis was examined in vitro using the human breast cancer cell lines MCF-7 and MDA-MB231. In addition, the in vivo experiments were conducted using the Ehrlich solid phase carcinoma model. Key findingsIn vitro cytotoxicity experiments demonstrated that TMZ improved the potency of DOX in MCF-7 cell lines in a synergistic manner. In vivo testing confirmed that DOX/TMZ combination exhibits synergistic effect at both DOX/TMZ 1:10 and 1:5 ratios, where DOX was administered at one tenth and one fifth of its original dose, respectively. The co-treatment (1:5 ratio) significantly reduced tumor Nicotinamide adenine dinucleotide (NAD)+/NADH ratio (6.1-fold) and Adenosine triphosphate (ATP) levels (61 %) with concurrent activation of AMP-activated protein kinase (AMPK) (2.2-fold) and peroxisome proliferator-activated receptor-gamma coactivator (PGC)1-α (5.5-fold) protein expression versus control. The same treatment decreased the nuclear levels of NF-κB (p65) (57.5 %) and induced tumor apoptosis as evidenced by elevated Bax/Bcl-2 ratio (6.8-fold) along with active caspase-3 levels (6.6-fold) against control. SignificanceThe current investigation constitutes a proof-of-concept study that provided preclinical evidence for the anticancer activity of DOX/TMZ combination and warrants further investigation for repurposing TMZ in DOX protocols.

Design and Synthesis of an Hsp90 and HDAC Dual Inhibitor as Antitumor Agent

Background: Cancer incidence and mortality have been increasing, and cancer is still the leading cause of death all over the world. Therefore, expanding the arsenal of anticancer drugs with high efficiency and low toxicity is still one of the most challenging tasks. As a branch of antitumor drug design and discovery, dual-targeting drug candidates draw extensive attention. Objective:: In this work, we try to construct a multitarget drug candidate and evaluate its antitumor effects. Methods: Hsp90 and histone deacetylase were selected as two targets to design a dual targeting inhibitor w11. Enzyme inhibition work, cell viability assay, and docking simulation were carried out to evaluate the activity of the compound. Results: w11 could inhibit the activity of Hsp90α and HDAC6 with the IC50 of 50.1 nM and 8.1 nM, respectively. In cell viability assay, five human tumor cell lines Eca-109, FaDu, HN6, MCF-7 and MDAMB- 231 were used, results showed that w11 could potently inhibit the proliferation of three human lines with IC50 values in the nM range. Molecular docking experiments proved the rationality of structure design. Conclusion: Compound w11 was a potent Hsp90 and HDAC dual inhibitor for anticancer research.

Danthron, an Anthraquinone Isolated from a Marine Fungus, Is a New Inhibitor of Angiogenesis Exhibiting Interesting Antitumor and Antioxidant Properties.

The role played by a sustained angiogenesis in cancer and other diseases stimulates the interest in the search for new antiangiogenic drugs. In this manuscript, we provide evidence that 1,8- dihydroxy-9,10-anthraquinone (danthron), isolated from the fermentation broth of the marine fungus Chromolaenicola sp. (HL-114-33-R04), is a new inhibitor of angiogenesis. The results obtained with the in vivo CAM assay indicate that danthron is a potent antiangiogenic compound. In vitro studies with human umbilical endothelial cells (HUVEC) reveal that this anthraquinone inhibits certain key functions of activated endothelial cells, including proliferation, proteolytic and invasive capabilities and tube formation. In vitro studies with human breast carcinoma MDA-MB231 and fibrosarcoma HT1080 cell lines suggest a moderate antitumor and antimetastatic activity of this compound. Antioxidant properties of danthron are evidenced by the observation that it reduces the intracellular reactive oxygen species production and increases the amount of intracellular sulfhydryl groups in endothelial and tumor cells. These results support a putative role of danthron as a new antiangiogenic drug with potential application in the treatment and angioprevention of cancer and other angiogenesis-dependent diseases.

Abstract 6089: Identification of novel YAP/TAZ pathway regulators in the triple-negative breast cancer cell line MDA-MB231 using single-cell CRISPR screening

Abstract The Hippo signaling cascade is a major pathway that integrates a broad spectrum of mechanosensory signals at the plasma membrane and regulates response via control of cell proliferation, self-renewal, differentiation, and apoptosis. Dysregulation of this pathway has been observed across a range of cancer types and results in an altered activity of its primary downstream effectors, the oncogenic transcription factors YAP/TAZ. For example, both germline and somatic loss-of-function mutations in the tumor suppressor gene NF2, a component of Hippo, induce hyperactivation of YAP/TAZ, transcriptional changes and ultimately result in tumor growth. The Hippo signaling pathway is an attractive target for drug discovery efforts, however, it is highly complex and still incompletely understood. Hence it is indispensable to get a deeper insight into the Hippo - YAP/TAZ signaling axis. To this end, we performed a genome-wide CRISPR knockout screen in the triple-negative breast cancer (TNBC) cell line MDA-MB231 (NF2LOF) expressing a YAP/TAZ reporter construct. We identified both negative and positive regulators of YAP/TAZ in breast cancer cells. In a second step, screening hits were further characterized in a focused single-cell CRISPR screen (Perturb-Seq), aiming at better understanding of the effects on YAP/TAZ activity regulation and downstream effects on gene expression. Here we present the technical details of our screening approaches and the results of perturbing known and novel regulators of YAP/TAZ on single cell level. We discuss the use of Perturb-Seq in the initial validation of hits from genome-wide screens and provide data that may serve as a basis for future drug discovery efforts, seeking for novel and effective treatments for triple-negative breast cancers and other malignancies with Hippo pathway alterations. Citation Format: Mareike Berlak, Zuzanna Makowska, Filippos Klironomos, Julia Kuehnlenz, Atanas Kamburov, Andreas Steffen, Martin Lange, Barbara Nicke, Ralf Lesche, Peter Staller, Charlotte Kopitz, Jan Naujoks. Identification of novel YAP/TAZ pathway regulators in the triple-negative breast cancer cell line MDA-MB231 using single-cell CRISPR screening [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 6089.

Abstract 3697: Modulation of Nicotinamide N-Methyl Transferase (NNMT) expression in TNBC is associated with altered cell membrane cholesterol levels

Abstract Introduction: Cancer cells often develop a program of metabolic adaptability and epigenetic remodelling to promote gene expression changes causing cancerous cell proliferation and invasion. The metabolic-epigenetic axis as a an underlying mechanism of carcinogenesis could lead to new approaches to cancer treatment. Nicotinamide N-Methyl Transferase (NNMT) is a metabolic enzyme that catalyzes the transfer of a methyl group from S-Adenosyl methionine (SAM) to nicotinamide, thereby decreasing the overall methyl pools for RNA, histone and DNA methylation. High NNMT expression has been previously associated with a poor outcome in TNBC. Hypothesis: We hypothesized that high versus low NNMT activity and resulting oncogenic gene expression in TNBC may alter histone methylation is association with altered metabolic pathway activation. Methods: We used two TNBC cell lines models, MDA-MB 231 and SUM159, to study the association between high versus low NNMT activity and metabolic pathway activation. Surprisingly, we identified gene expression pathways suggestive of increased free cholesterol biosynthesis gene expression with low NNMT, and then used biochemical assays and filipin to measure free cholesterol in both total high and low NNMT TNBC cell lysates and relative cholesterol expression in the High vs Low NNMT cell membranes. Results: In vitro, NNMT knockdown resulted in increased cholesterol synthetic pathway enzyme expression in both TNBC cell lines. In vivo studies also showed that protein expression of cholesterol synthesis enzymes was also increased in NNMT-depleted MDA-MB 231 xenografted tumors. Interestingly, the increased cholesterol level upon NNMT depletion was mainly localized at the plasma membrane via Filipin III staining, suggesting a possible association with less cellular membrane fluidity and a decreased oncogenic phenotype. In ongoing experiments, we are evaluating whether changes in histone methylation in high vs low NNMT cells are associated with differential expression of cholesterol biosynthetic genes, and free cholesterol production. We hypothesize that NNMT might epigenetically decrease the methylation of histones regulating cholesterol production in TNBC. Citation Format: Woei-Yaw Chee, Deniz Nesli Dolcen, Lynda Bennett, Suzanne D. Conzen. Modulation of Nicotinamide N-Methyl Transferase (NNMT) expression in TNBC is associated with altered cell membrane cholesterol levels. [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 3697.

Abstract 397: The role of heparan sulfate in enhancing the chemotherapeutic response in triple-negative breast cancer

Abstract Among women worldwide, breast cancer has the highest incidence and is the leading cause of cancer-related death. Patients with the triple-negative breast cancer (TNBC) subtype have an inferior prognosis in comparison to other breast cancers because the current therapies do not facilitate long-lasting responses. Thus, there is a critical need for novel therapeutic strategies that induce stronger responses. In our previous research, we discovered that augmenting the concentration of extracellular ATP (eATP) greatly enhances the chemotherapeutic response of TNBC cell lines by activating purinergic receptors (P2RXs), leading to cell death through the induction of non-selective membrane permeability. However, eATP levels are limited by several classes of extracellular ATPases. One endogenous molecule of interest that can inhibit multiple classes of extracellular ATPases is heparan sulfate. Heparan sulfate itself is degraded by heparanase, an enzyme that is known to be highly expressed in various cancers, including breast cancer. Heparan sulfate has previously been shown to regulate several cancer-related processes such as fibroblast growth factor signaling, neoangiogenesis by sequestering vascular endothelial growth factors in the extracellular matrix, hedgehog signaling and cell adhesion. In this project, we identified an additional mechanism for a tumor suppressor role of heparan sulfate: inhibition of extracellular ATPases, leading to augmented levels of eATP. Several heparanase inhibitors have been previously identified, including OGT 2115, suramin, PI-88, and PG 545. We hypothesized that heparanase inhibitors would augment eATP concentrations in TNBC by increasing heparan sulfate in the tumor microenvironment, resulting in increased cell death in response to chemotherapy. We treated TNBC cell lines MDA-MB 231, Hs 578t, and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells with increasing concentrations of the chemotherapeutic agent paclitaxel in the presence of heparan sulfate and/or OGT 2115 with eATP content and cell viability being assessed. Moreover, to confirm that the effects of OGT 2115 are mediated through eATP, we utilized specific antagonists to the purinergic receptors P2RX4 and P2RX7. In addition, the protein expression of heparanase was compared in all the examined cell lines via Western blot analysis. We also evaluated the effects of this combination on the breast cancer-initiating cell population in the treated cells using flow cytometry and tumorsphere formation efficiency assays. These results demonstrate that inhibiting the degradation of heparan sulfate in the tumor microenvironment enhances the susceptibility of TNBC cell lines to chemotherapy by augmenting extracellular ATP concentrations. This strategy could potentially be used to induce deeper and more durable responses in triple-negative breast cancer patients. Citation Format: Jasmine M. Manouchehri, Jharna Datta, Mathew A. Cherian. The role of heparan sulfate in enhancing the chemotherapeutic response in triple-negative 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 397.

Abstract 398: The inhibition of sulfation of heparan sulfate augments the chemotherapeutic response in triple-negative breast cancer

Abstract The leading cause of cancer-related death among women worldwide is breast cancer. The triple-negative breast cancer (TNBC) subtype has limited therapeutic options that produce durable responses. As a result, it is associated with an especially poor prognosis compared to other breast cancer subtypes. Hence, the development of innovative therapeutic strategies that produce deeper and more durable responses is critically needed. We found that increasing extracellular ATP (eATP) can enhance the susceptibility of TNBC cell lines to chemotherapy through the activation of purinergic receptors (P2RXs) P2RX4 and P2RX7. However, eATP levels are limited by several different classes of eATPases, making the design of a single molecule that effectively inhibits all classes of eATPase complicated. Heparan sulfate, a carbohydrate moiety that is covalently linked to several cell surface and extracellular matrix proteins has previously been identified as an endogenous inhibitor of several classes of eATPases. Heparan sulfate is desulfated by sulfatases 1 and 2 at the 6-O-hydroxyl groups of glucosamine residues in heparan sulfate. Sulfatase 2 has been determined to be highly expressed in a variety of cancers including breast cancer, whereas sulfatase 1 is not. Several sulfatase inhibitors have been previously developed, such as OKN-007, which is a specific inhibitor of sulfatase 2. We hypothesized that OKN-007 would augment eATP levels in TNBC cell lines exposed to chemotherapy by increasing levels of heparan sulfate in the tumor microenvironment, leading to intensified cell death. TNBC cell lines MDA-MB 231, Hs 578t, and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells were treated with increasing concentrations of the chemotherapeutic agent paclitaxel in the presence of heparan sulfate and/or OKN-007, and eATP content and cell viability were evaluated. Inhibitors to the purinergic receptors P2RX4 and P2RX7 were used to further confirm that the mechanism of enhanced cell death induced by OKN-007 was through these receptors. In addition, basal protein and cell surface expression of sulfatases 1 and 2 were determined in all the examined cell lines via ELISA, Western blot, and flow cytometry analysis. To assess the effects on cancer-initiating cell properties, TNBC cell lines were treated with paclitaxel in the presence of heparan sulfate and/or OKN-007, and the tumorsphere formation efficiency assay used to assess for cancer-initiating cells. In parallel, we assessed for effects on cancer-initiating cells in treated cells using flow cytometry. Our results showed that inhibiting the 6-O desulfation of heparan sulfate increases eATP accumulation, thereby, enhancing TNBC cell lines’ response to chemotherapy. These findings may lead to therapies for TNBC based on sulfatase 2 inhibition that results in more effective responses with fewer side effects. Citation Format: Jasmine M. Manouchehri, Jharna Datta, Mathew A. Cherian. The inhibition of sulfation of heparan sulfate augments the chemotherapeutic response in triple-negative 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 398.

67 Lipid metabolism and synthesis pathway analysis of Sigma-2 Receptor/TMEM97 in breast cancer cells

OBJECTIVES/GOALS: Breast cancer has an increased requirement for lipids. The sigma-2 receptor plays a critical role in the effective uptake of lipoproteins by forming a complex with the LDL Receptor. We investigate the role of the sigma02 receptor in modulating lipid uptake pathways in breast cancers, and how this can be leveraged as a viable therapeutic strategy. METHODS/STUDY POPULATION: CRISPR/Cas9 will be used to ablate TMEM97 in the MDAMB231 and MCF7 cell lines. This study seeks to identify pathways that are dysregulated upon TMEM97 knockout (KO) by characterizing RNASeq data to identify differentially expressed genes and perform pathway analysis. RESULTS/ANTICIPATED RESULTS: Knockout of TMEM97 in breast cancer cells is is expected to decrease lipid uptake. Treatment with statins in these knockout cells is expected to result in decreased cell viability and result in a quiescent cell population. DISCUSSION/SIGNIFICANCE: This is an important mechanistic study to understand the importance of lipid homeostasis in cancer cell proliferation and how it can be targeted to improve therapeutic anti-tumor strategies. Understanding the pathways that TMEM97 modulates is vital for therapeutic strategies to curb the proliferation of breast cancer cells.

Negative regulation of CD44st by miR-138-5p affects the invasive ability of breast cancer cells and patient prognosis after breast cancer surgery

ObjectiveTo investigate how the negative regulation of CD44st by miR-138-5p affects the invasive ability of breast cancer cell lines and prognosis in postoperative breast cancer patients.MethodsRT-PCR, qRT-PCR, and western blot assays were used to detect the expression of CD44s, CD44v6, and CD44st at both mRNA and protein levels. The expression of miR-138-5p in breast cancer cell lines was also evaluated. The binding ability of miR-138-5p to CD44st was determined via a dual-luciferase assay. The CD44 protein expression in breast cancer tissues was detected using immunohistochemistry. A Transwell assay was used to detect the invasive ability of tumor cells. The correlation between CD44st and miR-138-5p mRNA expression in breast cancer tissues was evaluated using qRT-PCR, and the relationship between clinicopathological features was statistically analyzed.ResultsCD44s and CD44v6 were highly expressed in MDAMB-231 cell line, while CD44st was highly expressed in MCF-7/Adr and Skbr-3 cells. None of the CD44 isoforms were expressed in MCF-7 cells. The miR-138-5p was highly expressed in MCF-7 cells, but not in MCF-7/Adr, Skbr-3, and MDAMB-231 cells. The dual-luciferase assay suggested that miR-138-5p could bind to wild-type CD44st 3'-UTR, miR-138-5p overexpression significantly inhibited the expression level of CD44 protein in MCF-7/Adr cells, and miR-138-5p + CD44st (3'-UTR)-treated MCF-7/Adr and Skbr-3 cells were significantly less invasive than those in the control group (P < 0.05). RT-PCR results for 80 postoperative breast cancer patients showed that the mRNA expression rate for CD44st was higher in cancer tissues than in paracancerous tissues, and the expression rate of miR-138-5p was higher in paracancerous tissues than in cancerous tissues (P < 0.01). In cancer tissues, CD44st was negatively correlated with miR-138-5p expression, with correlation coefficient r = -0.76 (Pearson’s correlation), coefficient of determination R2 = 0.573, F = 106.89, and P < 0.001. The median overall survival value for patients in the low miR-138-5p expression group was 40.39 months [95% confidence interval (CI): 35.59–45.18 months] and 56.30 months (95% CI: 54.38–58.21 months) for patients in the high-expression group, with a log rank (Mantel-Cox) of 13.120, one degree of freedom, and P < 0.001.ConclusionIn breast cancer cell lines, miR-138-5p negatively regulated expression of CD44st and affected the invasive ability of tumor cells and patient prognosis after breast cancer surgery.

Patient-Derived Breast Cancer Bone Metastasis In Vitro Model Using Bone-Mimetic Nanoclay Scaffolds

The unavailability of reliable models for studying breast cancer bone metastasis is the major challenge associated with poor prognosis in advanced-stage breast cancer patients. Breast cancer cells tend to preferentially disseminate to bone and colonize within the remodeling bone to cause bone metastasis. To improve the outcome of patients with breast cancer bone metastasis, we have previously developed a 3D in vitro breast cancer bone metastasis model using human mesenchymal stem cells (hMSCs) and primary breast cancer cell lines (MCF-7 and MDAMB231), recapitulating late-stage of breast cancer metastasis to bone. In the present study, we have tested our model using hMSCs and patient-derived breast cancer cell lines (NT013 and NT023) exhibiting different characteristics. We investigated the effect of breast cancer metastasis on bone growth using this 3D in vitro model and compared our results with previous studies. The results showed that NT013 and NT023 cells exhibiting hormone-positive and triple-negative characteristics underwent mesenchymal to epithelial transition (MET) and formed tumors in the presence of bone microenvironment, in line with our previous results with MCF-7 and MDAMB231 cell lines. In addition, the results showed upregulation of Wnt-related genes in hMSCs, cultured in the presence of excessive ET-1 cytokine released by NT013 cells, while downregulation of Wnt-related genes in the presence of excessive DKK-1, released by NT023 cells, leading to stimulation and abrogation of the osteogenic pathway, respectively, ultimately mimicking different types of bone lesions in breast cancer patients.

Abstract P6-14-16: Claudin-9 and its subcoat anchorage proteins ZO-1 and ZO-3 in breast cancer, the clinical and therapeutic significance

Abstract Introduction. Claudin-9 (CLDN9), a member of the claudin protein family, is thought to play a role in the control of tight junctions (TJ) in various cell types. CLDN9 protein needs intracellular subcoat proteins, primarily the Zonula Occluden (ZO)-1 and ZO-3 to anchor to the cytoskeleton in order to form TJ with other proteins, including occludin. In the body, CLDN9 is highly expressed in endocrine tissues but is relatively low in mammary tissues. The role of CLDN9 is not well understood in cancer. In the present study, we have for the first time examined the pattern of CLDN9 expression at protein and transcript levels in breast cancer and explored its clinical and therapeutic implications. Methods. CLDN9 protein and CLDN9 transcript in fresh frozen human mammary tissues were evaluated by immunohistochemistry and quantitative transcript analyses and, together with the ZO family members and occludin in our database, correlated with clinical indicators. Assessment of expression in a range of cell lines was also determined. The levels of CLDN9 transcript, was also assessed against the therapeutic responses of the patients to chemotherapies by using a dataset from TCGA database. Results. Breast tumour tissues had high levels of CLDN9 transcript in tumours versus normal tissues. However, high grade breast tumours had significantly lower levels than low grade (p=0.02 and p=0.003, grade-2 and 3 vs grade-1 respectively). Patients with metastasis also had significantly lower levels than those without (p=0.0075). Patients who died of breast cancer had higher levels of the transcript than those who survived, although this was not significant. CLDN9 expression was significantly correlated with ZO-1 (r=0.20, p&amp;lt; 0.001) and ZO-3 (r=0.179, p&amp;lt; 0.01), but not ZO-2 in our cohort. There was significant correlation with another key TJ molecule, occludin (r=0.236, p=0.11). CLDN9, together with ZO-1 and ZO-3 significantly linked to the survival of patients (1444.6 months for the low expressing group versus 1138.2 months for the high expression group, p=0.013). The expression profile of the CLDN9/ZO1/ZO3 complex also indicated potential as an independent prognostic indicator (p=0.004, HR=2.033). The prognostic value was highly applicable to non-triple negative breast cancer patients. CLDN9 transcript also appeared to have a significant impact on treatment responses; patients who were sensitive to chemotherapies had a significantly lower levels of CLDN9 transcript than those who were resistant to treatment (p&amp;lt; 0.000001). In human cell lines, expression levels of CLDN-9 in ER (+) breast cancer cell lines, MDAMB361, MCF7 and BT474 were high; in a ER(-) breast cancer cell line MDAMB231, the expression level of CLDN-9 was lower. Conclusion. In conclusion, CLDN9 expression was differentially expressed in human breast cancer cells lines and appeared to reflect changes in ER status. It was apparent from levels of CLDN9 in a breast cancer cohort that expression was associated with grade and metastatic disease. Of significant notice was the finding that expression of CLDN9 in an expression profile with ZO1 and ZO3 has potential as a prognostic indicator, particularly in non-triple negative patients and in those who are chemotherapy resistant. Citation Format: Xinguo Zhuang, Wen G. Jiang, Eleri Davies, Bing Xu, Tracey A. Martin. Claudin-9 and its subcoat anchorage proteins ZO-1 and ZO-3 in breast cancer, the clinical and therapeutic significance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-16.