MDA-MB231 Cells Research Articles

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.

Nanomolar pyrophosphate detection in water using a zinc-terpyridine receptor and its applications in antiproliferative and antioxidant activity

A new fluorescent 4′-(3,4-diethoxyphenyl)-2,2′:6′,2′'-terpyridine ligand L and its 1:1 zinc complex ZnCl2L have been synthesized, characterized with the help of NMR, Mass spectroscopy, CHN analysis, and DFT study. In-situ prepared non-fluorescent ZnCl2L receptor was utilized for selective detection of pyrophosphate anion (P2O74-, PPi) in water (10 mM HEPES buffer, pH 7.4) in the vicinity of other competing analytes (10 equivalents of HPO42-, H2PO4-, CO32–, HCO3–, Cl−, OH–, Br−, CH3CO2–, F−, I-, NO3–, SO42-, SO32-, S2O32-, N3–) by employing UV–Visible spectroscopy and fluorescence “ON–OFF–ON” mechanism. ZnCl2L receptor formed a fluorescent 2:1 adduct with PPi in water during its detection at a nanomolar concentration (LOD ∼27.89 nM). The receptor is also successfully employed in low-cost PPi detection through real-time easily accessible paper strip application. Further, ZnCl2L exhibited significant cytotoxic activity against MDAMB-231 cells with an IC50 value of 53.85 µM and excellent antioxidant activity having an antioxidant percentage of 73.75%.

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.

Synergistic Effect of the Association of BR2 Peptide with 2-Aminoethyl Dihydrogen Phosphate in Triple-Negative Breast Cancer Cells

Background: Breast cancer is one of the most common diseases among women worldwide. The triple negative subtype is the most aggressive, with low tumor-free survival and the worst clinical evolution, requiring the development of more effective and targeted therapies. The present study investigated the in vitro pharmacological effects of the association of BR2 peptide with 2-aminoethyl dihydrogen phosphate (2-AEH2P) on MDA-MB-231 and 4T1 triple-negative breast cancer cells. Methods: The physical-chemical analysis of the peptide was performed using the Heliquest software, the cell viability was assessed using the MTT colorimeter method and the predictive pharmacological effect was evaluated using the Synergy Finder software. Results: The results showed the BR2 tumor penetration peptide and the 2-AEH2P+BR2 association significantly increased cytotoxicity in the MDA MB-231 and 4T1 tumor lines, without compromising the viability of the normal fibroblastic cells. The results also showed that depending on the time and concentration, a synergistic effect was observed for the association with tumor cells, with a therapeutic window between 0.8 and 50µm for MDA-MB-231 tumor cells in 48h. Conclusion: The results demonstrated in vivo antitumor and antiproliferative efficiency for MDA-MB-231 and 4T1 tumor cells with low toxicity for normal fibroblast cells, with MDA MB-231 cells being more sensitive to treatments.

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.

Antitumor Effect of Iscador on Breast Cancer Cell Lines with Different Metastatic Potential

Studies were performed for the first time on the effect of Iscador Qu and Iscador M on phototoxicity, cytotoxicity, antiproliferative activity, changes in ξ-potential of cells, membrane lipid order, actin cytoskeleton organization and migration on three breast cancer lines with different metastatic potential: MCF10A (control), MCF-7 (low metastatic) and MDA-MB231 (high metastatic) cells. The tested Iscador Qu and M did not show any phototoxicity. The antiproliferative effect of Iscador species appeared to be dose-dependent and was related to the metastatic potential of the tested cell lines. A higher selectivity index was obtained for Iscador Qu and M towards the low metastatic MCF-7 cell line compared to the high metastatic MDA-MB-231. Iscador Qu demonstrated higher selectivity for both cancer cell lines compared to Iscador M. The malignant cell lines exhibited a decrease in fibril number and thickness regardless of the type of Iscador used. The strongest effect on migration potential was observed for the low metastatic cancer cell line MCF-7 after Iscador treatment. Both Iscador species induced a slight increase in the percentage of cells in early apoptosis for the low and high metastatic cell lines, MCF-7 and MDA-MB-231, unlike control cells. Changes in the zeta potential and membrane lipid order were observed for the low metastatic MCF-7 cell line in contrast to the high metastatic MDA-MB-231 cells. The presented results reveal a higher potential of Iscador as an antitumor agent for the low metastatic cancer cell line MCF-7 compared to the high metastatic one. Iscador Qu appears to be more potent compared to Iscador M, but at this point, the exact mechanism of action is still unclear and needs further investigations.

Valproic acid inhibits cell growth in both MCF-7 and MDA-MB231 cells by triggering different responses in a cell type-specific manner

BackgroundBreast cancer is the second leading cause of death among women after lung cancer. Despite the improvement in prevention and in therapy, breast cancer still remains a threat, both for pre- and postmenopausal women, due to the development of drug resistance. To counteract that, novel agents regulating gene expression have been studied in both hematologic and solid tumors. The Histone Deacetylase (HDAC) inhibitor Valproic Acid (VA), used for epilepsy and other neuropsychiatric diseases, has been demonstrated a strong antitumoral and cytostatic activity. In this study, we tested the effects of Valproic Acid on the signaling pathways involved in breast cancer cells viability, apoptosis and in Reactive Oxygen Species (ROS) production using ER-α positive MCF-7 and triple negative MDA-MB-231 cells.MethodsCell proliferation assay was performed by MTT Cell cycle, ROS levels and apoptosis were analyzed by flow cytometry, protein levels were detected by Western Blotting.ResultsCell treatment with Valproic Acid reduced cell proliferation and induced G0/G1 cell cycle arrest in MCF-7 and G2/M block in MDA-MB-231 cells. In addition, in both cells the drug enhanced the generation of ROS by the mitochondria. In MCF-7 treated cells, it has been observed a reduction in mitochondrial membrane potential, a down regulation of the anti-apoptotic marker Bcl-2 and an increase of Bax and Bad, leading to release of cytochrome C and PARP cleavage. Less consistent effects are recorded in MDA-MB-231 cells, in which the greater production of ROS, compared to MCF-7cells, involves an inflammatory response (activation of p-STAT3, increased levels of COX2).ConclusionsOur results have demonstrated that in MCF-7 cells the Valproic Acid is a suitable drug to arrest cell growth, to address apoptosis and mitochondrial perturbations, all factors that are important in determining cell fate and health. In a triple negative MDA-MB 231 cells, valproate directs the cells towards the inflammatory response with a sustained expression of antioxidant enzymes. Overall, the not always unequivocal data between the two cellular phenotypes indicate that further studies are needed to better define the use of the drug, also in combination with other chemotherapy, in the treatment of breast tumors.

Narirutin downregulates lipoxygenase-5 expression and induces G0/G1 arrest in triple-negative breast carcinoma cells

BackgroundTriple-negative breast cancer (TNBC) accounts for 20% of breast cancer that does not express HER2, progesterone and estrogen receptors. It is associated with a high mortality rate, morbidity, metastasis, recurrence, poor prognosis and resistance to chemotherapy. Lipoxygenase-5 (LOX-5), cyclooxygenase-2 (COX-2), cathepsin-D (CATD), ornithine decarboxylase (ODC) and dihydrofolate reductase (DHFR) are involved in breast cancer carcinogenesis; hence, there is a pressing need to identify novel chemicals that targets these enzymes. Narirutin, a flavanone glycoside abundantly present in citrus fruits, is reported to have immune-modulatory, anti-allergic and antioxidant potential. Still, the cancer chemopreventive mechanism against TNBC has not been explored. MethodsIn vitro experiments, enzyme activity, expression analysis, molecular docking and MD simulation were carried out. ResultsNarirutin suppressed the growth of MDA-MB-231 and MCF-7 in a dose-proportional manner. The pronounced effect with >50% inhibition was observed in SRB and MTT assays for MDAMB-231 cells. Unexpectedly, narirutin suppressed the proliferation of normal cells (24.51%) at 100 μM. Further, narirutin inhibits the activity of LOX-5 in cell-free (18.18 ± 3.93 μM) and cell-based (48.13 ± 7.04 μM) test systems while moderately affecting COX-2, CATD, ODC and DHFR activity. Moreover, narirutin revealed a down-regulation of LOX-5 expression with a fold change of 1.23. Besides, MD simulation experiments confirm that narirutin binding forms a stable complex with LOX-5 and improves the stability and compactness of LOX-5. In addition, the prediction analysis demonstrates that narirutin could not cross the blood-brain barrier and did not act as an inhibitor of different CYPs. Conclusions and significanceNarirutin could be a potent cancer chemopreventive lead for TNBC, further paving the way for synthesizing novel analogues.

Abstract P6-12-01: ALCAM (Activated Leukocyte Cell Adhesion Molecule) is associated with HGF/MET signalling pathway in different subtypes of breast cancer

Abstract Introduction. ALCAM (Activated Leukocyte Cell Adhesion Molecule), also known as CD166, is a cell adhesion molecule which belongs to the immunoglobulin superfamily and is widely expressed in various human tissues. It has been demonstrated that ALCAM plays an important role in the progression of malignant diseases and tumour metastasis in multiple cancer types including breast cancer. However, the molecular mechanism of ALCAM and cancer progression is currently unclear. The present study performed protein array analyses using ALCAM genetically manipulated cell models to select potential protein partners of ALCAM. The study focused on MET (Hepatocyte Growth Factor (HGF) Receptor), a prominent ALCAM interacting protein kinase and a protooncogene contributing to cancer progression and spread. Method. Human breast cancer cell lines MCF-7 and MDA-MB-231 were selected to create ALCAM knockdown cell models. A range of other breast cancer cell line with differing hormone receptor status were also used. Protein samples of transfected cells were used to perform Kinexus protein kinase microarray analysis. The protein interaction between ALCAM and other prospective protein kinases including MET was verified by the method of immunoprecipitation. Additionally, the ALCAM knockdown model was also assessed for the impact of ALCAM and HGF/MET on the biological functions by Electric Cell-substrate Impedance Sensing (ECIS). Results. MCF-7 and MDA MB-231 cells both were strongly expressed ALCAM. Cells models with ALCAM knocking down were successfully created by way of anti-ALCAM shRNA. We have shown on the protein kinase microarray analysis that the hepatocyte growth factor (HGF) receptor, MET was one of the kinases significantly affected following ALCAM knocking down. It was also found that the alteration of protein interaction between the MET protein kinase and ALCAM protein showed an opposite pattern between ER positive and ER negative ALCAM knocking down cells. This protein interaction was observed by immunoprecipitation in MDA-MB-361 and MDA-MB-231 cell lines, but not in MCF-7 cells. The biological analyses using the ECIS technologies showed that MET kinase inhibitors and exogenous recombinant HGF affected the ALCAM-mediated cell adhesion in different breast cancer subtypes. Conclusion. ALCAM is associated with HGF/MET signalling and the interaction between ALCAM and MET is different amongst subtypes of breast cancer which have different hormone receptor status. Citation Format: Yiming Yang, Andrew J. Sanders, Wen G. Jiang. ALCAM (Activated Leukocyte Cell Adhesion Molecule) is associated with HGF/MET signalling pathway in different subtypes of breast cancer [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 P6-12-01.

Design, synthesis and characterization of a series of 6-substituted-4-hydroxy-1-(2-substitutedthiazol-4-yl)quinolin-2(1H)-one derivatives and evaluation of their in vitro anticancer and antibacterial activity

The current research work deals with the design, synthesis and characterization of a series of 6-substituted-4-hydroxy-1-(2-substitutedthiazol-4-yl)quinolin-2(1H)-one derivatives [III(a-d)(1–3)] and evaluation of their in-vitro anticancer activity against MDA-MB (Breast cancer) and A549 (Lung cancer) cell lines based upon MTT assay and in-vitro antibacterial by the measurement of zone of inhibition and determining the Minimum Inhibitory Concentration (MIC). All the synthesized compounds were characterized by UV, IR, 1H NMR and 13C NMR spectral data.Molecular docking studies of the title compounds were carried out using Molegro Virtual Docker (MVD-2013, 6.0) software. The synthesized compounds exhibited well conserved hydrogen bond interactions with one or more amino acid residues in the active pocket of EGFRK tyrosine kinase domain (PDB ID: 1m17) for docking study on anticancer activity and S. aureus DNA Gyrase domain complexed with a ciprofloxacin inhibitor (PDB ID: 2XCT) for antibacterial docking study. All synthesized derivatives were potent against A549 (Lung cancer) cell line as compared to MDA-MB (Breast cancer) cell line. Compound 2-(4-(4-hydroxy-6-methyl-2-oxoquinolin-1(2H)-yl)thiazol-2-yl)hydrazin-1-ium iodide (IIId-2) was found to be the most cytotoxic as compared to the other synthesized derivatives, with IC50 values of 346.12 μg/mL against A549 (Lung cancer) cell line, however all synthesized derivatives were found to be a poor antibacterial agent when compared with standard ciprofloxacin.Thus, the synthesized derivatives possessed a potential to bind with some of the residues of the active site and can be further developed into potential pharmacological agents.

Abstract PD5-06: PD5-06 CCN6 suppresses spindle metaplastic breast carcinoma in part via antagonizing Wnt/β-catenin signaling

Abstract Background: Metaplastic breast carcinomas (mBrCAs) are a rare and highly aggressive subtype of triple negative breast cancer, with histological evidence of non-glandular differentiation and frequent activation of the canonical (-catenin-dependent) Wnt pathway. Our laboratory has reported that CCN6 is expressed in normal mammary epithelium, but CCN6 expression is lost in 68% of spindle mBrCAs. We found mice with mammary epithelial cell-specific conditional deletion of Ccn6 (MMTV-Cre; Ccn6fl/fl mice) develop mammary tumors that recapitulate human spindle mBrCAs, including upregulation of Wnt pathway genes. We investigated if and how secreted CCN6 protein functions in tumor suppression in spindle mBrCA via effects on the canonical Wnt pathway. Methods: To investigate CCN6 binding to the Wnt co-receptors LRP6 and FZD8 proteins, we performed Flag-IPs on MDA-MB231 mesenchymal-like breast cancer cells expressing Flag-CCN6 or vector. Effects of CCN6 on b-catenin subcellular localization and gene and protein expression were studied by IHC, IF, qRT-PCR and immunoblot in human cell lines and MMTV-Cre;Ccn6fl/fl tumors. To test effects of recombinant CCN6 on canonical Wnt signaling, we used the Leading-Light Wnt Reporter Assay and also tested CCN6 effects in WNT3A- and WNT10B-mediated Wnt signaling activation and on MDA-MB231 cell invasion. To study b-catenin/TCF function in invasive growth of CCN6-deficient cancer cells, we employed two independent approaches: i) expression of a dominant-negative Tcf4 (dnTcf4) versus control vector in MMTV-Cre; Ccn6fl/fl tumor-derived cells; and ii) expression of a constitutively active mutant (S33Y) b-catenin in concert with treatment with recombinant human CCN6 (rhCCN6; 500 ug/ml) versus BSA control. Syngeneic orthotopic mammary tumor transplants of MMTV-Cre;Ccn6fl/fl were used in vivo for rescue experiments with i.p. injections of rhCCN6 or BSA. We monitored tumor growth and morphology, and performed IHC to determine b-catenin localization and expression. Results: We found in co-IPs that CCN6 interacts with LRP6 and FZD8 to form a complex that antagonizes canonical Wnt signaling. CCN6 ectopic expression in MDA-MB231 cells led to reduced nuclear and increased membrane localization of b-catenin and decreased invasive growth in vitro. In vivo, CCN6 protein administration to MMTV-Cre; Ccn6fl/fl mice reduced tumor growth and was linked to decreased nuclear b-catenin in the tumors. Conclusion: CCN6 antagonizes canonical Wnt/b-catenin in part by binding Wnt ligands, leading to reduced active b-catenin in the cytoplasm and nucleus. Our data indicate a critical role for b-catenin activation for CCN6-deficient mBrCA tumor phenotypes. In vivo, rhCCN6 protein reduces tumorigenesis in MMTV-Cre; Ccn6fl/fl mBrCA tumors, highlighting how CCN6 restoration or b-catenin inhibition could be new therapeutic approaches for mBrCAs. Citation Format: Maria E. Gonzalez, Eric R. Fearon, Celina Kleer. PD5-06 CCN6 suppresses spindle metaplastic breast carcinoma in part via antagonizing Wnt/β-catenin signaling [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 PD5-06.

Abstract P6-14-17: Endomucins and their expression in breast cancer and the cellular and therapeutic impact

Abstract Introduction. Endomucin-1 and endomucin-2 are two proteins structurally related, yet with low homology. The membrane bound sialoglycoproteins appear to play a key role in interfering with the formation of focal adhesion complexes (FAC) and matrix adhesiveness of cells, by mechanisms independent of the MUC1 repeat, which the endomucins do not possess. Endomucins are thought to be expressed at high levels in endothelium and haematopietic cell lineages, although the levels in mammary tissues also seem high. Despite the seeming importance of endomucins in the adhesion and migration of cells, including cancer cells, the clinical value of endomucin in clinical cancer, including breast cancer, is largely unknown. The present study examined the expression profile of endomucins, together with the focal adhesion kinase FAK, in breast cancer and aimed to explore the cellular impact of endomucin on cancer cells. Methods. Human breast cancer cells MCF-7 and MDA MB-231 and a range of other cell types were used. An endomucin overexpression cell model was created and subsequently used to evaluate the function of the cells. The expression profile of the endomucin-1 and endomucin-2 transcripts and FAK, in an existing fresh frozen breast cancer tissue cohort, were quantified. Results. High levels of endomucins, particularly endomucin-1, are good indicators for the overall survival of the patient, p=0.021 for endomucin-1 and p=0.15 for endomucin-2. When expression levels of FAK were integrated into the survival analysis model, patients with high levels of both endomucins and low levels of FAK had the most favourable outcome, compared with those with most unfavourable outcome who had low level of endomucin and high FAK (survival during the follow up period respectively at 100% and 54%, p=0.013, Harzoud Ratio 0.298). Together with the Nottingham Prognostic Index, which independently predicts a poor outcome (p=0.009, HR=7.6), the integrated expression profile of endomucin/FAK represents an independent prognostic indicator for favourable overall survival (p=0.003, HR=0.13), and indeed for a favourable disease free survival (p=0.008, HR=0.17). Mammary tissues, and indeed breast cancer cell lines, expressed high levels of endomucin-2 transcripts and low levels of endomucin-1 transcripts. High levels of endomucin-2 were also seen in fibroblasts and vascular endothelial cells. We created a breast cancer cell submodel with MCF-7, by overexpressing endomucin. It was shown that although endomucin over-expression had some marginal impact on the adhesiveness of breast cancer cells, the over-expression however, had significant impact on cells’ sensitivity to FAK inhibitor, with a markedly reduced adhesiveness to matrix (p&amp;lt; 0.001 control versus endomucin overexpression cells). Discussion. Endomucins have a reduced expression in breast cancers and the reduction, together with low levels of focal adhesion kinase, facilitate a favourable outcome for the patients. Together with the findings of in vitro cell models, it would suggest that the expression profile of endomucins and FAK may be a good indicator, not only for evaluating clinical outcomes, but also for choice of target therapies. Citation Format: Amber Xinyu Li, Tracey A. Martin, Lin Ye, Andrew J. Sanders, Fiona Ruge, Jane Lane, Eleri Davies, Wen G. Jiang. Endomucins and their expression in breast cancer and the cellular and therapeutic impact [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 P6-14-17.

Abstract P2-19-02: Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors

Abstract Background: Epithelial-mesenchymal transition (EMT) phenotype is a complex process and plays a central role in tumor progression, aggression, invasion, metastasis, and resistance to therapy. Role of androgen receptor (AR) and androgens in inducing EMT has been well established in experimental systems of prostate cancer. AR is widely expressed in all subtypes of breast cancer (BC) and is considered to have context dependent role based on the steroid hormone microenvironment. Previous studies have shown AR as the only sex steroid receptor detectable in BC metastases and in metastatic lesion. We investigated the role of androgens in driving the EMT phenotype and association of AR induced genes with EMT in BC tumors using public dataset. Methods: We treated AR positive breast cancer cell line MDA-MB-453 with the androgen, dihydrotestosterone (DHT) followed by AR antagonist bicalutamide (Bic) and examined the expression levels of AR induced genes by quantitative real time PCR (qPCR) and AR induced protein GCDFP-15 by western blot. AR driven genes (SEC14L2, C1orf116, FKBP5, UGT2B11, UGT2B28, KCNMA1, PIP and ABCC11) were identified by bioinformatic pipeline using a systematic bioinformatic approach from public microarray data sets derived from AR positive breast cancer cell lines (S Rajarajan et al, SABCS 2021). Mesenchymal phenotype of the cell lines under DHT/Bic was evaluated morphologically and expression levels of EMT markers (ZEB1, Slug, Vimentin) and E-cadherin was estimated by qPCR, western blot and immunofluorescence. Migratory potential of the cell line under treatment was evaluated by wound healing assay and proliferation was examined by the MTT assay. Association of AR induced genes and levels of enzymes involved in intracrinal testosterone metabolism (SRD5A1 &amp; SRD5A3) with EMT in breast tumors was evaluated using transcript data from METABRIC database. EMT scores were derived from previous publications (Byers et al, 2013, Mak et al, 2016) and correlation was tested with AR induced genes. Results: The MDAMB-453 cell line was treated with 10nM DHT for 72hrs and this increased the expression of AR downstream protein GCDFP-15 by 31.47% (p=0.022) when compared to the control. Further, a significant increase in the expression levels of AR driven genes with a fold change &amp;gt;2 was observed in the DHT treated cells (p&amp;lt; 0.05). The expression of GCDP15 protein (p=0.028) and the AR downstream genes (p&amp;lt; 0.05) were significantly repressed upon treatment with bicalutamide. DHT treatment also showed a significant higher expression of EMT master regulator, SLUG (fold change&amp;gt;3, p=0.045) and a wound healing assay showed a 22% increase in migration (p=0.027). The expression of SLUG was significantly repressed (p=0.003) and the migratory ability of the DHT treated cells reduced upon treatment with bicalutamide (p=0.145). We also observed a significant loss of E-cadherin protein (by 23.65%; p=0.024) with DHT treatment and this was completely reversed upon treatment with bicalutamide (p=0.047). No significant change in proliferation was observed among the different treatment conditions. A significant positive correlation was observed between AR induced genes and the EMT scores (p&amp;lt; 0.05). Expression level of SRD5A3 was also positively correlated with EMT score (p=0.043) and AR induced genes (p&amp;lt; 0.05), except SEC14L2. Conclusion: Androgen receptor plays an important role in the biology of breast cancer and is considered a useful marker for prognosis. However, antiandrogen therapies haven’t seen the expected success in clinical trial settings. Our results support the controversial role played by AR in higher androgenic environment within breast cancer and warrant the consideration of the intratumoral levels of sex steroid hormones. Citation Format: Savitha Rajarajan, Snijesh VP, Madhumathy G Nair, Apoorva D, Chandrakala M, Vidya P Nimbalkar, Maalavika Pillai, Mohit Kumar Jolly, Jyothi S Prabhu. Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors [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 P2-19-02.

Cycloartane triterpenoid from Euphorbia macrostegia modulates ER stress signaling pathways to induce apoptosis in MDA-MB231 and MCF-7 breast cancer cell lines.

Unfolded protein response (UPR) is involved in breast cancer (BC) progression and drug resistance. Many natural products (NPs) could modulate UPR and used for therapeutic purposes. Herein, we aimed to investigate the molecular mechanism of Cycloart-23E-ene-3β, 25-diol (Cycloart-E25), cytotoxicity, as a NP extracted from Euphorbia macrostegia and focused on endoplasmic-reticulum stress (ERS) and UPR signaling pathways. Reactive oxygen species (ROS) were probed by DCFDA fluorescence dye. Apoptosis was assayed by annexin V/propidium iodide (PI), immunoblotting of anti- and proapoptotic, Bcl-2 and Bax proteins, and mitochondrial transmembrane potential (ΔΨm) changes. Thioflavin T (ThT) staining and immunoblotting of UPR signaling components (CHOP, PERK, ATF6, BiP, and XBP1) were recruited for the assessment of ERS. Our results indicated that Cycloart-E25 noticeably increases ROS levels in both MB-231 MDA-MB-231 and MCF-7 cell lines, p>0.05. Flow cytometry assessments revealed an increase in the cell population undergoing apoptosis. Also, the Bax/Bcl-2 ratio increased in a dose-dependent manner following Cycloart-E25 treatment, significantly, p>0.05. Mitochondrial involvement could be deduced by significant decreases in ΔΨm, p>0.05. Cycloart-E25 potently induces protein aggregation and upregulated CHOP, PERK, ATF6, BiP, and XBP1 factors in both MDA-MB-231 MB-231 and MCF-7 cell lines, indicating the involvement of ERS in Cycloart-E25-mediated apoptosis. In conclusion, Cycloart-E25 increased the accumulation of misfolded proteins and upregulated UPR components. Therefore, induction of ERS may be involved in the trigger of apoptosis in BC cell lines. Cycloart-E25 induced apoptosis in breast cancer cell lines through ERS. More assessments are needed to confirm its in vivo anti-tumoral effects.

Discovery of Novel Bioactive Tanshinones and Carnosol Analogues against Breast Cancer.

The abietane diterpenoids ferruginol (1), tanshinone IIA (3), and carnosol (4) are well-known for their interesting pharmacological properties, including antitumor, similar to other natural and semisynthetic abietanes. In this study, a pair of semisynthetic C18-functionalized analogues of 3 and 4 were prepared from the commercially available (+)-dehydroabietylamine or readily obtained methyl dehydroabietate. Semisynthetic ferruginol (1) and some selected analogues, together with the synthesized analogues, were tested in vitro for the inhibition of proliferation in four breast cancer cell lines, SUM149, MDA-MB231, T47D, and MCF07. As a result, several tested abietane analogues decreased cell proliferation and enhanced cell death, with IC50 in the range 1.3-18.7 μM. This work demonstrates the antitumor activities of two tested compounds, making these molecules interesting for the development of new anticancer agents.

Guided Isolation of An Uncommon Cembranoid Orthoester, Sarcotortin A, and Three Skeletal Diverse Terpenoids from the Hainan Soft Coral Sarcophyton tortuosum Based on Molecular Networking Strategy.

Applying the emerging molecular networking strategy, an uncommon cembranoid orthoester, sarcotortin A (1), featuring a 3/14/8/5-fused scaffold, an unusual eunicellane-type diterpenoid, sarcotorolide A (2), and two new biscembranoids, ximaolides M and N (7 and 8), along with nine known terpenoids 3-6 and 9-13 were isolated from the Hainan soft coral Sarcophyton tortuosum. The structure and absolute configuration of all new compounds were established by a combination of spectroscopic data, X-ray diffraction analysis, and/or quantum chemical computational approaches. The plausible biogenetic relationship among these skeletally different terpenoids was proposed and discussed. In in vitro bioassay, new compound 7 exhibited a remarkable inhibitory activity against protein tyrosine phosphatases 1B (PTP1B) with the IC50 value of 8.06 μM. In addition, compounds 4 and 10 displayed significant inhibitory effects on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 macrophages cells with the IC50 values of 19.13 and 16.45 μM, respectively. Compound 9 showed interesting cytotoxicity against H1975, MDA-MB231, A549, and H1299 cancer cell lines with IC50 values of 31.59, 34.96, 43.87, and 27.93 μM, respectively.

PGRMC1 promotes triple-negative breast cancer cell growth via suppressing ferroptosis

Objective Triple-negative breast cancer (TNBC) is the most malignant form of breast cancer with increasing incidence and mortality worldwide. The progesterone receptor membrane component-1 (PGRMC1) is a well-identified hormone receptor with unknown functions in TNBC. The current study aims to explore the involvement of PGRMC1 in regulation of glutathione metabolism and ferroptosis during development of TNBC, providing new therapy options for TNBC patients. Methods Bioinformatic analysis, cell proliferation assay, western blot assay and other biochemistry methods were performed in TNBC cells. Results Our results revealed that the expression of PGRMC1 is higher in TNBC than the other subtypes of breast cancer. Interestingly, as an iron binding protein, increased PGRMC1 expression in TNBC cells leads to resistance to ferroptosis inducer. On the contrary, silenced PGRMC1 expression enhanced sensitivity of MDA-MB231 cells to Erastin. Mechanistically, overexpression of PGRMC1 decreased the intracellular free iron concentration, which was reduced by AG205 treatment. Conclusions PGRMC1 increases the possibility of TNBC development through binding to intracellular iron and suppressing ferroptosis, providing the molecular basis of combined treatment for TNBC.

Cytotoxic Effect of the Alcoholic Extract of Conocarpus erectus Leaves on MDA-MB 231 and MCF7 Breast Cancer Cell Lines

Currently, there is a growing interest in medicinal plants extracts as some plants have shown antitumor potential. The goal of this study was to test the anticancer activity of methanol extract of Conocarpous erectus leaves in breast cancer cells. Cytotoxicity was tested in vitro on breast cancer cell lines, MCF7 [Estrogen receptor + (ER+)] and MDA-MB231 [Estrogen receptor - (ER-)], in addition to normal fibroblast cells (REF). MTT assay was utilized to measure the growth inhibitory effects after 48 hours exposure to extracts. Viability results indicated that MDA-MB231 were sensitive (GI50 = 56.1µg/ml).However, no sensitivity was seen in both MCF7 and REF cells (GI50&gt;100 µg/ml). Interestingly the sensitivity seen in MDA-MB231 cells was associated with a significant reduction in cell number and size. It can be concluded from this study that leaves extracts may provide a candidate therapy against breast cancer cells and sensitivity was not linked with ER expression. No effect was observed in normal cells.

Synthesis and Anti-Angiogenic Activity of Novel c(RGDyK) Peptide-Based JH-VII-139-1 Conjugates

Peptide-drug conjugates are delivery systems for selective delivery of cytotoxic agents to target cancer cells. In this work, the optimized synthesis of JH-VII-139-1 and its c(RGDyK) peptide conjugates is presented. The low nanomolar SRPK1 inhibitor, JH-VII-139-1, which is an analogue of Alectinib, was linked to the ανβ3 targeting oligopeptide c(RGDyK) through amide, carbamate and urea linkers. The chemostability, cytotoxic and antiangiogenic properties of the synthesized hybrids were thoroughly studied. All conjugates retained mid nanomolar-level inhibitory activity against SRPK1 kinase and two out of four conjugates, geo75 and geo77 exhibited antiproliferative effects with low micromolar IC50 values against HeLa, K562, MDA-MB231 and MCF7 cancer cells. The activities were strongly related to the stability of the linkers and the release of JH-VII-139-1. In vivo zebrafish screening assays demonstrated the ability of the synthesized conjugates to inhibit the length or width of intersegmental vessels (ISVs). Flow cytometry experiments were used to test the cellular uptake of a fluorescein tagged hybrid in MCF7 and MDA-MB231 cells that revealed a receptor-mediated endocytosis process. In conclusion, most conjugates retained the inhibitory potency against SRPK1 as JH-VII-139-1 and demonstrated antiproliferative and antiangiogenic activities. Further animal model experiments are needed to uncover the full potential of such peptide conjugates in cancer therapy and angiogenesis-related diseases.

Engineered Hybrid Nanosystem for Homologous Targeting of EMT Induced Triple Negative Breast Cancer Cells.

The increased mortality rate due to metastatic breast cancer with poor prognosis has raised concern over its effective therapy. Though various therapies and anticancer drugs have been approved, there is still a lack in the targeting of metastatic triple negative breast cancer (TNBC). We have developed a hybrid nanosystem that was synthesized by fusing exosomes from MCF-7 cells and nanovesicles from the MDA MB-231 cells that would be targeted. The developed nanosystem was characterized by various techniques like Western blotting, AFM, FETEM, DLS, CD, and fluorescence spectroscopy. The hybrid system was used for the delivery of an HDAC inhibitor, Trichostatin A (TSA), in combination with lapatinib (a tyrosine kinase inhibitor) for cotherapy of epithelial to mesenchymal transition (EMT) induced TNBC. This targeted cotherapy module had higher efficiency and effectivity in the reduction of metastatic ability and proliferation of EMT induced MDA MB-231 cells as compared to free inhibitor treatment or untargeted cotherapy. Reduction in the expression of the Wnt/β-catenin signaling pathway molecules like β-catenin (by 0.7 fold), Gsk3β (by 0.6 fold), and pGsk-3β (0.3 fold) was observed upon treatment. This subsequently resulted in the suppression of EMT markers, thereby resulting in reversing EMT to MET and suppressing metastatic breast cancer.