Metastatic Triple-negative Breast Cancer Cell Research Articles

Abstract 5634: Tristetraprolin induces an antitumorigenic phenotype in triple negative breast cancer via a novel non-canonical mechanism

Abstract The ultimate cause of death for most triple negative breast cancer (TNBC) patients is metastatic disease, which has a five-year survival rate of 12.8%. This low survival rate is partly due to the lack of targeted treatments for metastatic TNBC, resulting from the heterogeneity of the disease. As such, further research is needed to identify novel molecular mechanisms that can be targeted to suppress TNBC malignancy. Tristetraprolin (TTP) is an RNA-binding protein that binds to AU-rich elements (AREs) in the 3’ UTRs of select mRNAs, including many that encode proteins involved in cancer-related processes, and targets these mRNAs for degradation. Loss of TTP in tumors correlates with increased disease severity and decreased survival, suggesting a role as a tumor suppressor in multiple cancers, including breast cancer. While previous research has shown that TTP can suppress proliferation in breast cancer, the mechanism remains weakly defined. To elucidate how TTP impacts tumorigenic phenotypes in advanced breast cancer, we stably transfected a FLAG-TTP-expressing construct into three aggressive and metastatic TNBC cell lines. We analyzed TTP-induced changes in gene expression patterns among these cell lines using RNA-sequencing and observed that TTP alters the expression levels of many genes. Gene Set Enrichment Analysis (GSEA) of our datasets revealed that TTP affects multiple pathways, including significant suppression of pathways that promote cell growth, various metabolic processes contributing to tumor growth, and the response to hypoxia. As these pathways highly associated with tumorigenesis, we hypothesized that TTP would attenuate multiple tumor phenotypes. Our subsequent functional analyses revealed that TTP potently suppressed four tumor phenotypes in these cell models: cell proliferation, stem cell frequency, migration, and invasiveness. We hypothesized that inhibition of proliferation was mediated by cell cycle alterations, however, flow cytometry analysis revealed no substantial cell cycle perturbations between TTP-expressing cells and controls. Further analysis of cell cycle regulators revealed minimal changes in expression of proteins encoded by known or suspected TTP target mRNAs. Actinomycin D assays surprisingly revealed that TTP had no effect on the decay kinetics of several known TTP targets, suggesting that TTP’s antitumorigenic properties observed in TNBC cells are independent of its RNA-destabilizing function. To confirm this finding, we constructed cell models expressing an RNA-binding defective mutant of TTP, C147R, which recapitulated suppression of the four tumorigenic phenotypes seen with the wild-type protein. Collectively, these findings reveal that expression of TTP can potently suppress several oncogenic phenotypes in TNBC cells via a mechanism independent of its canonical RNA-binding/destabilizing functions. Citation Format: Julia L. Rutherford, Megan B. Stemberger, Rohaan Mahmud, Christina R. Ross, Elizabeth J. White, Gerald M. Wilson. Tristetraprolin induces an antitumorigenic phenotype in triple negative breast cancer via a novel non-canonical mechanism [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 5634.

Abstract 1570: Lipid metabolism alterations promote TNBC metastasis and immunotherapy resistance via PD-L1 internalization and signaling

Abstract Programmed death-ligand 1, PD-L1 (CD274), is an immune checkpoint protein upregulated by cancer cells to facilitate immune evasion. PD-L1 also performs oncogenic functions within cancer cells, but the underlying mechanisms of PD-L1 internalization and signaling remain largely unknown. Here, we report a new mechanism whereby altered ceramide metabolism via ceramide synthase 4 (CerS4) drives PD-L1 internalization to potentiate a TGFβ/Shh/β-catenin signaling axis to drive triple-negative breast cancer (TNBC) migration and metastasis. In response to altered ceramide metabolism, we observed that PD-L1 is internalized via clathrin-mediated endocytosis and depends on S278/S279 in its cytoplasmic domain. Once internalized, PD-L1 directly interacts with Caprin-1, an RNA-binding protein previously unassociated with PD-L1 signaling. Through our analysis of clinical specimens, we established a positive association between PD-L1/Caprin-1 interaction and metastatic progression in human TNBC. In terms of mechanism, we determined that the PD-L1/Caprin-1 complex plays a pivotal role in mediating CerS4-dependent cell migration. This complex selectively binds and stabilizes mRNA, resulting in heightened TGFβ/Shh/β-catenin signaling. Conversely, CerS4 restoration in metastatic TNBC cells impaired cellular migration and the interaction between PD-L1 and Caprin-1. Genetic depletion of CerS4 enhanced lung metastasis derived from 4T1 orthotopic allografts and transgenic MMTV-PyMT breast tumors concomitant with PD-L1/Caprin-1 interaction and TGFβ/Shh/β-catenin signaling. Bioinformatics analysis of clinical datasets was used to validate the impact of the CerS4/PD-L1/Caprin-1 axis on metastatic progression in human TNBC. In alignment with enhanced tumor progression, CerS4 depletion in tumors promoted regulatory T-cell infiltration, suggesting an immunosuppressive tumor microenvironment (TME). Treatment with sonidegib, an FDA-approved Hedgehog (Hh) inhibitor, effectively prevented PD-L1 internalization and its interaction with Caprin-1. We hypothesized that sonidegib could synergize with immune checkpoint blockade to sensitize TNBC to immunotherapy, which typically has low response rates. Sonidegib, in combination with αPD-L1 therapy, was significantly more effective in controlling TNBC primary and metastatic tumor growth, especially when Cers4 was downregulated, compared to αPD-L1 monotherapy. Our data also suggested that this potent anti-tumor effect was due to immunotherapy sensitization after combined flow cytometry, multiplexed immunofluorescence, and bulk RNA-sequencing analyses. Altogether, these findings demonstrate a mechanism to define intracellular PD-L1 signaling in response to altered ceramide metabolism, providing a new therapeutic strategy to target TNBC metastasis and improve response to immunotherapy. Citation Format: Wyatt O. Wofford, Jisun Kim, Dosung Kim, Alhaji H. Janneh, Han Gyul Lee, F. Cansu Atilgan, Natalia Oleinik, Elizabeth Hill, Ozge Saatci, Salih Gencer, Paramita Chakraborty, Shikhar Mehrotra, Ozgur Sahin, Besim Ogretmen. Lipid metabolism alterations promote TNBC metastasis and immunotherapy resistance via PD-L1 internalization and signaling [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 1570.

Antibodies toward Na+,HCO3--cotransporter NBCn1/SLC4A7 block net acid extrusion and cause pH-dependent growth inhibition and apoptosis in breast cancer.

Na+,HCO3--cotransporter NBCn1/Slc4a7 accelerates murine breast carcinogenesis. Lack of specific pharmacological tools previously restricted therapeutic targeting of NBCn1 and identification of NBCn1-dependent functions in human breast cancer. We develop extracellularly-targeted anti-NBCn1 antibodies, screen for functional activity on cells, and evaluate (a) mechanisms of intracellular pH regulation in human primary breast carcinomas, (b) proliferation, cell death, and tumor growth consequences of NBCn1 in triple-negative breast cancer, and (c) association of NBCn1-mediated Na+,HCO3--cotransport with human breast cancer metastasis. We identify high-affinity (KD ≈ 0.14 nM) anti-NBCn1 antibodies that block human NBCn1-mediated Na+,HCO3--cotransport in cells, without cross-reactivity towards human NBCe1 or murine NBCn1. These anti-NBCn1 antibodies abolish Na+,HCO3--cotransport activity in freshly isolated primary organoids from human breast carcinomas and lower net acid extrusion effectively in primary breast cancer tissue from patients with macrometastases in axillary lymph nodes. Inhibitory anti-NBCn1 antibodies decelerate tumor growth in vivo by ~50% in a patient-derived xenograft model of triple-negative breast cancer and pH-dependently reduce colony formation, cause G2/M-phase cell cycle accumulation, and increase apoptosis of metastatic triple-negative breast cancer cells in vitro. Inhibitory anti-NBCn1 antibodies block net acid extrusion in human breast cancer tissue, particularly from patients with disseminated disease, and pH-dependently limit triple-negative breast cancer growth.

MAP7D3, a novel prognostic marker for triple-negative breast cancer, drives cell invasiveness and cancer-initiating cell properties to promote metastatic progression

BackgroundPatients with triple-negative breast cancer (TNBC) tend to develop visceral metastasis within five years, making them the most challenging BC patients to treat. The MAP7 protein family is a group of microtubule-binding proteins with a well-known role in microtubule-related cell migration, but its role in metastasis-related properties of TNBC remains unclear.MethodsqRT-PCR and western blot were used to validate mRNA and protein expression of the MAP7 family in the isogenic pairs of TNBC cell lines with low and high metastasis potential. Functional characterization of MAP7D3 was carried out using cell-based and mouse models. The clinical association between MAP7D3 and TNBC was established using datasets in the public domain.ResultsMAP7D3 expression was consistently upregulated in the metastatic subline IV2 and 468-LN at both mRNA and protein levels. Knockdown of MAP7D3 inhibited the 3D colony-forming ability, cell migration, and invasion ability of IV2 and 468-LN, indicating its significant contribution to the metastasis phenotypes. Mechanistically, inhibition of MAP7D3 could significantly increase the sensitivity of metastatic TNBC cells to docetaxel and gemcitabine treatment by reducing the expression of proteins related to breast cancer-initiating cells (BCICs) and drug resistance, as well as suppressing the activity of Rac1. The animal study showed that the depletion of MAP7D3 drastically reduced TNBC tumor growth and impaired the metastatic capability of TNBC cells. Elevated expression of MAP7D3 was found in the metastatic lymph nodes and was significantly associated with advanced stage and higher grade TNBC. Moreover, MAP7D3 expression was significantly correlated with the TNBC population, and its high expression was significantly associated with lymph node metastasis and poor survival outcomes of patients with TNBC.ConclusionOur study indicates that targeting MAP7D3 could be a promising therapeutic strategy for addressing the progression of TNBC, and MAP7D3 may serve as a novel predictive biomarker for the survival outcomes of triple-negative breast cancer.

Abrogating Metastatic Properties of Triple-Negative Breast Cancer Cells by EGFR and PI3K Dual Inhibitors.

Triple-negative breast cancer (TNBC) is a devastating BC subtype. Its aggressiveness, allied to the lack of well-defined molecular targets, usually culminates in the appearance of metastases that account for poor prognosis, particularly when they develop in the brain. Nevertheless, TNBC has been associated with epidermal growth factor receptor (EGFR) overexpression, leading to downstream phosphoinositide 3-kinase (PI3K) signaling activation. We aimed to unravel novel drug candidates for TNBC treatment based on EGFR and/or PI3K inhibition. Using a highly metastatic TNBC cell line with brain tropism (MDA-MB-231 Br4) and a library of 27 drug candidates in silico predicted to inhibit EGFR, PI3K, or EGFR plus PI3K, and to cross the blood-brain barrier, we evaluated the effects on cell viability. The half maximal inhibitory concentration (IC50) of the most cytotoxic ones was established, and cell cycle and death, as well as migration and EGFR pathway intervenient, were further evaluated. Two dual inhibitors emerged as the most promising drugs, with the ability to modulate cell cycle, death, migration and proliferation, morphology, and PI3K/AKT cascade players such as myocyte enhancer factor 2C (MEF2C) and forkhead box P1 (FOXP1). This work revealed EGFR/PI3K dual inhibitors as strong candidates to tackle brain metastatic TNBC cells.

Calcium phosphate hybrid micelles inhibit orthotopic bone metastasis from triple negative breast cancer by simultaneously killing cancer cells and reprogramming the microenvironment of bone resorption and immunosuppression.

Triple negative breast cancer (TNBC) is prone to develop drug resistance and metastasis. Bone is the most common distant metastasis site of breast cancer cell. Patients with bone metastasis from TNBC suffer from unbearable pain due to the growth of bone metastasis and bone destruction. Simultaneously blocking the growth of bone metastasis and reprogramming the microenvironment of bone resorption and immunosuppression is a promising strategy to treat bone metastasis from TNBC. Herein, we prepared a pH and redox responsive drug delivery system, named DZ@CPH, by encapsulating docetaxel (DTX) with hyaluronic acid-polylactic acid micelle then reinforcing with calcium phosphate and zoledronate for targeting to bone metastasis from TNBC. DZ@CPH reduced the activation of osteoclast and inhibited bone resorption by decreasing the expression of nuclear factor κB receptor ligand and increasing the expression of osteoprotegerin in drug-resistant bone metastasis tissue. At the same time, DZ@CPH inhibited the invasion of bone metastatic TNBC cells by regulating the apoptosis-related and invasion-related protein expression. It also increased the sensitivity of orthotopic drug-resistant bone metastasis to DTX by inhibiting the expression of P-glycoprotein, Bcl-2 and transforming growth factor-β in tissue of drug-resistant bone metastasis. Moreover, the ratio between M1 type macrophage to M2 type macrophage in bone metastasis tissue was increased by DZ@CPH. In a word, DZ@CPH blocked the growth of bone metastasis from drug-resistant TNBC through inducing the apoptosis of drug-resistant TNBC cells and reprogramming the microenvironment of bone resorption and immunosuppression. DZ@CPH has a great potential in clinical application for the treatment of bone metastasis from drug-resistant TNBC. STATEMENT OF SIGNIFICANCE: Triple negative breast cancer (TNBC) is prone to develop bone metastasis. Now bone metastasis is still an intractable disease. In this study, docetaxel and zoledronate co-loaded calcium phosphate hybrid micelles (DZ@CPH) were prepared. DZ@CPH reduced the activation of osteoclasts and inhibited bone resorption. At the same time, DZ@CPH inhibited the invasion of bone metastatic TNBC cells by regulating the expression of apoptosis and invasion related protein in bone metastasis tissue. Moreover, the ratio between M1 type macrophages to M2 type macrophages in bone metastases tissue was increased by DZ@CPH. In a word, DZ@CPH blocked vicious cycle between the growth of bone metastasis and bone resorption, which greatly improved the therapeutic effect on bone metastasis from drug-resistant TNBC.

HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple‑negative breast cancer metastasis via the inhibition of CXCR4 expression.

Triple‑negative breast cancer (TNBC), a highly metastatic subtype of breast cancer, and it has the worst prognosis among all subtypes of breast cancer. However, no effective systematic therapy is currently available for TNBC metastasis. Therefore, novel therapies targeting the key molecular mechanisms involved in TNBC metastasis are required. The present study examined whether the expression levels of human epidermal growth factor receptor 3 (HER3) were associated with the metastatic phenotype of TNBC, and evaluated the potential of HER3 as a therapeutic target invitro and invivo. A new highly metastatic 4T1 TNBC cell line, termed 4T1‑L8, was established. The protein expression levels in 4T1‑L8 cells were measured using luminex magnetic bead assays and western blot analysis. The HER3 expression levels and distant metastasis‑free survival (DMFS) in TNBC were analyzed using Kaplan‑Meier Plotter. Transwell migration and invasion assays were performed to detect migration and invasion. The anti‑metastatic effects were determined in an experimental mouse model of metastasis. The results revealed that the increased expression of the HER3/Akt/mTOR pathway was associated with a greater level of cell migration, invasion and metastasis of TNBC cells. In addition, it was found that high expression levels of HER3 were associated with a poor DMFS. The inhibition of the HER3/Akt/mammalian target of rapamycin (mTOR) pathway decreased the migration, invasion and metastasis of TNBC cells by decreasing the expression of C‑X‑C chemokine receptor type 4 (CXCR4). Furthermore, treatment of metastatic TNBC cells with everolimus inhibited their migration, invasion and metastasis by decreasing CXCR4 expression. Thus, targeting the HER3/Akt/mTOR pathway opens up a new avenue for the development of therapeutics against TNBC metastasis; in addition, everolimus may prove to be an effective therapeutic agent for the suppression of TNBC metastasis.

ICAM2 initiates trans-blood-CSF barrier migration and stemness properties in leptomeningeal metastasis of triple-negative breast cancer

Leptomeningeal metastasis (LM) occurs when tumor cells spread to the leptomeningeal space surrounding the brain and the spinal cord, thereby causing poor clinical outcomes. The triple-negative breast cancer (TNBC) has been associated with symptoms of LM and mechanism remained unclear. Through proteomic analysis, we identified high expression of ICAM2 in leptomeningeal metastatic TNBC cells, which promoted the colonization of the spinal cord and resulted in poor survival in vivo. Two-way demonstration indicated that high levels of ICAM2 promoted blood–cerebrospinal fluid barrier (BCB) adhesion, trans-BCB migration, and stemness abilities and determined the specificity of LM in vivo. Furthermore, pull-down and antibody neutralizing assay revealed that ICAM2 determined the specificity of LM through interactions with ICAM1 in the choroid plexus epithelial cells. Therefore, neutralizing ICAM2 can attenuate the progression of LM and prolong survival in vivo. The results suggested that targeting ICAM2 is a potential therapeutic strategy for LM in TNBC.

Serum-Stable Gold(III) Bisphosphine Complex Induces Mild Mitochondrial Uncoupling and In Vivo Antitumor Potency in Triple Negative Breast Cancer.

The preparation of cyclometalated complexes offers a path to stable materials, catalysts, and therapeutic agents. Here, we explore the anticancer potential of novel biphenyl organogold(III) cationic complexes supported by diverse bisphosphine ligands, Au-1-Au-5, toward aggressive glioblastoma and triple negative breast cancer cells (TNBCs). The [C^C] gold(III) complex, Au-3, exhibits significant tumor growth inhibition in a metastatic TNBC mouse model. Remarkably, Au-3 displays promising blood serum stability over a relevant therapeutic window of 24 h and alteration in the presence of excess L-GSH. The mechanism-of-action studies show that Au-3 induces mitochondrial uncoupling, membrane depolarization, and G1 cell cycle arrest and prompts apoptosis. To the best of our knowledge, Au-3 is the first biphenyl gold-phosphine complex to uncouple mitochondria and inhibit TNBC growth in vivo.

Highly Metastatic Subpopulation of TNBC Cells Has Limited Iron Metabolism and Is a Target of Iron Chelators.

Excess iron is known to be a risk factor of carcinogenesis. Although iron chelators show anti-cancer effects, they have not been used successfully to treat cancer patients. Triple-negative breast cancer (TNBC) is a disease with poor prognosis without effective treatments. Thus, we aimed to evaluate a possibility of iron chelators as a therapy for TNBC. Deferasirox (DFX), an iron chelator, suppressed the growth of 4T1 murine TNBC cell line cells in vitro and in vivo. Lung metastasis was further significantly reduced, leading to the hypothesis that iron metabolism between metastatic and non-metastatic cells may be different. An analysis of existing database demonstrated that the expression of iron-uptake genes was significantly suppressed in TNBC cells that metastasized to lymph nodes or lungs compared to those in primary tumors. A highly metastatic clone of the murine 4T1 TNBC cells (4T1-HM) did not proliferate well under iron-rich or iron-depleted conditions by iron chelators compared to a low-metastatic clone (4T1-LM). Bulk RNA-seq analysis of RNA from 4T1-HM and 4T1-LM cells suggested that the PI3K-AKT pathway might be responsible for this difference. Indeed, DFX suppressed the proliferation via the AKT-mTOR pathway in 4T1-HM and the human MDA-MB-231 cells, a human mesenchymal-like TNBC cell line. DFX also suppressed the growth of 4T1-HM tumors in comparison to 4T1-LM tumors, and reduced lung metastases after surgical resection of primary 4T1 tumors. These results indicated, for the first time, that highly metastatic TNBC cells have limited iron metabolism, and they can be more effectively targeted by iron chelators.

Camptothecin loaded casein nanosystem for tuning the therapeutic efficacy against highly metastatic triple-negative breast cancer cells.

The heterogenic of TNBC and the side effects of chemo drugs lead to the failure of therapy. Protein-based nanoplatforms have emerged as an important domain in protein-engineered biomedicine for delivering anticancer therapeutics. Protein-based nanosystems are biocompatible and biodegradable, with a long half-life and high purity. TNBC is sensitive to DNA-damaging chemo drugs. In this study, we used 10-hydroxy camptothecin, which causes DNA damage in cancer cells. However, the inappropriate solubility and toxic side effects limit its application in cancer therapy. We encapsulated 10-Hydroxycamptothecin in biocompatible casein by synthesizing nanoparticles from it. The synthesized CS and CCS NPs showed excellent biocompatibility in fibroblast cell lines L929, NIH-3T3, and zebrafish embryos. Enhanced uptake of CCS NPs in zebrafish embryos and 4T1 cells, cancer cell toxicity of nearly 80-85%, sub-cellular mitochondrial localization, alterations of mitochondrial membrane potential, lysosomal localization, and reactive oxygen species generation that causes cancer cell apoptosis have been observed. Growth inhibition of 4T1 cell colonies and antimetastatic activity were also noted. Further upregulation of γ-H2AX which causes DNA damage, downregulation of the PARP protein related to DNA repair, and increased level of the CHOP protein marker for endoplasmic reticulum stress-mediated cell death were observed. The 3-D model of 4T1 cells exhibited deep tumor penetration with significant therapeutic efficacy for CCS NPs. These results imply that casein-based nanoformulation could open a new scope for safe and affordable cancer therapy in TNBC.

TFA-mediated stereoselective aza-Michael addition for the synthesis of 3β-arylamine derivatives of withaferin A and evaluation of their anticancer potential

A simple and efficient protocol for the aza-Michael addition of various aromatic anilines to ring A of withaferin A has been developed. Stereoselectivity, functional group tolerance, broad substrate scope, short reaction time and moderate to high yield are the merits of the protocol. One of the synthesized compounds 11 shows an IC 50 value of 3.8 μM against aggressive, highly metastatic triple-negative breast cancer cell line MDA-MB-231.

Antitumor Effect of Demethylzeylasteral (T-96) on Triple-Negative Breast Cancer via LSD1-Mediate Epigenetic Mechanisms.

Background and Purpose. Breast cancer ranks first in the incidence of female tumors. Triple-negative breast cancer (TNBC), one type of breast cancer, is more aggressive and has a worse prognosis. Demethylzeylasteral (T-96) is isolated from Tripterygium wilfordii Hook F. Our previous study found that T96 could inhibit TNBC invasion via suppressing the canonical and noncanonical TGF-β signaling pathways. However, the antitumor effects and mechanisms of T-96 on TNBC have not been studied. This study is aimed at investigating the antitumor effect and mechanism of T-96 on breast cancer. Experimental approach. MTT assay, Live and Dead cell assay, and TUNEL were used to observe the antitumor effect of breast cancer cells treated with T-96. siRNA of LSD1, Co-IP, and molecular docking were used to explore the direct target and mechanism of T-96. Subcutaneous murine xenograft models were used to detect the efficacy of T-96 antitumor activity in vivo. Key Results. T-96 was more susceptible to inducing the apoptosis of highly metastatic TNBC cell lines (SUM-1315). An abnormal level of histone methylation is a crucial characteristic of metastatic cancer cells. LSD1 is a histone demethylase. We found that T-96 could significantly decrease the protein expression of LSD1, increase its target protein PTEN expression and enhance histone methylation. T-96 could also down-regulate the PI3K/AKT signaling pathway, which could be blocked by PTEN. Knockdown of LSD1 by siRNA blocked the pharmacological activity of T-96. And the molecular docking predicted T-96 processed affinity toward LSD1 through hydrogen bonding. Finally, T-96 was evaluated in a murine xenograft model of SUM-1315 cells. And T-96 could significantly inhibit tumor growth without showing marked toxicity. Conclusions & Implications. The results illustrated that T-96 exerted antitumor activity in highly metastatic TNBC by inactivating the LSD1 function.

Ethanolic extract of Origanum syriacum L. leaves exhibits potent anti-breast cancer potential and robust antioxidant properties.

Background: Breast cancer (BC) is the second most common cancer overall. In women, BC is the most prevalent cancer and the leading cause of cancer-related mortality. Triple-negative BC (TNBC) is the most aggressive BC, being resistant to hormonal and targeted therapies. Hypothesis/Purpose: The medicinal plant Origanum syriacum L. is a shrubby plant rich in bioactive compounds and widely used in traditional medicine to treat various diseases. However, its therapeutic potential against BC remains poorly investigated. In the present study, we screened the phytochemical content of an ethanolic extract of O. syriacum (OSEE) and investigated its anticancer effects and possible underlying mechanisms of action against the aggressive and highly metastatic human TNBC cell line MDA-MB-231. Methods: MTT, trans-well migration, and scratch assays were used to assess cell viability, invasion, or migration, respectively. Antioxidant potential was evaluated in vitro using the DPPH radical-scavenging assay and levels of reactive oxygen species (ROS) were assessed in cells in culture using DHE staining. Aggregation assays were used to determine cell-cell adhesion. Flow cytometry was used to analyze cell cycle progression. Protein levels of markers of apoptosis (BCL-2, pro-Caspase3, p53), proliferation (p21, Ki67), cell migration, invasion, or adhesion (FAK, E-cadherin), angiogenesis (iNOS), and cell signaling (STAT3, p38) were determined by immunoblotting. A chorioallantoic Membrane (CAM) assay evaluated in ovo angiogenesis. Results: We demonstrated that OSEE had potent radical scavenging activity in vitro and induced the generation of ROS in MDA-MB-231 cells, especially at higher OSEE concentrations. Non-cytotoxic concentrations of OSEE attenuated cell proliferation and induced G0/G1 cell cycle arrest, which was associated with phosphorylation of p38 MAPK, an increase in the levels of tumor suppressor protein p21, and a decrease of proliferation marker protein Ki67. Additionally, only higher concentrations of OSEE were able to attenuate inhibition of proliferation induced by the ROS scavenger N-acetyl cysteine (NAC), indicating that the anti-proliferative effects of OSEE could be ROS-dependent. OSEE stimulated apoptosis and its effector Caspase-3 in MDA-MB-231 cells, in correlation with activation of the STAT3/p53 pathway. Furthermore, the extract reduced the migration and invasive properties of MDA-MB-231 cells through the deactivation of focal adhesion kinase (FAK). OSEE also reduced the production of inducible nitric oxide synthase (iNOS) and inhibited in ovo angiogenesis. Conclusion: Our findings reveal that OSEE is a rich source of phytochemicals and has robust anti-breast cancer properties that significantly attenuate the malignant phenotype of MD-MB-231 cells, suggesting that O. syriacum may not only act as a rich source of potential TNBC therapeutics but may also provide new avenues for the design of novel TNBC drugs.

Thioridazine combined with carboplatin results in synergistic inhibition of triple negative breast cancer by targeting cancer stem cells

Cancer stem cells (CSCs) in triple-negative breast cancer (TNBC) are closely related to tumorigenesis and metastasis. Thioridazine (THZ) is a usual phenothiazine antipsychotic drug that can destroy CSCs. We aimed to explore whether THZ could sensitize metastatic TNBC cells, especially the CSCs, to carboplatin (CBP) treatment. Metastatic TNBC cells, 4T1 cells, and tumor-bearing mice were treated with THZ and CBP as monotherapy or combination therapy. MTT, flow cytometry, electron microscopy, immunohistochemistry and western blotting were applied to assess the cell viability, apoptosis, mitochondrial morphology and the relevant protein levels, respectively. Tumor size and lung metastasis under different treatments as well as tumorigenesis of residual tumor cells from each group were monitored. THZ combined with CBP inhibited 4T1 tumor cell proliferation and induced apoptosis by inhibiting the PI3K-AKT-mTOR pathway and activating estrogen receptor stress. THZ also showed strong activity against breast CSCs, THZ combined with CBP significantly destroyed cancer cells, inhibited lung metastasis and relieved the tumor burden; Our data demonstrated that THZ can sensitize TNBC cells to CBP treatment and this combination therapy may provide a bright strategy for TNBC treatment by targeting both cancer cells and CSCs.

Biocompatible, Multi-Mode, Fluorescent, T2 MRI Contrast Magnetoelectric-Silica Nanoparticles (MagSiNs), for On-Demand Doxorubicin Delivery to Metastatic Cancer Cells.

There is a need to improve current cancer treatment regimens to reduce systemic toxicity, to positively impact the quality-of-life post-treatment. We hypothesized the negation of off-target toxicity of anthracyclines (e.g., Doxorubicin) by delivering Doxorubicin on magneto-electric silica nanoparticles (Dox-MagSiNs) to cancer cells. Dox-MagSiNs were completely biocompatible with all cell types and are therapeutically inert till the release of Doxorubicin from the MagSiNs at the cancer cells location. The MagSiNs themselves are comprised of biocompatible components with a magnetostrictive cobalt ferrite core (4–6 nm) surrounded by a piezoelectric fused silica shell of 1.5 nm to 2 nm thickness. The MagSiNs possess T2-MRI contrast properties on par with RESOVIST™ due to their cobalt ferrite core. Additionally, the silica shell surrounding the core was volume loaded with green or red fluorophores to fluorescently track the MagSiNs in vitro. This makes the MagSiNs a suitable candidate for trackable, drug nanocarriers. We used metastatic triple-negative breast cancer cells (MDAMB231), ovarian cancer cells (A2780), and prostate cancer cells (PC3) as our model cancer cell lines. Human umbilical vein endothelial cells (HUVEC) were used as control cell lines to represent blood-vessel cells that suffer from the systemic toxicity of Doxorubicin. In the presence of an external magnetic field that is 300× times lower than an MRI field, we successfully nanoporated the cancer cells, then triggered the release of 500 nM of doxorubicin from Dox-MagSiNs to successfully kill >50% PC3, >50% A2780 cells, and killed 125% more MDAMB231 cells than free Dox.HCl. In control HUVECs, the Dox-MagSiNs did not nanoporate into the HUVECS and did not exhibited any cytotoxicity at all when there was no triggered release of Dox.HCl. Currently, the major advantages of our approach are, (i) the MagSiNs are biocompatible in vitro and in vivo; (ii) the label-free nanoporation of Dox-MagSiNs into cancer cells and not the model blood vessel cell line; (iii) the complete cancellation of the cytotoxicity of Doxorubicin in the Dox-MagSiNs form; (iv) the clinical impact of such a nanocarrier will be that it will be possible to increase the current upper limit for cumulative-dosages of anthracyclines through multiple dosing, which in turn will improve the anti-cancer efficacy of anthracyclines.

MiR-623 Targets Metalloproteinase-1 and Attenuates Extravasation of Brain Metastatic Triple-Negative Breast Cancer Cells.

BackgroundMost breast cancer-related deaths result from metastasis. Understanding the molecular basis of metastasis is needed for the development of effective targeted and preventive strategies. Matrix metalloproteinase-1 (MMP1) plays an important role in brain metastasis (BM) of triple-negative breast cancer (TNBC) by promoting extravasation of cancer cells across the brain endothelium (BE). MMP1 expression is controlled by endogenous microRNAs. Preliminary bioinformatics analysis has revealed that miR-623, known to target the 3ʹUTR of MMP1, is significantly downregulated in brain metastatic tumors compared to primary BC tumors. However, the involvement of miR-623 in MMP1 upregulation in breast cancer brain metastatic cells (BCBMC) remains unexplored. Here, we investigated the role of miR-623 in MMP1 regulation and its impact on the extravasation of TNBC cells through the BE in vitro.Materials and MethodsA loss-and-gain of function method was employed to address the effect of miR-623 modulation on MMP1 expression. MMP1 regulation by miR-623 was investigated by real-time PCR, western blot, luciferase and transwell migration assays using an in vitro human BE model.ResultsOur results confirmed that brain metastatic TNBC cells express lower levels of miR-623 compared with cells having low propensity to spread toward the brain. miR-623 binds to the 3′-untranslated region of MMP1 transcript and downregulates its expression. Restoring miR-623 expression significantly decreased MMP1 expression, preserved the endothelial barrier integrity, and attenuated transmigration of BCBMC through the BE.ConclusionOur study elucidates, for the first time, the crucial role of miR-623 as MMP1 direct regulator in BCBMC and sheds light on miR-623 as a novel therapeutic target that can be exploited to predict and prevent brain metastasis in TNBC. Importantly, the presents study helps in unraveling a brain metastasis-specific microRNA signature in TNBC that can be used as a guide to personalized metastasis prediction and preventive approach with better therapeutic outcome.

Abstract 7: Modeling cell-cell crosstalk in TNBC brain metastasis

Abstract Brain metastasis (BrM) is a difficult clinical problem in triple-negative breast cancer (TNBC) patients: it occurs in 25% to 40% of metastatic patients while two-year survival rate stubbornly remains a single digit. TNBC usually spreads to the brain rapidly at earlier stages or before the diagnosis of primary cancer. The shorter development time of BrM in TNBC and shorter survival time after BrM diagnosis may indicate a unique innate ability of TNBC tumor cells in adapting to the brain. Indeed, emerging data including ours indicates that TNBC tumor cells have intrinsic brain-like traits resulting in brain-tropism metastasis. Our preliminary studies identified enriched and diverse expressions of receptors and transporters of various neuro-active substances in brain metastatic TNBC cells, including neurotransmitters, gliotransmitters, neurotrophic factors and neural cytokines. By immunopanning astrocytes and microglia/macrophages from mouse brains bearing TNBC brain metastatic tumors at day 3 post tumor cell injection through left cardiac ventricle, and use the CCCExplorer software in modeling the interactions among tumor cells, astrocytes, and microglia/macrophage with cell-type specific RNAseq data, we identified several novel crosstalk signaling, including tumor autocrine TPH1-HTR2, astrocyte→tumor PCDH7-EGFR, IL6-IL6R, CCL5-CCR5 and VGF-C3AR1, and microglia/macrophage→tumor ARTN-GFAR3. Functional experiments demonstrated that cell type-specific depletion of the secreted neuro-active substances deactivated the activities of corresponding receptor and downstream signaling in co-cultured BrM tumor cells. Given that more complex paracrine/autocrine signaling network may dynamically evolve during BrM development, we are characterizing the spatiotemporal cell-cell crosstalk in the BrM metastatic niche that will have important implications for novel therapeutics development. Citation Format: Hong Zhao, Xin Wang, Nikola Liu, Jianting Sheng, Stephen Wong. Modeling cell-cell crosstalk in TNBC brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 7.

Atovaquone Suppresses the Growth of Metastatic Triple-Negative Breast Tumors in Lungs and Brain by Inhibiting Integrin/FAK Signaling Axis.

Triple-negative breast cancer (TNBC) is considered to be the most aggressive and malignant neoplasm and is highly metastatic in nature. In the current study, we investigated the anti-metastatic potential of atovaquone, a protozoal drug prescribed for Pneumocystis pneumonia. We showed that atovaquone induced apoptosis and reduced the survival of several aggressive metastatic TNBC cell lines including metastatic patient-derived cells by reducing the expression of integrin α6, integrin β4, FAK, Src, and Vimentin. In order to study the efficacy of atovaquone in suppressing metastasized breast tumor cells in brain and lungs, we performed three in vivo experiments. We demonstrated that oral administration of 50 mg/kg of atovaquone suppressed MDA-MB-231 breast tumor growth by 90% in lungs in an intravenous metastatic tumor model. Anti-metastatic effect of atovaquone was further determined by intracardiac injection of 4T1-luc breast tumor cells into the left ventricle of mouse heart. Our results showed that atovaquone treatment suppressed the growth of metastatic tumors in lungs, liver and brain by 70%, 50% and 30% respectively. In an intracranial model, the growth of HCC1806-luc brain tumors in atovaquone treated mice was about 55% less than that of control. Taken together, our results indicate the anti-metastatic effects of atovaquone in vitro and in vivo in various breast tumor metastasis models.

Combinatorial targeting of microRNA-26b and microRNA-101 exerts a synergistic inhibition on cyclooxygenase-2 in brain metastatic triple-negative breast cancer cells.

Extravasation of triple-negative (TN) metastatic breast cancer (BC) cells through the brain endothelium (BE) is a critical step in brain metastasis (BM). During extravasation, metastatic cells induce alteration in the inter-endothelial junctions and transmigrate through the endothelial barrier. Transmigration of metastatic cells is mediated by the upregulation of cyclooxygenase-2 (COX-2) that induces matrix metalloproteinase-1 (MMP-1) capable of degrading inter-endothelial junctional proteins. Despite their important role in BM, the molecular mechanisms upregulating COX-2 and MMP-1 in TNBC cells remain poorly understood. In this study, we unraveled a synergistic effect of a pair of micro-RNAs (miR-26b-5p and miR-101-3p) on COX-2 expression and the brain transmigration ability of BC cells. Using a gain-and-loss of function approach, we modulated levels of miR-26b-5p and miR-101-3p in two TNBC cell lines (the parental MDA-MB-231 and its brain metastatic variant MDA-MB-231-BrM2), and examined the resultant effect on COX-2/MMP-1 expression and the transmigration of cancer cells through the BE. We observed that the dual inhibition of miR-26b-5p and miR-101-3p in BC cells results in higher increase of COX-2/MMP-1 expression and a higher trans-endothelial migration compared to either micro-RNA alone. The dual restoration of both micro-RNAs exerted a synergistic inhibition on COX-2/MMP-1 by targeting COX-2 and potentiated the suppression of trans-endothelial migration compared to single micro-RNA. These findings provide new insights on a synergism between miR-26-5p and miR-101-3p in regulating COX-2 in metastatic TNBC cells and shed light on miR-26-5p and miR-101-3p as prognostic and therapeutic targets that can be exploited to predict or prevent BM.