Transition In Triple-negative Breast Cancer Research Articles

Tabersonine enhances cisplatin sensitivity by modulating Aurora kinase A and suppressing epithelial–mesenchymal transition in triple-negative breast cancer

Context Tabersonine has been investigated for its role in modulating inflammation-associated pathways in various diseases. However, its regulatory effects on triple-negative breast cancer (TNBC) have not yet been fully elucidated. Objective This study uncovers the anticancer properties of tabersonine in TNBC cells, elucidating its role in enhancing chemosensitivity to cisplatin (CDDP). Materials and methods After tabersonine (10 μM) and/or CDDP (10 μM) treatment for 48 h in BT549 and MDA-MB-231 cells, cell proliferation was evaluated using the cell counting kit-8 and colony formation assays. Quantitative proteomics, online prediction tools and molecular docking analyses were used to identify potential downstream targets of tabersonine. Transwell and wound-healing assays and Western blot analysis were used to assess epithelial–mesenchymal transition (EMT) phenotypes. Results Tabersonine demonstrated inhibitory effects on TNBC cells, with IC50 values at 48 h being 18.1 μM for BT549 and 27.0 μM for MDA-MB-231. The combined treatment of CDDP and tabersonine synergistically suppressed cell proliferation in BT549 and MDA-MB-231 cells. Enrichment analysis revealed that the proteins differentially regulated by tabersonine were involved in EMT-related signalling pathways. This combination treatment also effectively restricted EMT-related phenotypes. Through the integration of online target prediction and proteomic analysis, Aurora kinase A (AURKA) was identified as a potential downstream target of tabersonine. AURKA expression was reduced in TNBC cells post-treatment with tabersonine. Discussion and conclusions Tabersonine significantly enhances the chemosensitivity of CDDP in TNBC cells, underscoring its potential as a promising therapeutic agent for TNBC treatment.

MiRNA-200c-3p deficiency promotes epithelial-mesenchymal transition in triple-negative breast cancer by activating CRKL expression

Epithelial-mesenchymal transition (EMT) plays an important role in malignant progression of Triple-negative breast cancer (TNBC). Many studies have confirmed that miRNA-200c-3p is related to EMT. And we found that it is involved in the regulation of EMT, but the exact mechanism is unclear. CRKL is highly expressed in a variety of tumors and plays a role in EMT. In this study, the potential targets of miRNA-200c-3p were searched in miRPathDB, Targetscan and PicTar. And there are 68 potential targets at the intersection of the three databases. Then, bioinformatics and text mining performed by Coremine Medica, and found that among 68 potential targets, CRKL has the strongest correlation with EMT in TNBC. Therefore, we speculated that miRNA-200c-3p involvement in EMT might be related to CRKL. To verify miRNA-200c-3p inhibits the malignant phenotype of TNBC by regulating CRKL, RT‒PCR, western blotting, Clonal formation assays,CCK-8 proliferation assays, transwell invasion assays, Luciferase reporter assay and nude mouse transplantation tumor assay were performed. In this study, we found that miRNA-200c-3p is under-expressed and EMT-related genes are up-regulated in TNBC, and miRNA-200c-3p can inhibit cancer cell proliferation, invasion and the expression of EMT-related genes and proteins in TNBC. Further research confirmed that miRNA-200c-3p could inhibit EMT by inhibiting the expression of CRKL that directly combining CRKL gene.

Retraction: Silencing of Prrx1b suppresses cellular proliferation, migration, invasion and epithelial-mesenchymal transition in triple-negative breast cancer.

Zhi-Dong Lv, Zhao-Chuan Yang, Xiang-Ping Liu, Li-Ying Jin, Qian Dong, Hui-Li Qu, Fu-Nian Li, Bin Kong, Jiao Sun, Jiao-Jiao Zhao, Hai-Bo Wang, Silencing of Prrx1b suppresses cellular proliferation, migration, invasion and epithelial-mesenchymal transition in triple-negative breast cancer. Journal of Cellular and Molecular Medicine, 20: 1640-1650. https://doi.org/10.1111/jcmm.12856 The above article, published online on 29 March 2016 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the journal Editor-in-Chief, Stefan Constantinescu, The Foundation for Cellular and Molecular Medicine and John Wiley and Sons Ltd. In 2022, the authors asked to retract the article as they found that some images were mistakenly duplicated from their previously published papers. Subsequently, an investigation into concerns raised by a third party, revealed inappropriate duplications of image panels within the article (Figures2A,B and 4D) as well as between this (Figure1B) and three other articles (with the same first or corresponding author) that were published previously in a different scientific context. Due to the number and the level of errors identified in the published figures, the editors consider the conclusions of this manuscript substantially compromised. Although the authors initially asked for the retraction, they did not agree to the retraction wording.

Immunohistochemical analysis of CD9, CD29 and epithelial to mesenchymal transition in triple-negative breast cancer

Immunohistochemical analysis of CD9, CD29 and epithelial to mesenchymal transition in triple-negative breast cancer

Bioinformatic miRNA-mRNAs Analysis Revels to miR-934 as a Potential Regulator of the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer and has the worst prognosis. In patients with TNBC tumors, the tumor cells have been reported to have mesenchymal features, which help them migrate and invade. Various studies on cancer have revealed the importance of microRNAs (miRNAs) in different biological processes of the cell in that aberrations, in their expression, lead to alterations and deregulations in said processes, giving rise to tumor progression and aggression. In the present work, we determined the miRNAs that are deregulated in the epithelial-mesenchymal transition process in breast cancer. We discovered that 25 miRNAs that regulate mesenchymal genes are overexpressed in patients with TNBC. We found that miRNA targets modulate different processes and pathways, such as apoptosis, FoxO signaling pathways, and Hippo. We also found that the expression level of miR-934 is specific to the molecular subtype of the triple-negative breast cancer and modulates a set of related epithelial-mesenchymal genes. We determined that miR-934 inhibition in TNBC cell lines inhibits the migratory abilities of tumor cells.

Tetrandrine Inhibits Cancer Stem Cell Characteristics and Epithelial to Mesenchymal Transition in Triple-Negative Breast Cancer via SOD1/ROS Signaling Pathway.

Targeting the stemness of triple-negativebreastcancer (TNBC)is a potential therapeutic approach for treating TNBC.Tetrandrine, a natural plant alkaloid, has several anticancer effects. Here, we aimed to evaluate the efficacy of tetrandrine in cancer stemness and epithelial to mesenchymal transition (EMT) in TNBC, and to explore the underlying mechanisms. The effects of tetrandrine on cell growth, cell viability, cell stemness capacity, cell migration, and cell invasion, as well as the molecules involved in these processes, were investigated in a cell culture system. An in vivo xenograft tumor and lung metastasis study was performed using nude mice to verify the effects and mechanisms of tetrandrine. Tetrandrine exhibited antiproliferative and cell cycle arrest activities in TNBC cell lines, significantly reduced aldehyde dehydrogenase and CD44[Formula: see text]CD24[Formula: see text] characteristic subpopulation, and successfully prevented mammosphere formation. It suppressed migration and invasion, enhanced anoikis, and regulated the expression of proteins involved in the EMT, including E-cadherin, Vimentin, and Occludin, in both TNBC cells and MDA-MB-231 spheroid cells. Further studies revealed that tetrandrine downregulated the expression of superoxide dismutase 1 (SOD1) and catalase and induced reactive oxygen species (ROS) production, which subsequently contributed to the inhibition of cell EMT and stemness. The in vivo studies also showed that tetrandrine inhibited tumor growth and metastasis of both adherent normal cells, and flow cytometry sorted specific CD44[Formula: see text]CD24[Formula: see text] breast cancer stem cells, which could be rescued by SOD1 overexpression. The results of this study suggest that tetrandrine could effectively inhibit breast cancer stem cell characteristics and the EMT process via the SOD1/ROS signaling pathway. Therefore, tetrandrine can be considered a promising anti-TNBC agent.

Intercellular Communication Reveals Therapeutic Potential of Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer.

(1) Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high intra-tumoral heterogeneity. The epithelial-mesenchymal transition (EMT) is one of the inducers of cancer metastasis and migration. However, the description of the EMT process in TNBC using single-cell RNA sequencing (scRNA-seq) remains unclear. (2) Methods: In this study, we analyzed 8938 cellular gene expression profiles from five TNBC patients. We first scored each malignant cell based on functional pathways to determine its EMT characteristics. Then, a pseudo-time trajectory analysis was employed to characterize the cell trajectories. Furthermore, CellChat was used to identify the cellular communications. (3) Results: We identified 888 epithelium-like and 846 mesenchyme-like malignant cells, respectively. A further pseudo-time trajectory analysis indicated the transition trends from epithelium-like to mesenchyme-like in malignant cells. To characterize the potential regulators of the EMT process, we identified 10 dysregulated transcription factors (TFs) between epithelium-like and mesenchyme-like malignant cells, in which overexpressed forkhead box protein A1 (FOXA1) was recognized as a poor prognosis marker of TNBC. Furthermore, we dissected the cell-cell communications via ligand-receptor (L-R) interactions. We observed that tumor-associated macrophages (TAMs) may support the invasion of malignant epithelial cells, based on CXCL-CXCR2 signaling. The tumor necrosis factor (TNF) signaling pathway secreted by TAMs was identified as an outgoing communication pattern, mediating the communications between monocytes/TAMs and malignant epithelial cells. Alternatively, the TNF-related ligand-receptor (L-R) pairs showed promising clinical implications. Some immunotherapy and anti-neoplastic drugs could interact with the L-R pairs as a potential strategy for the treatment of TNBC. In summary, this study enhances the understanding of the EMT process in the TNBC microenvironment, and dissections of EMT-related cell communications also provided us with potential treatment targets.

ESR2 Drives Mesenchymal-to-Epithelial Transition in Triple-Negative Breast Cancer and Tumorigenesis In Vivo.

Estrogen receptors (ERs) have pivotal roles in the development and progression of triple-negative breast cancer (TNBC). Interactions among cancer cells and tumor microenvironment are orchestrated by the extracellular matrix that is rapidly emerging as prominent contributor of fundamental processes of breast cancer progression. Early studies have correlated ERβ expression in tumor sites with a more aggressive clinical outcome, however ERβ exact role in the progression of TNBC remains to be elucidated. Herein, we introduce the functional role of ERβ suppression following isolation of monoclonal cell populations of MDA-MB-231 breast cancer cells transfected with shRNA against human ESR2 that permanently resulted in 90% reduction of ERβ mRNA and protein levels. Further, we demonstrate that clone selection results in strongly reduced levels of the aggressive functional properties of MDA-MB-231 cells, by transforming their morphological characteristics, eliminating the mesenchymal-like traits of triple-negative breast cancer cells. Monoclonal populations of shERβ MDA-MB-231 cells undergo universal matrix reorganization and pass on a mesenchymal-to-epithelial transition state. These striking changes are encompassed by the total prevention of tumorigenesis in vivo following ERβ maximum suppression and isolation of monoclonal cell populations in TNBC cells. We propose that these novel findings highlight the promising role of ERβ targeting in future pharmaceutical approaches for managing the metastatic dynamics of TNBC breast cancer.

Inhibition of endoplasmic reticulum stress alleviates triple-negative breast cancer cell viability, migration, and invasion by Syntenin/SOX4/Wnt/β-catenin pathway via regulation of heat shock protein A4

ABSTRACT Endoplasmic reticulum stress (ER stress) is a double-edged sword in the occurrence and development of malignant cancer. The aim of this study was to explore the roles of ER stress in metastasis and epithelial-mesenchymal transitionin triple-negative breast cancer (TNBC) and potential mechanisms. In this study, 4-PBA was administrated to inhibit the ER stress. Cell viability was evaluated using a cell counting kit-8 assay. Cell migration and invasion were identified by wound healing and transwell assay, respectively. Levels of MMP2 and MMP9 were measured by enzyme-linked immunosorbent assay and immunohistochemical staining. Western blot assay was used to assess the levels of ER stress-related proteins, Syndecan-1 (SDC-1)/Syntenin-1 (SDCBP-1)/SRY-related HMG-box 4 (SOX4) signaling and Wnt/β-catenin signaling. Moreover, a xenograft mice model was conducted to confirm the role of ER stress in TNBC. The data indicate that the ability of viability and metastasis of breast cancer cells were stronger than normal mammary epithelial cells. More aggressiveness was manifested in TNBC cells than that in non-TNBC cells. 4-PBA significantly suppressed the viability, migration, and invasion in BC cells and inhibited the SDC/SDCBP/SOX4 axis and Wnt/β-catenin signaling. Furthermore, heat shock protein A4 (HSPA4) overexpression stimulated ER stress and activated the SDC-1/SDCBP-1/SOX4 pathway and Wnt/β-catenin signaling. Animal experiments showed similar results that 4-PBA repressed tumor growth and inactivated the two pathways, while HSPA4 overexpression reversed the effects of 4-PBA. In summary, inhibition of ER stress inhibited TNBC viability, migration, and invasion by Syntenin/SOX4/Wnt/β-catenin pathway via regulation of HSPA4 in vivo and in vitro.

SMARCA4 Depletion Induces Cisplatin Resistance by Activating YAP1-Mediated Epithelial-to-Mesenchymal Transition in Triple-Negative Breast Cancer.

Simple SummarySMARCA4 mutations were over-representative in cisplatin resistance and metastatic triple-negative breast cancer (TNBC). Additionally, SMARCA4 inactivation induced the mesenchymal-like subtype TNBC. The epithelial-to-mesenchymal transition and Hippo-YAP/TAZ pathways were activated in SMARCA4 inactivation samples of both SMARCA4 knockout cell lines and TNBC patients. In SMARCA4 knockout cells, the YAP1 inhibitor verteporfin suppressed YAP1 target genes. This study depicts the clinical importance of SMARCA4 depletion in TNBC and suggests YAP/TAZ as a novel target for cisplatin-resistant patients.The role of SMARCA4, an ATPase subunit of the SWI/SNF chromatin remodeling complex, in genomic organization is well studied in various cancer types. However, its oncogenic role and therapeutic implications are relatively unknown in triple-negative breast cancer (TNBC). We investigated the clinical implication and downstream regulation induced by SMARCA4 inactivation using large-scale genome and transcriptome profiles. Additionally, SMARCA4 was knocked out in MDA-MB-468 and MDA-MB-231 using CRISPR/Cas9 to identify gene regulation and a targetable pathway. First, we observed an increase in SMARCA4 mutations in cisplatin resistance and metastasis in TNBC patients. Its inactivation was associated with the mesenchymal-like (MSL) subtype. Gene expression analysis showed that the epithelial-to-mesenchymal transition (EMT) pathway was activated in SMARCA4-deficient patients. Next, the Hippo pathway was activated in the SMARCA4 inactivation group, as evidenced by the higher CTNNB1, TGF-β, and YAP1 oncogene signature scores. In SMARCA4 knockout cells, EMT was upregulated, and the cell line transcriptome changed from the SL to the MSL subtype. SMARCA4 knockout cells showed cisplatin resistance and Hippo-YAP/TAZ target gene activation. The YAP1 inhibitor verteporfin suppressed the expression of YAP1 target genes, and decreased cell viability and invasiveness on SMARCA4 knockout cells. SMARCA4 inactivation in TNBC endowed the resistance to cisplatin via EMT activation. The YAP1 inhibitor could become a novel strategy for patients with SMARCA4-inactivated TNBC.

Nonphosphorylatable PEA15 mutant inhibits epithelial-mesenchymal transition in triple-negative breast cancer partly through the regulation of IL-8 expression

BackgroundTriple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks targeted therapies. Patients with TNBC have a very poor prognosis because the disease often metastasizes. New treatment approaches addressing drivers of metastasis and tumor growth are crucial to improving patient outcomes. Developing targeted gene therapy is thus a high priority for TNBC patients. PEA15 (phosphoprotein enriched in astrocytes, 15 kDa) is known to bind to ERK, preventing ERK from being translocated to the nucleus and hence blocking its activity. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the function and impact of phosphorylation status of PEA15 in the regulation of TNBC metastasis and in epithelial-to-mesenchymal transition (EMT) are not well understood.MethodsWe established stable cell lines overexpressing nonphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) mutants. To dissect specific cellular mechanisms regulated by PEA15 phosphorylation status, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to determine the effects of PEA15 phosphorylation on tumor growth and metastasis.ResultsWe found that the nonphosphorylatable mutant PEA15-AA prevented formation of mammospheres and expression of EMT markers in vitro and decreased tumor growth and lung metastasis in in vivo experiments when compared to control, PEA15-WT and phosphomimetic PEA15-DD. However, phosphomimetic mutant PEA15-DD promoted migration, mesenchymal marker expression, tumorigenesis, and lung metastasis in the mouse model. PEA15-AA-mediated inhibition of breast cancer cell migratory capacity and tumorigenesis was the partial result of decreased expression of interleukin-8 (IL-8). Further, we identified that expression of IL-8 was possibly mediated through one of the ERK downstream molecules, Ets-1.ConclusionsOur results show that PEA15 phosphorylation status serves as an important regulator for PEA15’s dual role as an oncogene or tumor suppressor and support the potential of PEA15-AA as a therapeutic strategy for treatment of TNBC.

Curcumol enhances the anti-tumor effects of metformin via suppressing epithelial-mesenchymal transition in triple-negative breast cancer.

BackgroundTriple-negative breast cancer (TNBC) is a severe disease with a high mortality rate. Metformin has been found to possess anti-tumor properties. Curcumol, an active ingredient extracted from curcuma, exerts the protective effect in TNBC cells through inducing apoptosis. However, the effects of curcumol combined with metformin on the treatment of TNBC have yet to be fully established.MethodsTNBC cells MDA-MB-231 and MDA-MB-468 cells were used in the study. TNBC cells were treated with curcumol and metformin alone or treated with curcumol combined with metformin. Cell viability was determined using Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The levels of proteins were measured using Western blot. Wound healing assay and Transwell invasion assays were used to determine cell migration and invasion ability, respectively. A xenograft model was established to investigate the tumor growth ability. Immunohistochemistry was performed to determine the expression of Ki-67 and Vascular endothelial growth factor (VEGF).ResultsIn the study, the administration of curcumol alone had no significant effects on the TNBC cells. However, the anti-proliferation, anti-metastasis, and anti-epithelial-mesenchymal transition (EMT) effects of metformin were enhanced by the addition of curcumol. Further, curcumol reversed TNBC cell proliferation, migration, invasion, and EMT induced by rucaparib, and enhanced the effect of metformin on rucaparib-induced TNBC cells. The combination of curcumol and metformin also suppressed tumor growth, EMT marker expression, and the activation of Wnt2/β-Catenin signaling during in vivo experiments.ConclusionsThe combination of curcumol and metformin enhances the anti-tumor effects of metformin on TNBC via inhibiting EMT. Curcumol combined with metformin may hold promise as a therapeutic strategy for TNBC.

Lignin-Graft-Plga Drug-Delivery System Improves Efficacy of MEK1/2 Inhibitors in Triple-Negative Breast Cancer Cell Line

Aim: Few targeted therapies are available for triple-negative breast cancer (TNBC) patients. Here, we propose a novel alkaline-lignin-conjugated-poly(lactic-co-glycolic acid) (L-PLGA) nanoparticle drug delivery system to improve the efficacy of targeted therapies.Materials & methods: L-PLGA nanoparticles (NPs) loaded with the MEK1/2 inhibitor GDC-0623 were characterized, tested in vitro on MDA-MB-231 TNBC cell line and compared with loaded PLGA NPs. Results: Loaded L-PLGA NPs were less than half the size of PLGA NPs, had slower drug release and improved the efficacy of GDC-0623 when tested in vitro. We demonstrated that GDC-0623 reversed epithelial-to-mesenchymal transition in TNBC. Conclusion: Our findings indicate that L-PLGA NPs are superior to PLGA NPs in delivering GDC-0623 to cancer cells for improved efficacy in vitro.

Oncolytic virotherapy armed with an engineered interfering lncRNA exhibits antitumor activity by blocking the epithelial mesenchymal transition in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) has special characteristics of significant aggressiveness, and strong potential for metastasis and recurrence; currently there are no targeted drugs for TNBC. Abnormal activation of epithelial-mesenchymal transition (EMT) plays an important role in these malignant behaviors of TNBC. In the crosstalk among the multiple EMT-associated signaling pathways, many miRNAs participate in regulating pathway activity, where they act as "traffic lights" at the intersection of these pathways. In this study, we used miRNA microarray technology to detect differentially expressed miRNAs related to EMT in TNBC, and we identified and verified 9 highly expressed oncogenic miRNAs (OncomiRs). High expression of these OncomiRs in clinical breast cancer tissues affected the prognosis of patients, and inhibition of their expression blocked EMT in TNBC cell lines and suppressed cancer cell proliferation and migration. We constructed an oncolytic adenovirus (AdSVP-lncRNAi9) armed with an artificially-designed interfering lncRNA (lncRNAi9), which exhibited an activity to block EMT in TNBC cells by disrupting the functions of multiple OncomiRs; the efficacy of such a treatment for TNBC was demonstrated in cytology and animal experiments. This research provides a new candidate oncolytic virotherapy for treating highly malignant refractory TNBC.

Abstract P6-07-04: Mechanosensing calcium channel PIEZO2 accelerates epithelial-mesenchymal transition in triple-negative breast cancer

Abstract Introduction: Breast cancer is the most common female malignancy. Despite improvements in therapeutic options, metastatic disease remains incurable with limited treatments especially in triple-negative breast cancer (TNBC). Clinical outcomes in patients with TNBC continue to be worse as compared to other breast cancer subtypes. Therefore, novel therapeutic targets for TNBC are in urgent need. PIEZO2, which is a transmembrane calcium channel, is known to function as a channel-type mechanosensor in various aspects of mechanotransduction. Recently there have been reports that PIEZO2 may be involved in carcinogenesis and cancer progression. However, the role of PIEZO2 in breast cancer has not been elucidated. We hypothesized that PIEZO2 plays significant roles in progression of TNBC. Methods: To investigate the role of PIEZO2 in human breast cancer patients, The Cancer Genome Atlas (TCGA) breast cancer cohort, in which there are mRNA expression data from RNA-seq and clinical data, was utilized. PIEZO2 expression level was compared among breast cancer subtypes. Patients’ survival was compared based on PIEZO2 expression in each subtype. We used gene set enrichment analysis (GSEA) algorithm to evaluate pathways associated with PIEZO2 expression. To further elucidate the mechanisms of PIEZO2 roles, TNBC cell line, MDA-MB-231 was utilized for in vitro experiments. PIEZO2 expression was downregulated by small interfering RNA (siRNA). Cell viability was quantified by MTT assay. Protein expression was compared by western blot. Results: Breast cancer showed the highest PIEZO2 expression level, among various types of cancers in TCGA. In the breast cancer cohort, PIEZO2 expression level was lower in the TNBC subtype compared to other subtypes (p<0.001). Thus we hypothesized that PIEZO2 has different roles in each subtype. Survival analyses revealed that patients with high expression of PIEZO2 tumors showed significantly worse overall survival (p=0.016) in the TNBC cohort. However, there was no significant difference in the hormone receptor (HR)-positive (p=0.555) and the HER2-positive (p=0.380) cohorts. To explore underlying mechanisms how PIEZO2 high expressing tumors associate with worse prognosis in TNBC, GSEA was conducted. PIEZO2 high expressing tumors enriched epithelial-mesenchymal transition (EMT; NES=1.90, p=0.012) and Angiogenesis (NES=1.91, p<0.001) gene sets, as well as TAZ/YAP gene set (NES=1.57, p=0.028). PIEZO2 knockdown by siRNA significantly suppressed cell viability. This finding was validated by PIEZO inhibitor GSMTx-4 treatment. Further, expression levels of EMT related proteins, such as Vimentin and Snail, were downregulated in the PIEZO2 knockdown MDA-MB-231 cells compared to control cells. Conclusion: We describe a novel signaling pathway mediated by PIEZO2 leading to worse clinical outcomes in patients with TNBC by activation of EMT. Further work is ongoing to validate PIEZO2 as a novel therapeutic target for TNBC. Citation Format: Eriko Katsuta, Marija Vujcic, Shipra Gandhi, Arthur Beyder, Kazuaki Takabe, Mateusz Opyrchal. Mechanosensing calcium channel PIEZO2 accelerates epithelial-mesenchymal transition in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-07-04.

Metastasis suppressor 1 acts as a tumor suppressor by inhibiting epithelial-to-mesenchymal transition in triple-negative breast cancer.

This study aimed to analyze the function of metastasis suppressor 1 (MTSS1) in triple negative breast cancer (TNBC). MTSS1 expression in 30 TNBC and paracancerous tissues was measured by quantitative reverse transcriptase polymerase chain reaction. The prognostic value of MTSS1 was assessed by Kaplan-Meier analysis followed by the log-rank test. MCF7 cells were transfected with si-MTSS1, while MDA-MB-231 cells were transfected with pcDNA3.1-MTSS1. Cell proliferation assay and transwell assay were performed to investigate the effects of MTSS1 on the biological behavior of breast cancer cells. Immunofluorescence and western blot were used to detect the influence of MTSS1 on epithelial-mesenchymal transition (EMT) markers. MTSS1 expression was significantly lower in TNBC tissues compared with that in paracancerous tissues (0.012 vs. 0.370; P = 0.006). A lower MTSS1 expression level was also found in tumor tissues of patients with lymph node metastasis (P = 0.002) or tumor node metastasis stage (P = 0.010). Patients with low expression of MTSS1 (⩽ 0.009) had shorter disease-free survival (47.4 vs. 56.0 months; P = 0.012). The knockdown of MTSS1 in MCF7 cells inhibited cell proliferation, enhanced cell migration and invasion capacities, decreased the E-cadherin level, and increased the vimentin level, whereas overexpression of MTSS1 in MDA-MB-231 cells had the opposite effects (P < 0.05). Our findings demonstrated that MTSS1 regulates proliferation, invasion, migration, and EMT in TNBC, and that decreased MTSS1 is associated with shorter disease-free survival.

Polysaccharides of Huaier Suppress Metastasis and Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer by Inducing Autophagy to Degrade Snail

Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype and lacks targeted therapies. Trametes robiniophila Murr (Huaier) has been widely used in clinical anti-cancer treatments in China. Previously, we found that Huaier can effectively improve 5-year overall survival and disease free survival in stage III TNBC patients. The Polysaccharides of Huaier (PS-T) were identified as the major components of Huaier. Further study will be needed to elucidate the efficacy of PS-T as potential anti-tumor adjuvant strategy in TNBC. Methods: Transwell assay, scratch assay and mice lung metastasis model were used to observe cell invasion and migration in vitro and in vivo. Western blotting was used to determine the expression of epithelial-mesenchymal transition (EMT) and autophagy related proteins. Immunofluorescence analysis was used to detect autophagosome-lysosome fusion and protein expression of LC3 and Snail. A cell line MDA-MB-231-siATG5 in which autophagy key protein ATG5 was stably interfered was constructed. An E-cadherin promoter reporter gene plasmid and a Snail overexpression plasmid were sequentially introduced into MDA-MB-231 cells. Immunohistochemistry and Database analysis online(the Human Protein Atlas, UALCAN, Kaplan-Meier plotter) were used to explore the distinct prognostic and potential therapeutic value of Snail in breast cancer patients. Findings: PS-T inhibited cell invasion and migration in vitro and in vivo, and reversed the EMT while induced autophagy. Utilization of autophagy inhibitor LY294002 or knockdown of ATG5 by lentivirus infection suppressed the inhibitory effects of PS-T. Snail, a key transcription factor that controls EMT initiation was found to be degraded by PS-T induced autophagy. Overexpression of Snail reversed the inhibitory effects of PS-T. IHC for breast cancer tissues of 33 cases of TNBC patients and Database analysis online verified that high expression of Snail was associated with poor prognostics. Interpretation: We demonstrate that PS-T can inhibit EMT in breast cancer cell by the induction of autophagy to degrade Snail protein which contributes to both the EMT and metastasis. This study provides a new insight into the mechanism by which PS-T reduce the recurrence of TNBC and new ideas for comprehensive treatment strategies for TNBC patients. Funding Statement: National Natural Science foundation of China (No. 81802664). Declaration of Interests: All authors declare no competing interest. Ethics Approval Statement: Animals were treated according to protocols established by the ethics committee of Army Medical University and the experiments in vivo were conducted according to the approved guidelines and approved by the ethics committee of Army Medical University (AMUWEC2019199).

Long non-coding RNA ZEB2-AS1 promotes the proliferation, metastasis and epithelial mesenchymal transition in triple-negative breast cancer by epigenetically activating ZEB2.

The triple‐negative breast cancer is the most malignant type of breast cancer. Its pathogenesis and prognosis remain poor despite the significant advances in breast cancer diagnosis and therapy. Meanwhile, long noncoding RNAs (LncRNAs) play a pivotal role in the progression of malignant tumors. In this study, we found that LncRNA‐ZEB2‐AS1 was dramatically up‐regulated in our breast cancer specimens and cells (MDA231), especially in metastatic tumor specimens and highly invasive cells, and high lncRNA‐ZEB2‐AS1 expression is associated with clinicopathologic features and short survival of breast cancer patients. LncRNA‐ZEB2‐AS1 promotes the proliferation and metastasis of MDA231 cells in SCID mice. Thus, it is regarded as an oncogene in triple‐negative breast cancer. It is mainly endo‐nuclear and situated near ZEB2, positively regulating ZEB2 expression and activating the epithelial mesenchymal transition via the PI3K/Akt/GSK3β/Zeb2 signaling pathway. Meanwhile, EGF‐induced F‐actin polymerization in MDA231 cells can be suppressed by reducing lncRNA‐ZEB2‐AS1 expression. The migration and invasion of triple‐negative breast cancer can be altered through cytoskeleton rearrangement. In summary, we demonstrated that lncRNA‐ZEB2‐AS1 is an important factor affecting the development of triple‐negative breast cancer and thus a potential oncogene target.

Abstract P6-11-10: Preclinical efficacy of the novel PIM2 kinase inhibitor, JP11646 in triple negative breast cancer models

Abstract Background: Triple negative breast cancer (TNBC) patients have poorer prognosis and there remains a lack of novel targeted therapies for their treatment. PIM2 (Proviral Integrations of Moloney virus 2) belongs to a family of three kinases that have been implicated in the survival and progression of hematologic malignancies and solid tumors. PIM2 has been linked to epithelial to mesenchymal transition in TNBC, which can lead to metastasis and chemotherapeutic resistance. We hypothesized that PIM2 may present as a therapeutic target in TNBC. Materials and Methods: The study involved both in vitro and in vivo studies involving a novel PIM2 inhibitor JP11646 (obtained from Jasco Pharmaceuticals). TNBC cell lines MDA-MB-231 and BT-549 were obtained for our in vitro studies. Cell viability was evaluated using MTT assay. Western Blot assay was used to evaluate relative protein expression. For in vivo studies, female SCID mice were inoculated in the mammary fat pads with 1 × 106 MDA-MB-231 cells. When tumor volumes reached 100 mm3, the mice were treated with JP11646 at the dosage 15mg/kg intraperitoneally for 2 consecutive days weekly for total of 4 weeks as determined from previous experiments. Control animals received vehicle only. The mice were euthanized once tumors reached ∼1,700 mm3. Results: BT-549 cells treated in vitro with 3 different available PIM kinase inhibitors AZD 1208, LGB321 and JP12641 showed only modest reduction in cell viability. However, treatment of both MDA-MB-231 and BT-549 with JP 11646 demonstrated significant reduction in cell viability with IC50 ranging from 40 to 71.6 nM. Treatment with JP11646 demonstrated a novel mechanism of action resulting in downregulation of PIM2 in both cell lines. Treatment with JP11646, but not other PIM kinase inhibitors, resulted in activation of apoptosis as measured by cleaved PARP (cPARP) levels. Anti-PIM2 siRNA treatment but not treatment with non-specific PIM kinase inhibitor AZD1208 resulted in cPARP induction. Inhibition of proteolysis by bortezomib resulted in preservation of PIM2 and inhibition of apoptosis as demonstrated by decreased cPARP levels after treatment with JP11646. PIM2 over-expressing clone of MDA-MB-231 cells showed enhanced proliferation and migration properties both in vitro and in vivo.Treatment of mice with orthotopically implanted MDA-MB-231 tumors with JP 11646 resulted in significant reduction in the tumor growth (p=0.0019) and increased overall survival (p=0.018) as compared to control mice. Conclusions: PIM2 upregulation in TNBC cell line resulted in more aggressive phenotype. JP11646, through novel mechanism of action resulting in degradation of PIM2, showed robust activity in TNBC cell lines both in vitro and in vivo. Further correlative studies in tumors harvested from in vivo experiments are ongoing. These results encourage further exploration of use of JP11646 as a targeted agent in treatment of TNBC. Citation Format: Mehta R, Kothai Guruswamy Sangameswaran D, Bezbatchenko K, Moore J, Gil M, Khoury T, Baldino C, Caserta J, Fetterly, Jr. G, Lee K, Adjei A, Opyrchal M. Preclinical efficacy of the novel PIM2 kinase inhibitor, JP11646 in triple negative breast cancer models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-10.

MiR-212-5p Suppresses the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer by Targeting Prrx2

Background/Aims: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Our study investigated the functional role of miR-212-5p in TNBC. Methods: Realtime PCR was used to quantify miR-212-5p expression levels in 30 paired TNBC samples and adjacent normal tissues. Wound healing and Transwell assays were used to evaluate the effects of miR-212-5p expression on the invasiveness of TNBC cells. Luciferase reporter and Western blot assays were used to verify whether the mRNA encoding Prrx2 is a major target of miR-212-5p. Results: MiR-212-5p was downregulated in TNBC, and its expression levels were related to tumor size, lymph node status and vascular invasion in breast cancer. We also observed that the miR-212-5p expression level was significantly correlated with a better prognosis in TNBC. Ectopic expression of miR-212-5p induced upregulation of E-cadherin expression and downregulation of vimentin expression. The expression of miR212-5p also suppressed the migration and invasion capacity of mesenchymal-like cancer cells accompanied by a morphological shift towards the epithelial phenotype. Moreover, our study observed that miR-212-5p overexpression significantly suppressed Prrx2 by targeting its 3’-untranslated region (3’-UTR) region, and Prrx2 overexpression partially abrogated miR-212-5p-mediated suppression. Conclusions: Our study demonstrated that miR-212-5p inhibits TNBC from acquiring the EMT phenotype by downregulating Prrx2, thereby inhibiting cell migration and invasion during cancer progression.