Invasion Of Triple-negative Breast Cancer Cells Research Articles

The miR-124-3p/Neuropilin-1 Axis Contributes to the Proliferation and Metastasis of Triple-Negative Breast Cancer Cells and Co-Activates the TGF-β Pathway.

Triple-negative breast cancer (TNBC) accounts for 90% of breast cancer-associated mortality. Neuropilin-1 (NRP-1) acts as a non-tyrosine kinase receptor for several cellular signaling pathways involved in the proliferation and metastasis of cancer cells. However, the miRNAs that regulate NRP-1 expression and the underlying mechanisms in TNBC cells remain unclear. In the present study, we found that TNBC cells expressed higher levels of NRP-1 than non-TNBC cells. Stable transfectants depleted of NRP-1 were generated from two TNBC cell lines, human MDA-MB-231 and mouse 4T1 cells. NRP-1 depletion significantly suppressed the proliferation of TNBC cells by arresting the cell cycle at phase G0/G1 by upregulating p27 and downregulating cyclin E and cyclin-dependent kinase 2. NRP-1 depletion also repressed cell migration and epithelial-mesenchymal transition (EMT) by inducing the upregulation of E-cadherin and the downregulation of N-cadherin, matrix metalloproteinase (MMP)-2 and MMP-9, and reducing MMP-2 and MMP-9 activities as detected by gelatin zymography assay. By applying multiple miRNA-target prediction tools, we screened potential miRNAs with binding sites with the 3’-untranslated region of the NRP-1 gene and selected 12 miRNA candidates, among which miR-124-3p displayed the most vigorous activity to downregulate NRP-1 as validated by luciferase assay and miRNA transfection assay. By downregulating NRP-1, miR-124-3p mimics inhibited the proliferation, migration, and invasion of TNBC cells, and antagomiR-124-3p could partially abolish the effects of NRP-1 depletion. In the animal experiments, NRP-1 depletion inhibited tumorigenesis and liver metastasis of TNBC cells, while miR-124-3p mimics inhibited the growth of established TNBC tumors. In the mechanistic exploration, we revealed that NRP-1 co-interacted with transforming growth factor (TGF)-β to activate the TGF-β pathway, which regulates EMT-related molecules. In summary, the present results indicate that the miR-124-3p/NRP-1 axis contributes to the proliferation and metastasis of TNBC cells and co-activates the TGF-β pathway, suggesting that these molecules may present as potential therapeutic targets and valuable biomarkers for TNBC.

LncRNA GSEC Promotes the Progression of Triple Negative Breast Cancer (TNBC) by Targeting the miR-202-5p/AXL Axis.

BackgroundThis study aimed to explore the biological functions of G-quadruplex-forming sequence containing lncRNA (GSEC) in triple negative breast cancer (TNBC).MethodsThe expression of GSEC in TNBC tissues was evaluated by qRT-PCR. Cell viability was evaluated by Cell Counting Kit-8 assay. Cell proliferation was evaluated by 5-ethynyl-20-deoxyuridine (EdU) staining assay. Cell invasion and migration were evaluated by Transwell assay. Gain- and loss-function assays were performed to assess the biological functions of GSEC in TNBC. The interactions between GSEC, miR-202-5p and AXL were determined by luciferase report assay and RNA immunoprecipitation (RIP) assay. In addition, a nude mouse xenograft model was used to confirm the oncogenic role of GSEC in TNBC.ResultsGSEC was significantly upregulated in TNBC tissues and cancer cell lines, and high level of GSEC was associated with advanced tumor stage, positive lymph-node metastasis and the poor prognosis of TNBC patients. Knockdown of GSEC effectively inhibited TNBC cell proliferation, invasion and migration in vitro. GSEC regulated the expression of AXL by directly sponging miR-202-5p. Downregulation of miR-202-5p attenuated GSEC knockdown-induced inhibition on TNBC cell proliferation, invasion and migration in vitro. Meanwhile, overexpression of AXL obviously reversed the inhibitory effects of miR-202-5p mimics in TNBC progression in vitro.ConclusionGSEC functioned as a potential oncogene and promoted AXL-mediated TNBC progression by sponging miR-202-5p, which might be a novel diagnostic and therapeutic target for TNBC.

LncRNA GHET1 Promotes Hypoxia-Induced Glycolysis, Proliferation, and Invasion in Triple-Negative Breast Cancer Through the Hippo/YAP Signaling Pathway.

ObjectiveThis study was to assess the specific impacts and mechanism of lncRNA GHET1 in the development of triple-negative breast cancer (TNBC).MethodsThe lncRNA GHET1 expression in TNBC tissues and adjacent healthy tissues was detected by qRT-PCR, and its expression was then measured at the cellular level, including TNBC cells and human normal breast epithelial cell line MCF10A. On the completion of transfection of negative shRNA or lncRNA GHET1 shRNA, the TNBC cells, HCC1937 and MDA-MB-468, were then cultured in a normoxia or hypoxia environment, respectively. 5-Ethynyl-2′-deoxyuridine (EdU) assay, colony formation assay, and transwell assay were applicable to the determination of cell proliferation, cell viability, and invasion in each group, respectively. Reagent kits were used for testing glucose consumption and lactate production levels. HCC1937 cells with knockdown or overexpression of lncRNA GHET1 were injected into the nude mice, followed by the examination of resulting tumor volume and weight. The distribution and expression of Hippo/YAP signaling pathway-related proteins were probed using western blotting.ResultsHighly expressed lncRNA GHET1 in TNBC tissues and cells and induction of lncRNA GHET1 by hypoxia were proved. Knockdown of lncRNA GHET1 significantly reduced proliferation, viability, and invasion of TNBC cells, and decreased glucose consumption and lactate production levels under the hypoxia condition. Furthermore, lncRNA GHET1 knockdown decreased HIF-1α expression in hypoxia and significantly inhibited tumor development in vivo. Knockdown of lncRNA GHET1 increased the phosphorylation levels of LATS1 and Yes-associated protein (YAP) to retain YAP within the cytoplasm, while the overexpression of lncRNA GHET1 or hypoxia promoted nuclear translocation of YAP and TNBC development.ConclusionLncRNA GHET1 expression can be induced by hypoxia, which leads to excessive activation of the Hippo/YAP signaling pathway, thus promoting TNBC progression.

Aquaporin 1 knockdown inhibits triple-negative breast cancer cell proliferation and invasion in vitro and in vivo.

Aquaporin 1 (AQP1) contributes to the progression of several cancer types, but its potential involvement in triple-negative breast cancer (TNBC) is unclear. The aim of the present study was to examine the role of AQP1 in cell proliferation and invasion in TNBC. Reverse transcription-quantitative PCR analysis and western blotting were used to detect AQP1 expression in different cell lines. A short hairpin (sh)RNA targeting AQP1 was established and transfected into MDA-MB-231 breast cancer cells. To investigate the effects of AQP1 knockdown, breast cancer cell proliferation, migration and invasion were evaluated by Cell Counting Kit-8 and Transwell assays. Furthermore, the volume and weight of tumor xenografts in mice were measured to evaluate breast cancer growth ability. The results revealed that the levels of AQP1 were higher in the MDA-MB-231 cell line compared with those in other breast cancer cell lines (MCF-7 and SK-BR-3) and a normal mammary epithelial cell line (MCF-10A). The shRNA targeting AQP1 effectively downregulated AQP1 expression at the mRNA and protein levels, and markedly suppressed TNBC cell proliferation, migration and invasion in vitro, and tumor growth in vivo. These results suggested that AQP1 may serve as a potential therapeutic target in TNBC.

MiR-770-5p regulates EMT and invasion in TNBC cells by targeting DNMT3A

MicroRNAs (miRNAs) are shown to regulate various processes in cancer like motility and invasion that are key features of the metastatic triple negative breast cancer (TNBCs). Epithelial-mesenchymal transition (EMT) is one of the well-defined cellular transitioning processes characterized with reduced E-cadherin expression and increased mesenchymal molecules such as Vimentin or Snail thereby gives the cells mobility and invasive character. Aberrant DNA methylation by DNA methyltransferases (DNMTs) plays an important role in carcinogenesis. It is well known that DNMTs are required for transcriptional silencing of tumor-associated genes. DNMT3A-induced promoter hypermethylation of E-cadherin has also been known to improve cancer metastasis. Our results indicated that miR-770-5p could downregulate Vimentin and Snail expression levels, while increasing or restoring the expression of E-Cadherin hence, leading to inhibition of EMT phenotypes along with motility and invasion. Specifically, we showed that overexpression of miR-770-5p restored the expression of E-Cadherin in MDA-MB-231 cells via directly targeting DNMT3A. We also observed the change in the spindled shapes showing the loss of mesenchymal characteristics and gain of epithelial phenotype in miR-770-5p overexpressing cells. When considered together, our results show that miR-770-5p could effectively inhibit invasion potential driven by EMT.

LncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis

Triple-negative breast cancer (TNBC) is a highly invasive subtype of breast cancer. This study investigated the molecular mechanism and influences of MIR503HG, miR-224-5p, and homeobox A9 (HOXA9) on TNBC cell growth and migration. Dual-luciferase reporter gene and RNA immunoprecipitation were performed to examine the regulation of MIR503HG, miR-224-5p, and HOXA9. Cell proliferation, apoptosis, migration, and invasion were evaluated by colony formation, flow cytometry, and Transwell assays. Finally, nude mice were employed to investigate the influence of MIR503HG on TNBC tumor growth. HOXA9 protein levels were detected by immunohistochemical staining. MIR503HG and HOXA9 expression were reduced in TNBC, while miR-224-5p was increased. Overexpression of MIR503HG or HOXA9 reduced the cell migration ability and proliferation and promoted apoptosis, and knockdown of MIR503HG or overexpression of miR-224-5p exhibited the opposite effects. Furthermore, MIR503HG promoted HOXA9 expression by inhibiting miR-224-5p. Overexpression of miR-224-5p reversed the effects of MIR503HG overexpression on TNBC cells, while overexpression of HOXA9 reversed the effect of MIR503HG knockdown. Additionally, an in vivo study proved that MIR503HG inhibited TNBC tumor growth via the miR-224-5p/HOXA9 axis. MIR503HG inhibited cell proliferation and promoted the apoptosis of TNBC cells via the miR-224-5p/HOXA9 axis, which may function as a novel target for the treatment of TNBC.

CTGF regulates cell proliferation, migration, and glucose metabolism through activation of FAK signaling in triple-negative breast cancer.

Connective tissue growth factor (CTGF), also known as CCN2, is a member of the CCN protein family of secreted proteins with roles in diverse biological processes. CTGF regulates biological functions such as cell proliferation, migration, adhesion, wound healing, and angiogenesis. In this study, we demonstrate a mechanistic link between CTGF and enhanced aerobic glycolysis in triple-negative breast cancer (TNBC). We found that CTGF is overexpressed in TNBC and high CTGF expression is correlated with a poor prognosis. Also, CTGF was required for in vivo tumorigenesis and in vitro proliferation, migration, invasion, and adhesion of TNBC cells. Our results indicate that extracellular CTGF binds directly to integrin αvβ3, activating the FAK/Src/NF-κB p65 signaling axis, which results in transcriptional upregulation of Glut3. Neutralization of CTGF decreased cell proliferation, migration, and invasion through downregulation of Glut3-mediated glycolytic phenotypes. Overall, our work suggests a novel function for CTGF as a modulator of cancer metabolism, indicating that CTGF is a potential therapeutic target in TNBC.

A novel anthraquinone‑quinazoline hybrid7B blocks breast cancer metastasis and EMT via targeting EGFR and Rac1.

At present, effective therapeutic drugs for triple‑negative breast cancer (TNBC) are lacking due to the absence of identified or available targets. Therefore, the present study aimed to identify key molecular targets and a specific targeted therapeutic drug to aid with the development of novel therapeutic strategies for TNBC. Based on the high expression of EGFR and Rac1 in TNBC and inspired by a novel antitumor strategy termed combi‑targeting, novel anthraquinone‑quinazoline hybrid7B was synthesized to simultaneously target EGFR and Rac1. It was hypothesized that hybrid7B may possess enhanced potency compared with its parent compounds. Breast cancer cell viability was detected by performing MTT assays. Flow cytometry was conducted to detect the effects of hybrid7B on the cell cycle, apoptosis and the mitochondrial outer membrane potential. Ultrastructural alterations were observed by transmission electron microscopy. Cell invasion and migration were assessed by performing Transwell and wound‑healing assays, respectively. The expression levels of epithelial‑mesenchymal transition (EMT) markers and metastasis‑related proteins were detected by western blotting. Compared with Rhein and gefitinib, hybrid7B displayed superior antiproliferative activity in MDA‑MB‑231 cells with an IC50 value of 2.31µM, which was 14‑fold higher compared with the EGFR tyrosine kinase inhibitor gefitinib. Further experiments demonstrated that hybrid7B significantly reduced the mitochondrial membrane potential, enhanced MDA‑MB‑231 cell apoptosis and induced cell cycle arrest at the G2/Mphase compared with the control group. Typical morphological alterations of apoptotic cells were observed in hybrid7B‑treated MDA‑MB‑231 and MCF‑7 cells. Compared with the control group, hybrid7B significantly inhibited MDA‑MB‑231 cell invasion and migration by downregulating Rac1, EGFR, matrix metalloproteinases, snail family transcriptional repressor1, Vimentin and β‑catenin protein expression levels, and upregulating E‑cadherin protein expression levels. The present study demonstrated that hybrid7B inhibited TNBC cell migration and invasion by reversing EMT and targeting EGFR and Rac1; therefore, hybrid7B may serve as a promising therapeutic agent for TNBC.

Abstract PD15-03: LGR4 engages a wnt-independent mechanism to enhance EGFR signaling and promote metastasis of triple-negative breast cancer

Abstract Leucine-rich repeat-containing G-protein-coupled receptors 4/5/6 (LGR4/5/6) have crucial functions in embryonic development, adult tissue homeostasis, and diseases. LGR4/5/6 also play important roles in cancer initiation and progression. LGR4 is highly expressed in multiple types of cancer and associated with poor patient outcome. LGR4 promotes tumorigenesis and metastasis and modulates cancer stem cells, while LGR5 and LGR6 mark cancer stem cells and progenitor cells that contribute to tumor initiation. The biological functions of LGR4/5/6 are primarily attributed to their roles in potentiating Wnt signaling, which requires the binding of their four R-spondin ligands (RSPO1-RSPO4). However, it is unclear whether these proteins have Wnt signaling-independent functions. We have previously reported that LGR4 is important in mammary gland development and breast cancer progression and metastasis. Here, we demonstrate that LGR4 enhances triple-negative breast cancer (TNBC) metastasis independently of Wnt signaling. We first found that LGR4 was highly expressed in the TNBC subtype and associated with poor metastasis-free survival in TNBC patients, while a well-validated Wnt signature failed to correlate with metastasis-free survival of this same subset of TNBC patients. These bioinformatic data suggested divergent functions of LGR4 versus Wnt signaling in TNBC progression. Then we showed that depletion of LGR4 inhibited, while inducible expression of LGR4 promoted TNBC cell migration and invasion, without added exogenous ligands and any detectable impact on baseline Wnt signaling. Importantly, blockade of Wnt signaling by Wnt inhibitors or by genetic depletion of Wntless cannot abolish the effect of LGR4 on TNBC cell metastatic ability. Furthermore, we have generated non-RSPO-binding LGR4 mutants that failed to activate Wnt signaling. These LGR4 mutants that are uncoupled from Wnt signaling can still promote TNBC cell migration and invasion in vitro and TNBC lung metastasis and bone metastasis in vivo, as potently as wild-type LGR4. To identify the molecular mechanisms by which LGR4 promotes TNBC metastasis, we did computational analyses of TCGA data to search for candidate proteins associated with LGR4 and proteomics screenings to uncover protein pathways regulated by LGR4 knockdown in TNBC cells. Both in silico and proteomic analyses identified EGFR as a downstream component of LGR4 signaling in TNBC. We confirmed LGR4 enhanced EGFR signaling by immunoblot analyses and found that EGFR was the crucial mediator of LGR4’s role in TNBC metastasis. Ectopic EGFR rescued migration and invasion of LGR4 knockout cells, while EGFR knockdown and the EGFR inhibitor erlotinib attenuated LGR4-induced cell migration and invasion. In an intra-iliac injection model of bone metastasis, erlotinib also suppressed LGR4-induced TNBC bone metastasis in vivo. Mechanistically, LGR4 did not affect EGFR mRNA levels but EGFR protein stability. LGR4 interacted with EGFR and blocked EGFR ubiquitination and degradation, resulting in persistent EGFR activation. Together, these data uncover a new signaling pathway controlled by LGRs with broad implications for cancer progression and targeted therapy. Citation Format: Fei Yue, Weiyu Jiang, Amy T Ku, Adelaide IJ Young, Weijie Zhang, Eric P Souto, Yankun Gao, Zhihan Yu, Yi Wang, Chad J Creighton, Chandandeep Nagi, Tao Wang, Xin-Hua Feng, Shixia Huang, Cristian Coarfa, Xiang H.-F. Zhang, Qingyun Liu, Xia Lin, Yi Li. LGR4 engages a wnt-independent mechanism to enhance EGFR signaling and promote metastasis of triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-03.

Overexpression of SERPINA3 promotes tumor invasion and migration, epithelial-mesenchymal-transition in triple-negative breast cancer cells

BackgroundRecent studies have indicated that serpin peptidase inhibitor, clade A, member 3 (SERPINA3) is a potential marker associated with tumor progression, which connoted that SERPINA3 is related to malignant phenotypes in cancer. However, the biological function of SERPINA3 in breast cancer (BC) remains unclear.MethodsBioinformatics data were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Immunohistochemical staining (IHC) was conducted to determine SERPINA3 expression. With strong aggressive abilities, triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, BT549 and MDA-MB-436) were obtained to examine SERPINA3 expression and functions. Wound healing and Transwell assays were performed to measure cell migration and invasion. Cell Counting Kit-8 (CCK-8) assay was conducted to detect cell proliferation abilities and cell viabilities.ResultsSERPINA3 was upregulated in BC tissues. Functional assays suggested that overexpression of SERPINA3 significantly promoted cell proliferation, where migration and invasion of TNBC cells were accelerated. Knockdown of SERPINA3 had the opposite effects. These results causing by overexpression of SERPINA3 were also confirmed in non-TNBC cell lines. Overexpression of SERPINA3 remarkably enhanced the epithelial–mesenchymal transition (EMT) by upregulating the EMT markers and EZH2. In addition, the overexpression of SERPINA3 reduced the sensitivity of TNBC cells to cisplatin.ConclusionSERPINA3 can regulate the migration, invasion and EMT of TNBC cells and increased expression of SERPINA3 confers resistance to cisplatin in TNBC cells. We discern it is required for the regulation of BC progression and is a critical target for the clinical treatment of BC.

Receptor-type protein tyrosine phosphatase alpha (PTPα) mediates MMP14 localization and facilitates triple-negative breast cancer cell invasion.

The ability of cancer cells to invade surrounding tissues requires degradation of the extracellular matrix (ECM). Invasive structures, such as invadopodia, form on the plasma membranes of cancer cells and secrete ECM-degrading proteases that play crucial roles in cancer cell invasion. We have previously shown that the protein tyrosine phosphatase alpha (PTPα) regulates focal adhesion formation and migration of normal cells. Here we report a novel role for PTPα in promoting triple-negative breast cancer cell invasion in vitro and in vivo. We show that PTPα knockdown reduces ECM degradation and cellular invasion of MDA-MB-231 cells through Matrigel. PTPα is not a component of TKS5-positive structures resembling invadopodia; rather, PTPα localizes with endosomal structures positive for MMP14, caveolin-1, and early endosome antigen 1. Furthermore, PTPα regulates MMP14 localization to plasma membrane protrusions, suggesting a role for PTPα in intracellular trafficking of MMP14. Importantly, we show that orthotopic MDA-MB-231 tumors depleted in PTPα exhibit reduced invasion into the surrounding mammary fat pad. These findings suggest a novel role for PTPα in regulating the invasion of triple-negative breast cancer cells.

UV radiation resistance-associated gene (UVRAG) promotes cell proliferation, migration, invasion by regulating cyclin-dependent kinases (CDK) and integrin-β/Src signaling in breast cancer cells.

Breast cancer is a highly heterogeneous group of human cancer with distinct genetic, biological and clinicopathological features. Triple-negative breast cancer (TNBC) is the most aggressive and metastatic type of breast cancer and associated with poor patient survival. However, the role of UV Radiation Resistance-Associated Gene (UVRAG) in TNBC remains unknown. Here, we report that UVRAG is highly upregulated in all TNBC cells and its knockdown leads to the inhibition of cell proliferation, colony formation and progression of cell cycle, which is associated with and reduced expression of cell cycle related protein expression, including Cyclin A2, B1, D1, cdc2 and cdk6 in TNBC cells. Inhibition of UVRAG also suppressed cell motility, migration and invasion of TNBC cells by inhibition of Integrin β1 and β3 and Src activity. Our findings suggest for the first time that UVRAG expression contributes to proliferation, cell cycle progression, motility/migration and invasion of TNBC cells. Thus, targeting UVRAG could be a potential strategy in breast cancer especially against TNBC.

NcRNA therapy with miRNA-22-3p suppresses the growth of triple-negative breast cancer

Deregulation of noncoding RNAs, including microRNAs (miRs), is implicated in the pathogenesis of many human cancers, including breast cancer. Through extensive analysis of The Cancer Genome Atlas, we found that expression of miR-22-3p is markedly lower in triple-negative breast cancer (TNBC) than in normal breast tissue. The restoration of miR-22-3p expression led to significant inhibition of TNBC cell proliferation, colony formation, migration, and invasion. We demonstrated that miR-22-3p reduces eukaryotic elongation factor 2 kinase (eEF2K) expression by directly binding to the 3′ untranslated region of eEF2K mRNA. Inhibition of EF2K expression recapitulated the effects of miR-22-3p on TNBC cell proliferation, motility, invasion, and suppression of phosphatidylinositol 3-kinase/Akt and Src signaling. Systemic administration of miR-22-3p in single-lipid nanoparticles significantly suppressed tumor growth in orthotopic MDA-MB-231 and MDA-MB-436 TNBC models. Evaluation of the tumor response, following miR-22-3p therapy in these models using a novel mathematical model factoring in various in vivo parameters, demonstrated that the therapy is highly effective against TNBC. These findings suggest that miR-22-3p functions as a tumor suppressor by targeting clinically significant oncogenic pathways and that miR-22-3p loss contributes to TNBC growth and progression. The restoration of miR-22-3p expression is a potential novel noncoding RNA-based therapy for TNBC.

Hsa_circ_0000199 facilitates chemo-tolerance of triple-negative breast cancer by interfering with miR-206/613-led PI3K/Akt/mTOR signaling.

Increasing attentions have been paid to the role of circRNAs in the etiology of triple-negative breast cancer (TNBC), and we strived to figure out the association of circRNA AKT3/miRNA axis with TNBC chemo-resistance. Altogether 207 BC patients were divided into TNBC group (n=83) and non-TNBC group (n=124), and MCF-10A, MDA-MB-231, MDA-MB-468, SK-BR-3 and MCF-7 cell lines were prepared in advance. Expressions of AKT3-derived circRNAs and relevant miRNAs in the TNBC tissues and cell lines were determined by employing real-time polymerase chain reaction (PCR). It was indicated that hsa_circ_0000199 expression was higher in TNBC tissues than in non-TNBC tissues, and high hsa_circ_0000199 expression was predictive of large tumor size, advanced TNM grade, high Ki-67 level and poor 3-year survival of TNBC patients (all P<0.05). Furthermore, miR-613 and miR-206 were sponged and negatively regulated by hsa_circ_0000199 (P<0.001), and PI3K/Akt/mTOR signaling was depressed by si-hsa_circ_0000199 in TNBC cell lines (P<0.01). Ultimately, miR-206/miR-613 inhibitor reversed impacts of si-hsa_circ_0000199 on PI3K/Akt/mTOR signaling, proliferation, migration, invasion, chemo-sensitivity and autophagy of TNBC cells (all P<0.01). Conclusively, silencing of hsa_circ_0000199 enhanced TNBC chemo-sensitivity by promoting miR-206/miR-613 expression and deactivating PI3K/Akt/mTOR signaling, which was conducive to improving chemotherapeutic efficacy of TNBC patients.

A novel humanized Frizzled-7-targeting antibody enhances antitumor effects of Bevacizumab against triple-negative breast cancer via blocking Wnt/\u03b2-catenin signaling pathway

BackgroundAnti-angiogenic therapy has been widely applied to the clinical treatment of malignant tumors. However, the efficacy of such treatments has been called into question, especially in triple-negative breast cancer (TNBC). Bevacizumab, the first anti-angiogenic agent approved by FDA, actually increases invasive and metastatic properties of TNBC cells, resulting from the activation of Wnt/β-catenin signaling in response to hypoxia. As a critical receptor of Wnt/β-catenin signaling, Frizzled-7 (Fzd7) is aberrantly expressed in TNBC, indicating Fzd7 a potential target for developing drugs to be combined with anti-angiogenic agents.MethodsHybridoma technique and antibody humanization technique were utilized to generate a Fzd7-targeting antibody (SHH002-hu1). Biolayer interferometry (BLI) assay and near infrared (NIR) imaging were conducted to detect the affinity and targeting ability of SHH002-hu1. Next, whether SHH002-hu1 could suppress the invasion and migration of TNBC cells induced by Bevacizumab were validated, and the underlying molecular mechanisms were elucidated by luciferase reporter and western blot assays. The nude-mice transplanted TNBC models were established to assess the anti-TNBC activities of SHH002-hu1 when combined with Bevacizumab. Then, the effects on putative TNBC stem-like cells and Wnt/β-catenin signaling were evaluated by immunofluorescence (IF). Further, the tumor-initiating and self-renew capacity of TNBC cells were studied by secondary nude mouse xenograft model and sphere formation assay. In addition, the effects of SHH002-hu1 on the adaptation of TNBC cells to hypoxia were evaluated by the detection of vasculogenic mimicry (VM) and hypoxia-inducible factor-1α (HIF-1α) transcriptional activity.ResultsThe novel humanized antibody targeting Fzd7 (SHH002-hu1) exhibited extremely high affinity with Fzd7, and specifically targeted to Fzd7+ cells and tumor tissues. SHH002-hu1 repressed invasion, migration and epithelial-mesenchymal cell transformation (EMT) of TNBC cells induced by Bevacizumab through abating Wnt/β-catenin signaling. SHH002-hu1 significantly enhanced the capacity of Bevacizumab to inhibit the growth of TNBC via reducing the subpopulation of putative TNBC stem-like cells, further attenuating Bevacizumab-enhanced tumor-initiating and self-renew capacity of TNBC cells. Moreover, SHH002-hu1 effectively restrained the adaptation of TNBC cells to hypoxia via disrupting Wnt/β-catenin signaling.ConclusionSHH002-hu1 significantly enhances the anti-TNBC capacity of Bevacizumab, and shows the potential of preventing TNBC recurrence, suggesting SHH002-hu1 a good candidate for the synergistic therapy together with Bevacizumab.

Overexpression of GLUT3 promotes metastasis of triple-negative breast cancer by modulating the inflammatory tumor microenvironment.

Triple-negative breast cancer (TNBC) exhibits a higher level of glycolytic capacity and are commonly associated with an inflammatory microenvironment, but the regulatory mechanism and metabolic crosstalk between the tumor and tumor microenvironment (TME) are largely unresolved. Here, we show that glucose transporter 3 (GLUT3) is particularly elevated in TNBC and associated with metastatic progression and poor prognosis in breast cancer patients. Expression of GLUT3 is crucial for promoting the epithelial-to-mesenchymal transition and enhancing invasiveness and distant metastasis of TNBC cells. Notably, GLUT3 is correlated with inflammatory gene expressions and is associated with M1 tumor-associated macrophages (TAMs), at least in part by C-X-C Motif Chemokine Ligand 8 (CXCL8). We found that expression of GLUT3 regulates CXCL8 production in TNBC cells. Secretion of CXCL8 participates in GLUT3-overexpressing TNBC cells-elicited activation of inflammatory TAMs, which further enhances GLUT3 expression and mobility of TNBC cells. Our findings demonstrate that aerobic glycolysis in TNBC not only promotes aggressiveness of tumor cells but also initiates a positive regulatory loop for enhancing tumor progression by modulating the inflammatory TME.

Regulatory Effect of miR497-5p-CCNE1 Axis in Triple-Negative Breast Cancer Cells and Its Predictive Value for Early Diagnosis.

ObjectiveTo explore the regulatory role of miR497-5p–CCNE1 axis in triple-negative breast cancer (TNBC) cells and its predictive value for early diagnosis.MethodsCancer tissue and adjacent tissue samples were collected from 86 patients with TNBC.RT-PCR was used to detect the expression of miR497-5p and CCNE1 (target gene) mRNA, determined by biological prediction in tissue and TNBC cells. ROC was used to analyze the diagnostic value of miR497-5p in TNBC. MTT, invasion, and flow cytometry were used to detect the proliferation, invasion, cycle, apoptosis rate, and expression of related proteins of TNBC cells with overexpression of miR497-5p or knockdown of CCNE1.ResultsRT-qPCR results showed that miR497-5p levels were significantly downregulated in TNBC tissue and cells, while CCNE1 expression was significantly upregulated, and miR497-5p expression was negatively correlated with that of CCNE1 (P<0.001). ROC analysis showed that the AUC of miR497-5p for TNBC was >0.9, which had better diagnostic value. The cell tests revealed that miR497-5p played a role in tumor inhibition, including inhibiting proliferation and invasion of TNBC cells, blocking the cell cycle, and promoting apoptosis. Bioinformatic prediction and subsequent experiments revealed that CCNE1 was the direct target of miR497-5p. Furthermore, after knocking down the expression of CCNE1 in TNBC cells, the proliferation and invasion of TNBC cells were significantly inhibited, the cell cycle blocked, and the apoptosis rate significantly increased (P<0.001), and expression of the proapoptosis-related proteins Bax and caspase 3 (cleaved) were upregulated, while expression of the antiapoptosis-related protein BCL2 was downregulated (P<0.001).ConclusionmiR497-5p inhibited the proliferation and invasion of TNBC cells by targeting CCNE1, blocked the cell cycle and promoted the apoptosis of TNBC cells, and had better diagnostic value for TNBC. miR497-5p can be used as a new potential target for the treatment of TNBC.

Hypoxia-associated circular RNA RPPH1 modulates triple-negative breast cancer cell growth via the miR-1296-5p/TRIM14 axis

Hypoxia affects the advancement, metastasis, and metabolism of breast cancer (BC). The circular RNA ribonuclease P RNA component H1 (circRPPH1) (has_circ_0000515) is implicated in tumor progression. Nevertheless, the regulatory mechanism related to circRPPH1 in hypoxia-mediated triple-negative breast cancer (TNBC) progression is indistinct. The expression levels of circRPPH1, miR-1296-5p, tripartite motif-containing 14 (TRIM14) mRNA in tissue samples and cells were examined through quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability, migration, and invasion were determined with Cell Counting Kit-8 (CCK-8) or transwell assays. The levels of glucose consumption and lactate production were assessed via the Glucose Assay Kit or Lactate Assay Kit. The protein levels of TRIM14, Glucose Transporter GLUT1 (GLUT1), and lactic dehydrogenase A (LDHA) were detected by western blot analysis. The targeting relationship between circRPPH1 or TRIM14 and miR-1296-5p was verified with dual-luciferase reporter assay. The role of circRPPH1 was confirmed via xenograft assay. We verified that circRPPH1 and TRIM14 expression were increased while miR-1296-5p expression was decreased in BC tissues and hypoxia-cultured TNBC cells. Functionally, circRPPH1 silencing reversed the promoting effect of hypoxia on viability, migration, invasion, and glycolysis of TNBC cells. CircRPPH1 knockdown repressed decreased TNBC cell growth in vivo. Mechanistically, circRPPH1 sponged miR-1296-5p to modulate TRIM14 expression. Also, miR-1296- 5p silencing restored circRPPH1 inhibition-mediated influence on the viability, migration, invasion, and glycolysis of hypoxia-treated TNBC cells. TRIM14 elevation overturned the inhibitory impact of miR-1296-5p mimic on viability, migration, invasion, and glycolysis of hypoxia-cultured TNBC cells. In conclusion, hypoxia-induced circRPPH1 fostered TNBC progression through regulation of the miR-1296-5p/TRIM14 axis, indicating that circRPPH1 was a promising target for TNBC treatment.

Proteomic Landscape of Exosomes Reveals the Functional Contributions of CD151 in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Patients with TNBC have poor overall survival because of limited molecular therapeutic targets. Recently, exosomes have been recognized as key mediators in cancer progression, but the molecular components and function of TNBC-derived exosomes remain unknown. The main goal of this study was to reveal the proteomic landscape of serum exosomes derived from ten patients with TNBC and 17 healthy donors to identify potential therapeutic targets. Using a tandem mass tag–based quantitative proteomics approach, we characterized the proteomes of individual patient-derived serum exosomes, identified exosomal protein signatures specific to patients with TNBC, and filtered out differentially expressed proteins. Most importantly, we found that the tetraspanin CD151 expression levels in TNBC-derived serum exosomes were significantly higher than those exosomes from healthy subjects, and we validated our findings with samples from 16 additional donors. Furthermore, utilizing quantitative proteomics approach to reveal the proteomes of CD151-deleted exosomes and cells, we found that exosomal CD151 facilitated secretion of ribosomal proteins via exosomes while inhibiting exosome secretion of complement proteins. Moreover, we proved that CD151-deleted exosomes significantly decreased the migration and invasion of TNBC cells. This is the first comparative study of the proteomes of TNBC patient–derived and CD151-deleted exosomes. Our findings indicate that profiling of TNBC-derived exosomal proteins is a useful tool to extend our understanding of TNBC, and exosomal CD151 may be a potential therapeutic target for TNBC.

Long Noncoding-RNA Component of Mitochondrial RNA Processing Endoribonuclease Promotes Carcinogenesis in Triple-Negative Breast Cancer Cells via the Competing Endogenous RNA Mechanism.

PurposeTriple-negative breast cancer (TNBC) is a subtype of breast cancer. Increasing evidence supports that dysregulation of long noncoding RNAs (lncRNAs) plays a vital role in cancer progression. RNA component of mitochondrial RNA processing endoribonuclease (RMRP), a lncRNA, is characterized as a tumor-propeller in some cancers, but its mechanism in TNBC remains poorly understood. This study aimed to determine whether and how RMRP functions in TNBC.MethodsCell proliferation was determined by cell counting kit-8 (CCK-8) and colony formation assays and cell apoptosis by flow cytometry analysis and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Cell migration and invasion were determined by transwell assays. RNA-binding protein immunoprecipitation (RIP), luciferase reporter, and RNA pulldown assays were implemented to assess the interaction of RMRP with other molecules in TNBC cells.ResultsRMRP expression was elevated in TNBC cells. RMRP knockdown repressed cell proliferation, migration, and invasion, but induced apoptosis in TNBC. In addition, RMRP was found to target microRNA-766-5p (miR-766-5p) in TNBC cells. Silencing miR-766-5p enhanced cell viability and decreased apoptosis, whereas miR-766-5p overexpression had opposite effects. Furthermore, miR-766-5p was found to bind to yes-associated protein 1 (YAP1). Moreover, miR-766-5p inhibition reversed the repressive effect of RMRP knockdown on the malignant progression of TNBC.ConclusionThe present study manifested that RMRP promotes the growth, migration, and invasion of TNBC cells via the miR-766-5p/YAP1 axis. These findings provide novel perspectives for TNBC treatment.