Metastasis Of Breast Cancer Cells Research Articles

Decreased LDHB expression in breast tumor cells causes NK cell activation and promotes tumor progression.

Abnormal metabolism is the underlying reason for breast cancer progression. Decreased lactate dehydrogenase B (LDHB) has been detected in breast cancer but the function of LDHB remains unknown. Western blot was used to analyze LDHB expression in breast cancer cells. The impact of LDHB on tumor cell migration and invasion was determined using Transwell assays, wound healing assays, and a mouse lung metastasis model. Subcutaneous tumor formation, a natural killer (NK) cell cytotoxicity assay, and flow cytometry evaluated NK cell activation. Immunofluorescence and quantitative real-time PCR detected NK cell activation markers. Kaplan-Meier analysis evaluated the effect of immune cell infiltration on prognosis. Single-sample gene set enrichment analysis determined NK cell activation scores. A support vector machine predicted the role of LDHB in NK cell activation. In this study we showed that LDHB inhibits the breast cancer cell metastasis and orchestrates metabolic reprogramming within tumor cells. Our results revealed that LDHB-mediated lactic acid clearance in breast cancer cells triggers NK cell activation within the tumor microenvironment. Our findings, which were confirmed in a murine model, demonstrated that LDHB in tumor cells promotes NK cell activation and ultimately results in the eradication of malignant cells. Clinically, our study further validated that LDHB affects immune cell infiltration and function. Specifically, its expression has been linked to enhanced NK cell-mediated cytotoxicity and improved patient survival. Furthermore, we identified LDHB expression in tumors as an important predictor of NK cell activation, with strong predictive ability in some cancers. Our results suggest that LDHB is a promising target for activating the tumor immune microenvironment in breast cancer, where LDHB-associated lactic acid clearance leads to increased NK cell activity. This study highlights the critical role of LDHB in regulating immune responses and its potential as a therapeutic target for breast cancer.

Abstract 4122: Integrin avb3 and HSPG targeting agents prevent extracellular vesicles uptake and breast cancer metastasis

Abstract INTRODUCTION: Metastasis is the major cause of cancer-related deaths (90%). Intercellular communication between cancer cells and with cells in their environment plays a significant function in tumor progression. In fact, secreted factors like cytokines and extracellular vesicles (EVs) can either halt or stimulate tumorigenesis as well as metastasis. We have previously reported, that the integrin B3 (ITGB3) is required for metastasis of breast cancer cells in vivo (Sese et al. Oncotarget, 2017). We furthermore demonstrated, that EVs mediated colony growth is dependent on ITGB3 and that endocytosis mediated uptake of EVs relies on the interaction of ITGB3 with integrin av (ITGav) and heparan sulphated glycoproteins (HSPGs) (Fuentes et al. Nat. Commun., 2021). OBJECTIVE: The objective of this work is focused on identifying inhibitors targeting the ITGav/B3/HSPGs complex to prevent extracellular vesicle uptake in cancer cells and to test their ability to prevent metastasis. METHODOLOGY. Inhibitors targeting ITGB3/ITGAV or HSPGs were either commercially available or obtained through collaborations and single chain antibodies (scFv) were produced in house. Inhibition of fluorescent labeled EVs was determined by FACS analysis or Incucyte based measurements and the efficacy of inhibitors was determined in a dose dependent manner. Toxicity of inhibitors was studied by crystal violet-based assay in normal and tumor cell lines. Tail vein injection of luciferase labeled MDA-MB-231 cells into nude mice was used as model to study metastatic diseases under treatment with the identified inhibitors. Metastasis formation was monitored by bioluminescence imaging and lungs were examined pathologically at end point. Overall survival was determined. RESULTS: Among the tested Integrin and HSPGs targeting compounds, we identified an integrin-binding antibody, an integrin binding scFv, an RGD-mimetic compound and an HSPGs-binding peptide as inhibitors of EV uptake. Inhibition was validated in a dose dependent manner and toxicity studies did not show adverse effects. Three of those inhibitors, targeting either ITGB3/ITGAV or HSPGs were tested in mice models in vivo and all inhibitors could significantly reduce the formation of metastasis and strongly increase the overall survival. CONCLUSION: We have identified four drug candidates that target the ITGav/B3/HSPGs complex and are able to prevent EV uptake, demonstrating that EV uptake is a druggable process. Even more, the inhibitors tested in vivo could strongly reduce metastasis formation and increase overall survival. This data supports our hypothesis, that targeting EV mediated intercellular communication is a valuable approach to combat cancer metastasis. Citation Format: Elena Muro-Blanc, Rocío Bayona-Ramón y Cajal, Marta Cano-Galietero, Sara García-Ortega, Chiara Falciani, Luisa Bracci, Alberto J. Schuhmacher, Stefan Hümmer, Santiago Ramón y Cajal. Integrin avb3 and HSPG targeting agents prevent extracellular vesicles uptake and breast cancer metastasis [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 4122.

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) 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 1392.

Abstract 4291: Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models

Abstract The adhesion G-protein coupled- receptor 1 ADGRB1/Bai1 was identified as a phosphatidylserine receptor that cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. This function of Bai1 was also shown to promote myoblasts fusion. Cell-cell fusion has been demonstrated to occur in normal physiological functions as well as in diseases. Studies have shown in cancer that cell-cell fusion increases tumor heterogeneity, drug resistance, and metastasis as hybrids acquire new properties that allow them to migrate and proliferate at new sites. Activation of ELMO-Dock signaling pathway has been associated with breast cancer metastasis. Moreover, ELMO1 has been identified as a modifier of breast cancer risk for BRCA mutation carriers. Rac1 was also shown to be overexpressed in proliferative breast disease, preinvasive and invasive breast carcinoma as well as lymph node metastases. Our recent studies showed that breast cancer cell fusion with bone marrow-derived mesenchymal stem cells was modulated by hypoxic conditions and promoted by apoptosis. Our hypothesis is that hypoxia stress induced apoptosis activates the phosphatidylserine receptor Bai1, and it signals through the ELMO/DOCK180/Rac to promote breast cancer cell fusion-driven tumor heterogeneity and metastasis. In this study, we determined the ability of Bai1 depleted breast cancer cells to induce tumor growth in immunocompromised mice. We used Bai1 sgRNA to inhibit Bai1 expression in the breast cancer cell MDA-MB-231. These cells (1x 106) together with bone marrow-derived mesenchymal stem cells (MSCs) (1x 106) were injected into the fat pad of the breast of 15 NOD SCID female mice. Control mice (15) received the same cells with no inhibition of Bai1. The mice were monitored for eight weeks. We found that Bai1 inhibition significantly reduced tumor growth in these mice in the course of time (P<0.05). The average tumor weight was also significantly reduced when Bai1 was inhibited (P<0.001). RNA sequencing of tumors generated from these mice revealed a significant increase in the expression of genes driving ferroptosis and a reduction in critical oncogenes in the experiment group compared to the control group. These findings will be helpful in the development of new classes of drugs effective in breast cancer treatment. Grant Support The NIH/NIMHD grant 5U54MD015929-03, the Society for Investigative Dermatology (SID) Freinkel Diversity Fellowship, and NSF Award Notice for Award ID 2142465 (awarded to FNK). Citation Format: Felicite Kamdem Noubissi, Oluwatowin Odubanjo, Hung-Chung Huang, Brenda Ogle, Paul Tchounwou. Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models [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 4291.

LSD1 modulates the bone metastasis of breast cancer cells through hnRNPA2B1-mediated sorting of exosomal miRNAs

Bone metastasis is a key contributor to morbidity and mortality of breast cancer patients. We have previously shown that exosomal miRNAs derived from LSD1 knockdown (KD) breast cancer cells inhibit osteoblast differentiation and promote osteoclast differentiation. However, how LSD1 regulates exosomal miRNAs and whether miRNAs promote bone metastasis through the formation of pre-metastatic niches remains unclear. In vivo experiments demonstrates that exosomes derived from LSD1 KD breast cancer cells significantly promoted bone metastasis. To explore the mechanism underlying the effect of LSD1 on exosomes in breast cancer cells, exosomal and cellular miRNAs from control, LSD1 KD, and rescue cells were sequenced. Interestingly, approximately 80% of LSD1-associated miRNAs were downregulated in exosomes from LSD1 KD cells. The consensus sequence UAGGGC, was identified in many miRNAs downregulated in LSD1 KD exosomes. We found that hnRNPA2B1 regulated the exosomal sorting of miR-6881-3p and some other miRNAs. LSD1 deficiency reduced hnRNPA2B1 expression in breast cancer cells by decreasing the level of H3K9me2 demethylation in the promoter region of the hnRNPA2B1 gene. Our study revealed that LSD1 plays a crucial role in the regulation of exosomal sorting of miRNA.

The deubiquitinase BRCC3 increases the stability of ZEB1 and promotes the proliferation and metastasis of triple-negative breast cancer cells.

Triple negative breast cancer (TNBC) has a high recurrence rate, metastasis rate and mortality rate. The aim of this study is to identify new targets for the treatment of TNBC. Clinical samples are used for screening deubiquitinating enzymes (DUBs). MDA-MB-231 cells and a TNBC mouse model are used for in vitro and in vivo experiments, respectively. Western blot analysis is used to detect the protein expressions of DUBs, zinc finger E-box binding homeobox 1 (ZEB1), and epithelial-mesenchymal transition (EMT)-related markers. Colony formation and transwell assays are used to detect the proliferation, migration and invasion of TNBC cells. Wound healing assay is used to detect the mobility of TNBC cells. Immunoprecipitation assay is used to detect the interaction between breast cancer susceptibility gene 1/2-containing complex subunit 3 (BRCC3) and ZEB1. ZEB1 ubiquitination levels, protein stability, and protein degradation are also examined. Pathological changes in the lung tissues are detected via HE staining. Our results show a significant positive correlation between the expressions of BRCC3 and ZEB1 in clinical TNBC tissues. Interference with BRCC3 inhibits TNBC cell proliferation, migration, invasion and EMT. BRCC3 interacts with ZEB1 and interferes with BRCC3 to inhibit ZEB1 expression by increasing ZEB1 ubiquitination. Interference with BRCC3 inhibits TNBC cell tumorigenesis and lung metastasis in vivo. In all, this study demonstrates that BRCC3 can increase the stability of ZEB1, upregulate ZEB1 expression, and promote the proliferation, migration, invasion, EMT, and metastasis of TNBC cells, providing a new direction for cancer therapy.

Curcumin suppresses metastasis of triple-negative breast cancer cells by modulating EMT signaling pathways: An integrated study of bioinformatics analysis.

This study aimed to use bioinformatics approaches for predicting the anticancer mechanisms of curcumin on triple-negative breast cancer (TNBC) and to verify these predictions through in vitro experiments. Initially, the Cell Counting Kit-8 (CCK8) assay was employed to rigorously investigate the influence of curcumin on the proliferative capacity of TNBC cells. Subsequently, flow cytometry was employed to meticulously assess the impact of curcumin on cellular apoptosis and the cell cycle regulation. Transwell assays were employed to meticulously evaluate the effect of curcumin on the motility of TNBC cells. RNA sequencing was conducted, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of differentially expressed genes, aiming to elucidate the potential anticancer mechanisms underlying curcumin's effects. To thoroughly elucidate the interactions among multiple proteins, we constructed a protein-protein interaction (PPI) network. Finally, the expression levels of several key proteins, including fibronectin, mTOR, β-Catenin, p-Akt, Akt, N-Cadherin, p-S6, and S6, were assessed using the western blot. The CCK8 assay results showed that curcumin significantly inhibited the proliferation of Hs578T and MDA-MB-231 cells. Flow cytometry results showed that curcumin induced apoptosis in these cells and arrested the cell cycle at the G2/M phase. Additionally, Transwell assay results showed that curcumin effectively reduced the motility of Hs578T and MDA-MB-231 cells. Enrichment analysis of RNA sequencing data showed that the mechanism of action of curcumin was significantly associated with signaling pathways such as pathways in cancer, focal adhesion, and PI3K-Akt signaling pathways. Subsequently, we constructed a protein-protein interaction network to elucidate the interactions among multiple proteins. Finally, Western blotting analysis showed that curcumin significantly decreased the expression levels of key proteins including Fibronectin, mTOR, β-Catenin, p-Akt, Akt, N-Cadherin, p-S6, and S6. Curcumin exhibits its therapeutic potential in TNBC by modulating multiple signaling pathways. It may inhibit the epithelial-mesenchymal transition process by downregulating the expression of proteins involved in the mTOR and PI3K-Akt signaling pathways, thereby suppressing the motility of TNBC cells. These findings provide experimental evidence for considering curcumin as a potential therapeutic strategy in the treatment of TNBC.

Association Between Temporomandibular Joint Disorders and Metastatic Breast Cancer—A Mapping Review

Aim: The authors of the present study speculate that metastasis of breast cancer cells to the temporomandibular joint (TMJ) region mimics classical signs and symptoms of temporomandibular disorders (TMD). The aim of this mapping review was to investigate the association between metastatic breast cancer (MBC) and TMD. Materials and Methods: The focused question was, “Is there an association between MBC and TMD?” Indexed databases (PubMed/Medline, EMBASE, OVID, Scopus, Google Scholar, and Web of Science) were electronically searched up to and including March 2022. These databases were searched using different combinations of the following keywords: cancer, carcinoma, temporomandibular disorders, temporomandibular joint, disc displacement disorder, nociception, and tumor. Original clinical studies that meet the inclusion criteria were included. The risk of bias (RoB) was assessed using the Joanna Briggs Institute Critical Appraisal Checklist for case reports. The structure of this current evidence-based review was tailored to primarily condense the relevant information. Results: Fifteen case reports were selected after screening the retrieved records from the electronic search. Fifteen out of 16 patients were women aged between 32 and 78 years. All patients were diagnosed with MBC, and most presented with adenocarcinoma. The symptoms identified in patients with TMD included: (a) preauricular pain; (b) preauricular swelling; (c) limited mouth opening; and (d) malocclusion. Fourteen of the included studies had a low RoB, while one had a moderate RoB. Conclusion: Based on the currently available evidence, a direct association between TMD and MBC or vice versa cannot be established. Routine visits to medical and oro-dental healthcare providers can contribute toward early diagnosis and management of TMD and MBC.

Expression and clinical significance of hypoxia-induced long non-coding RNA TCONS_I2_00001955 in breast cancer.

Long non-coding RNAs (lncRNAs) have been found to play important roles in occurrence, development, and metastasis of various tumors. We aimed to screen long non-coding RNAs (lncRNAs) that promote invasion and metastasis of breast cancer cells under hypoxia, and investigate the relationship between lncRNA expression and clinicopathological features and prognosis in invasive breast cancer. LncRNA microarray was used to screen the differentially expressed lncRNAs in MCF7, MDA-MB-231, and SKBR3 breast cancer cell lines cultured under normoxia and hypoxia, respectively. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to verify the microarray results. CCK8 and Transwell experiments were performed to identify the lncRNA that promote proliferation, migration, and invasion of breast cancer cells. Expression of the lncRNA and HIF-1α in invasive breast cancer was detected by RNAscope and immunohistochemistry, respectively. Correlation between the lncRNA expression and baseline characteristics was analyzed. Prognostic value of the lncRNA was evaluated using univariate and multivariate Cox regression. Expression of lncRNA TCONS_I2_00001955 in all the three breast cancer cells was increased under hypoxia. Overexpression of TCONS_I2_00001955 significantly enhanced proliferation, migration, and invasion of SKBR3 cells. Positive expression of TCONS_I2_00001955 was associated with recurrence, metastasis, and high expression of HIF-1α (P < 0.05), and it was an independent risk factor for poor disease-free survival of breast cancer. Hypoxia-induced lncRNA TCONS_I2_00001955 was associated with aggressive feature and poor prognosis of breast cancer.

MiR-622 Increases miR-30a Expression through Inhibition of Hypoxia-Inducible Factor 1α to Improve Metastasis and Chemoresistance in Human Invasive Breast Cancer Cells

Hypoxia-inducible factor 1α (HIF-1α) plays a pivotal role in the survival, metastasis, and response to treatment of solid tumors. Autophagy serves as a mechanism for tumor cells to eliminate misfolded proteins and damaged organelles, thus promoting invasiveness, metastasis, and resistance to treatment under hypoxic conditions. MicroRNA (miRNA) research underscores the significance of these non-coding molecules in regulating cancer-related protein synthesis across diverse contexts. However, there is limited reporting on miRNA-mediated gene expression studies, especially with respect to epithelial–mesenchymal transition (EMT) and autophagy in the context of hypoxic breast cancer. Our study reveals decreased levels of miRNA-622 (miR-622) and miRNA-30a (miR-30a) in invasive breast cancer cells compared to their non-invasive counterparts. Inducing miR-622 suppresses HIF-1α protein expression, subsequently activating miR-30a transcription. This cascade results in reduced invasiveness and migration of breast cancer cells by inhibiting EMT markers, such as Snail, Slug, and vimentin. Furthermore, miR-30a negatively regulates beclin 1, ATG5, and LC3-II and inhibits Akt protein phosphorylation. Consequently, this improves the sensitivity of invasive MDA-MB-231 cells to docetaxel treatment. In conclusion, our study highlights the therapeutic potential of inducing miR-622 to promote miR-30a expression and thus disrupt HIF-1α-associated EMT and autophagy pathways. This innovative strategy presents a promising approach to the treatment of aggressive breast cancer.

PTX3 promotes breast cancer cell proliferation and metastasis by regulating PKCζbreast cancer, pentraxin 3, protein kinase Cζ, proliferation, metastasis.

Breast cancer (BC) is the most commonly diagnosed cancer in women, providing a leading cause of death from malignancy. Pentraxin 3 (PTX3) and protein kinase C ζ (PKCζ) are both known to exert important roles in the progression of multiple types of tumors, including BC. The present study aimed to explore both their interaction and their role in promoting the proliferation and metastasis of BC. The expression level of PTX3 was found to be elevated both in patients with BC and in BC cells; furthermore, it was found to be associated with lymph node metastasis in patients with BC. Knockdown of PTX3 decreased the rate of cell proliferation and the effects of a series of metastasis-associated cellular processes, including cell chemotaxis, migration, adhesion and invasion, as well as diminishing actin polymerization of the MDA-MB-231 and MCF7 BC cells, and decreasing tumor pulmonary metastasis in vivo. Mechanistically, PTX3 and PKCζ were found to be colocalized intracellularly, and they were co-translocated to the cell membrane upon stimulation with epidermal growth factor. Following the knockdown of PTX3, both the phosphorylation and membrane translocation of PKCζ were significantly impaired, suggesting that PTX3 regulates the activation of PKCζ. Taken together, the findings of the present study have shown that PTX3 may promote the proliferation and metastasis of BC cells through regulating PKCζ activation to enhance cell migration, cell chemotaxis, cell invasion and cell adhesion.

Propranolol inhibits EMT and metastasis in breast cancer through miR-499-5p-mediated Sox6

PurposeThis study will focus on 4T1 cells, a murine mammary adenocarcinoma cell line, as the primary research subject. We aim to investigate the inhibitory effects and mechanisms of propranolol on epithelial–mesenchymal transition (EMT) in breast cancer cells, aiming to elucidate this phenomenon at the miRNA level.MethodsIn this study, the EMT inhibitory effect of propranolol was observed through in vitro and animal experiments. For the screening of potential target miRNAs and downstream target genes, second-generation sequencing (SGS) and bioinformatics analysis were conducted. Following the screening process, the identified target miRNAs and their respective target genes were confirmed using various experimental methods. To confirm the target miRNAs and target genes, Western Blot (WB), reverse transcription polymerase chain reaction (RT-PCR), and immunofluorescence experiments were performed.ResultsIn this study, we found that propranolol significantly reduced lung metastasis in 4T1 murine breast cancer cells (p < 0.05). In vitro and in vivo experiments demonstrated that propranolol inhibited the epithelial–mesenchymal transition (EMT) as evidenced by Western Blot analysis (p < 0.05). Through next-generation sequencing (SGS), subsequent bioinformatics analysis, and PCR validation, we identified a marked downregulation of miR-499-5p (p < 0.05), suggesting its potential involvement in mediating the suppressive effects of propranolol on EMT. Overexpression of miR-499-5p promoted EMT, migration, and invasion of 4T1 cells, and these effects were not reversed or attenuated by propranolol (Validated via Western Blot, wound healing assay, transwell migration, and invasion assays, p < 0.05). Sox6 was identified as a functional target of miR-499-5p, with its downregulation correlating with the observed EMT changes (p < 0.05). Silencing Sox6 or overexpressing miR-499-5p inhibited Sox6 expression, further promoting the processes of EMT, invasion, and migration in 4T1 cells. Notably, these effects were not alleviated by propranolol (validated via Western Blot, wound healing assay, transwell migration, and invasion assays, p < 0.05). The direct interaction between miR-499-5p and Sox6 mRNA was confirmed by dual-luciferase reporter gene assay.ConclusionThese results suggest that propranolol may have potential as a therapeutic agent for breast cancer treatment by targeting EMT and its regulatory mechanisms.

Tumor Cell-Derived Exosomal miR-191-5p Activates M2-Subtype Macrophages Through SOCS3 to Facilitate Breast Cancer.

Differential activation of macrophages is associated with poor progression of breast cancer (BC). Many reports have elucidated the important involvement of exosomes produced by cancer cells in remodeling the macrophage activation phenotype to promote tumor expansion and invasion. However, the underlying mechanisms by which exosomes secreted by BC cells facilitate macrophage M2 polarization remain enigmatic and worth exploring. In this study, quantitative real-time PCR (RT-qPCR) was used to investigate miR-191-5p expression in BC tumor tissues and cells. Cell counting kit 8 (CCK-8), transwell, and flow cytometry were applied to assess the functional role of miR-191-5p in BC. Isolated nano-vesicles were identified using transmission electron microscopy and western blotting. We also observed that miR-191-5p was significantly elevated in BC clinical samples and that inhibition of miR-191-5p hindered the growth and metastasis of BC cells. Importantly, BC cells successfully accelerated macrophage M2-like polarization by directly transferring exosomes to macrophages, resulting in increased miR-191-5p levels in macrophages. Mechanistically, exosomal miR-191-5p directly inhibited the suppressors of cytokine signaling 3 (SOCS3) expression in macrophages and aggravated macrophage M2 polarization. Similarly, si-SOCS3 transfected macrophages boosted BC cell migration and invasion in a positive feedback manner. Overall, our results manifested a pro-growth and pro-metastatic role between the two cells by elucidating the crucial role of exosomal miR-191-5p in stimulating M2 macrophage polarization and mediating communication between BC cells and macrophages. These findings opened up new horizons for the development of BC therapeutic strategies.

Breast cancer malignancy is governed by regulation of the macroH2A2/TM4SF1 axis, the AKT/NF-κB pathway, and elevated MMP13 expression.

The histone variant, macroH2A (mH2A) influences gene expression through epigenetic regulation. Tumor suppressive function of mH2A isoforms has been reported in various cancer types, but few studies have investigated the functional role of mH2A2 in breast cancer pathophysiology. This study aimed to determine the significance of mH2A2 in breast cancer development and progression by exploring its downstream regulatory mechanisms. Knockdown of mH2A2 facilitated the migration and invasion of breast cancer cells, whereas its overexpression exhibited the opposite effect. In vivo experiments revealed that augmenting mH2A2 expression reduced tumor growth and lung metastasis. Microarray analysis showed that TM4SF1 emerged as a likely target linked to mH2A2 owing to its significant suppression in breast cancer cell lines where mH2A2 was overexpressed among the genes that exhibited over twofold upregulation upon mH2A2 knockdown. Suppressing TM4SF1 reduced the migration, invasion, tumor growth, and metastasis of breast cancer cells in vitro and in vivo. TM4SF1 depletion reversed the increased aggressiveness triggered by mH2A2 knockdown, suggesting a close interplay between mH2A2 and TM4SF1. Our findings also highlight the role of the mH2A2/TM4SF1 axis in activating the AKT/NF-κB pathway. Consequently, activated NF-κB signaling leads to increased expression and secretion of MMP13, a potent promoter of metastasis. In summary, we propose that the orchestrated regulation of the mH2A2/TM4SF1 axis in conjunction with the AKT/NF-κB pathway and the subsequent elevation in MMP13 expression constitute pivotal factors governing the malignancy of breast cancer.

An antihypertensive drug-AT1 inhibitor attenuated BRCA development promoted by chronic psychological stress via Ang II/PARP1/FN1 pathway

Chronic psychological stress contributes to the occurrence of cancer and activates the renin-angiotensin system (RAS). However, the mechanisms by which RAS promotes the progression of breast cancer (BRCA) under chronic psychological stress are remain unknown. In this study, we observed elevated levels of Angiotensin II (Ang II) in both serum and BRCA tissue under chronic stress, leading to accelerated BRCA growth in a mouse model. An antihypertensive drug, candesartan (an AT1 inhibitor), effectively attenuated Ang II-induced cell proliferation and metastasis. Utilizing mass spectrometry and weighted gene co-expression network analysis (WGCNA), we identified fibronectin 1 (FN1) as the hub protein involved in chronic stress-Ang II/AT1 axis. Focal adhesion pathway was identified as a downstream signaling pathway activated during the progression of chronic stress. Depletion of FN1 significantly attenuated Ang II-induced proliferation and metastasis of BRCA cells. Poly (ADP-ribose) polymerase 1 (PARP1) was found to bind to the DNA promoter of FN1, leading to the transcription of FN1. Ang II upregulated PARP1 expression, resulting in increased FN1 levels. Recombinant FN1 partially restored the progress of BRCA malignancy induced by the Ang II/PARP1 pathway. In vivo, candesartan reversed the progressive effect of chronic psychological stress on BRCA. In clinical samples, Ang II levels in both serum and tumor tissues are higher in stressed patients compared to control patients. Serum Ang II levels were positively correlated with chronic stress indicators. In conclusion, our study demonstrated that chronic psychological stress accelerates the malignancy of BRCA, and the AT1 inhibitor candesartan counteracts these effects by suppressing the Ang II-AT1 axis and the downstream PARP1/FN1/focal adhesion pathway.

FKBP38 suppresses endometrial cancer cell proliferation and metastasis by inhibiting the mTOR pathway

Endometrial cancer (EC) is a common gynecological malignancy, and advanced-stage or recurrent EC is associated with a high mortality rate owing to the ineffectiveness of currently available treatments. FK506-binding protein 38 (FKBP38) is a member of the immunophilin family and inhibits melanoma and breast cancer cell metastasis. However, the functions of FKBP38 and its potential mechanism in EC remain unclear. Herein, we analyzed the expression levels of FKBP38 in EC cells and found that the FKBP38 expression was high in Ishikawa cells, and low in AN3CA cells, traditionally considered a low grade and a high grade cell line, respectively, in pathology classification. Moreover, FKBP38 inhibited cell proliferation, migration and invasion in EC cells, FKBP38 knockdown significantly promoted tumor growth of Ishikawa cells in a subcutaneous xenograft model and increased the number of lung metastases of Hec-1-A cells in a metastatic mouse model. Furthermore, FKBP38 suppressed several target proteins of epithelial-to-mesenchymal transition (EMT) and reduced the phosphorylation of ribosomal S6 protein (S6), eukaryotic initiation factor 4E-binding protein 1 (4EBP-1), indicating the potent inhibition of the mammalian target of rapamycin (mTOR) pathway. Meanwhile, the inhibition of mTOR neutralized the elevation of EC cell proliferation, migration and invasion after FKBP38 knockdown. In summary, FKBP38 would exert a tumor-suppressing role by modulating the mTOR pathway. Our results indicate that FKBP38 may be considered as a factor of EC metastasis and a new target for EC therapeutic intervention.

TC2N inhibits distant metastasis and stemness of breast cancer via blocking fatty acid synthesis

BackgroundTandem C2 domains, nuclear (TC2N) is a C2 domain-containing protein that belongs to the carboxyl-terminal type (C-type) tandem C2 protein family, and acts as an oncogenic driver in several cancers. Previously, we preliminarily reported that TC2N mediates the PI3K-Akt signaling pathway to inhibit tumor growth of breast cancer (BC) cells. Beyond that, its precise biological functions and detailed molecular mechanisms in BC development and progression are not fully understood.MethodsTumor tissues of 212 BC patients were subjected to tissue microarray and further assessed the associations of TC2N expression with pathological parameters and FASN expression. The protein levels of TC2N and FASN in cell lines and tumor specimens were monitored by qRT-PCR, WB, immunofluorescence and immunohistochemistry. In vitro cell assays, in vivo nude mice model was used to assess the effect of TC2N ectopic expression on tumor metastasis and stemness of breast cancer cells. The downstream signaling pathway or target molecule of TC2N was mined using a combination of transcriptomics, proteomics and lipidomics, and the underlying mechanism was explored by WB and co-IP assays.ResultsHere, we found that the expression of TC2N remarkedly silenced in metastatic and poorly differentiated tumors. Function-wide, TC2N strongly inhibits tumor metastasis and stem-like properties of BC via inhibition of fatty acid synthesis. Mechanism-wise, TC2N blocks neddylated PTEN-mediated FASN stabilization by a dual mechanism. The C2B domain is crucial for nuclear localization of TC2N, further consolidating the TRIM21-mediated ubiquitylation and degradation of FASN by competing with neddylated PTEN for binding to FASN in nucleus. On the other hand, cytoplasmic TC2N interacts with import proteins, thereby restraining nuclear import of PTEN to decrease neddylated PTEN level.ConclusionsAltogether, we demonstrate a previously unidentified role and mechanism of TC2N in regulation of lipid metabolism and PTEN neddylation, providing a potential therapeutic target for anti-cancer.

Sinensetin suppresses breast cancer cell progression via Wnt/β-catenin pathway inhibition.

Although there are many treatments for breast cancer, such as surgery, radiotherapy, chemotherapy, estrogen receptor antagonists, immune checkpoint inhibitors and so on. However, safer and more effective therapeutic drugs for breast cancer are needed. Sinensetin, a safer therapeutic drugs, come from citrus species and medicinal plants used in traditional medicine, while its role and underlying mechanism in breast cancer remain unclear. Our study aimed to investigate the role and mechanism of sinensetin in breast cancer. Cell Counting Kit-8 (CCK-8) was used to determine the safe concentration of sinensetin in MCF-10A, MCF7 and MDA-MB-231 cells; 120 μM sinensetin was used in subsequent experiments. Real time polymerase chain reaction (RT-PCR), Western blotting, Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay, Transwell invasion assay and Clone formation assay were used in this study to determine cell viability, mRNA expression, protein levels, apoptosis, proliferation, invasion and so on. Herein, our results showed that 120 μM sinensetin suppressed the cell viability and promoted apoptosis of MCF7 and MDA-MB-231 cells. Treatment with 120 µM sinensetin for 24 h showed no significant toxicity to normal mammary cells; 120 μM sinensetin decreased cell proliferation, invasion, and epithelial-mesenchymal transition (EMT), and downregulated β-catenin, lymphatic enhancing factor 1 (LEF1), T-cell factor (TCF) 1/TCF7, and TCF3/TCF7L1 expression in MCF7 and MDA-MB-231 cells. The Wnt agonist SKL2001 reversed the inhibitory effect of sinensetin on cell survival, metastasis, and EMT. Sinensetin-induced downregulation of β-catenin, LEF1, and TCF1/TCF7 expression were upregulated by SKL2001 in MCF7 and MDA-MB-231 cells. In summary, sinensetin suppressed the metastasis of breast cancer cell via inhibition of Wnt/β-catenin pathway and there were no adverse effects on normal breast cells. Our study confirmed the role of sinensetin in breast cancer cells and provided a better understanding of the underlying mechanism.

NOTCH3 inhibits transcription factor ZEB1 expression and metastasis of breast cancer cells via transcriptionally upregulating miR-223.

Background: NOTCH receptor 3 (NOTCH3) and zinc finger E-box binding protein 1 (ZEB1) play important roles in breast cancer respectively. NOTCH3 maintains the luminal phenotype and inhibits epithelial-mesenchymal transition (EMT) in breast cancer, while ZEB1 and NOTCH3 have the opposite effects. Methods: Public databases were used to predict the expression of NOTCH3 and ZEB1 in breast cancer cell lines. The regulatory effect of NOTCH3 on ZEB1 expression was verified by western blot and RT-PCR. MiRNAs regulating ZEB1 expression were identified by using multiple databases and confirmed by reporter gene experiments. Cellular function experiments were conducted to evaluate the role of NOTCH3/miR-223/ZEB1 in the proliferation and invasion of triple-negative breast cancer (TNBC). Results: NOTCH3 and ZEB1 have opposite expression pattern in MCF-7 cells that over-express LncATB or were incubated in TGF-β to induce EMT. Western blotting and RT-PCR showed that NOTCH3 could regulate expression of ZEB1. MiR-223 inhibited the proliferation and invasion of breast cancer cells via down-regulating the expression of ZEB1. NOTCH3 inhibited the proliferation and invasion of breast cancer cells via up-regulating the expression of miR-223. Clinically, high expression of NOTCH3, miR-223 or low expression of ZEB1 were related to good prognosis of breast cancer patients. Conclusion: The current study reports a novel NOTCH3/miR-223/ZEB1 axis, which can inhibit the proliferation and invasion of breast cancer cells, and may serve as a potential biomarker for the prognosis of breast cancer.

DNA Methylation-Regulated HOXC8's Role in HER2-Positive Breast Cancer Function and its Contribution to Herceptin Resistance.

The epidermal growth factor receptor 2 (HER2) is overexpressed in 30% of breast cancers, and this overexpression is strongly correlated with a poor prognosis. Herceptin is a common treatment for HER2-positive breast cancer; however, cancer cells tend to adapt gradually to the drug, rendering it ineffective. The study revealed an association between the methylation status of the Homeobox C8 (HOXC8) gene and tumor development. Therefore, it is of paramount importance to delve into the interaction between HOXC8 and HER2-positive breast cancer, along with its molecular mechanisms. This exploration holds significant implications for a deeper understanding of the pathophysiological processes underlying HER2-positive breast cancer. Tumor tissue and pathological data from patients with HER2-positive breast cancer were systematically collected. Additionally, the human HER2-positive breast cancer cell line, SKBR3, was cultured in vitro to assess both the expression level of HOXC8 and the degree of DNA methylation. The study aimed to explore the relationship between the relative expression of HOXC8 and the clinical characteristics of breast cancer patients. The expression level of HOXC8 and the promoter methylation of HOXC8 were verified by methylation treatment of SKBR3 breast cancer cells. The regulation of HOXC8 was meticulously carried out, leading to the division of the cells into distinct groups. The study further analyzed the expression levels and biological capabilities within each group. Finally, the in vitro and in vivo sensitivity of the cells to Herceptin, a common treatment for HER2-positive breast cancer, was measured to assess the efficacy of the drug. In HER2-positive breast cancer cases characterized by poor methylation, there was an up-regulation of HOXC8. Its expression was found to be correlated with key clinical factors such as tumor size, lymph node status, clinical tumor, node, metastasis (cTNM) staging, and Herceptin resistance (p < 0.05). Upon methylation of breast cancer cells, there was a significant decrease in HOXC8 expression (p < 0.05). The study revealed that overexpression of HOXC8 resulted in increased proliferation, cloning, and metastasis of HER2-positive breast cancer cells, along with a reduced apoptosis rate (p < 0.05). Conversely, interference with HOXC8 expression reversed this scenario (p < 0.05). A Herceptin-resistant substrain, POOL2, was established using SKBR3 cells. Animal studies demonstrated that overexpressing HOXC8 accelerated tumor development and enhanced POOL2 cells' resistance to Herceptin (p < 0.05). However, following interference with HOXC8, POOL2 cells exhibited increased responsiveness to Herceptin, leading to a gradual reduction in tumor size (p < 0.05). In HER2-positive breast cancer, the expression of HOXC8 is elevated in a manner dependent on DNA methylation, and this elevated expression is closely linked to the pathology of the patient. Interfering with HOXC8 expression demonstrates the potential to partially inhibit the development and spread of breast cancer, as well as to alleviate resistance to Herceptin.