Triple-Negative Breast Cancer Cells Recruit Neutrophils by Secreting TGF-β and CXCR2 Ligands

Tumor associated neutrophils (TANs) are frequently detected in triple-negative breast cancer (TNBC). Recent studies also reveal the importance of neutrophils in promoting tumor progression and metastasis during breast cancer. However, the mechanisms regulating neutrophil trafficking to breast tumors are less clear. We sought to determine whether neutrophil trafficking to breast tumors is determined directly by the malignant potential of cancer cells. We found that tumor conditioned media (TCM) harvested from highly aggressive, metastatic TNBC cells induced a polarized morphology and robust neutrophil migration, while TCM derived from poorly aggressive estrogen receptor positive (ER+) breast cancer cells had no activity. In a three-dimensional (3D) type-I collagen matrix, neutrophils migrated toward TCM from aggressive breast cancer cells with increased velocity and directionality. Moreover, in a neutrophil-tumor spheroid co-culture system, neutrophils migrated with increased directionality towards spheroids generated from TNBC cells compared to ER+ cells. Based on these findings, we next sought to characterize the active factors secreted by TNBC cell lines. We found that TCM-induced neutrophil migration is dependent on tumor-derived chemokines, and screening TCM elution fractions based on their ability to induce polarized neutrophil morphology revealed the molecular weight of the active factors to be around 12 kDa. TCM from TNBC cell lines contained copious amounts of GRO (CXCL1/2/3) chemokines and TGF-β cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-β, while the activity remained intact in the presence of either single receptor inhibitor. Together, our findings establish a direct link between the malignant potential of breast cancer cells and their ability to induce neutrophil migration. Our study also uncovers a novel coordinated function of TGF-β and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.

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TRIPLE-NEGATIVE BREAST CANCER CELLS RECRUIT NEUTROPHILS BY SECRETING TGF-β AND CXCR2 LIGANDS

10.1101/2021.01.28.428630 ◽

2021 ◽

Author(s):

Shuvasree SenGupta ◽

Lauren E. Hein ◽

Yang Xu ◽

Jason Zhang ◽

Jamie Konwerski ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Triple Negative Breast Cancer ◽

Breast Cancer Cells ◽

Triple Negative ◽

Neutrophil Migration ◽

Breast Tumors ◽

Malignant Potential ◽

Tnbc Cell

AbstractTumor associated neutrophils (TANs) are frequently detected in triple-negative breast cancer (TNBC). Recent studies also reveal the importance of neutrophils in promoting tumor progression and metastasis during breast cancer. However, the mechanisms regulating neutrophil trafficking to breast tumors are less clear. We sought to determine whether neutrophil trafficking to breast tumors is determined directly by the malignant potential of cancer cells. We found that tumor conditioned media (TCM) harvested from highly aggressive, metastatic TNBC cells induced a polarized morphology and robust neutrophil migration, while TCM derived from poorly aggressive estrogen receptor positive (ER+) breast cancer cells had no activity. In a three-dimensional (3D) type-I collagen matrix, neutrophils migrated toward TCM from aggressive breast cancer cells with increased velocity and directionality. Moreover, in a neutrophil-tumor spheroid co-culture system, neutrophils migrated with increased directionality towards spheroids generated from TNBC cells compared to ER+ cells. Based on these findings, we next sought to characterize the active factors secreted by TNBC cell lines. We found that TCM-induced neutrophil migration is dependent on tumor-derived chemokines, and screening TCM elution fractions based on their ability to induce polarized neutrophil morphology revealed the molecular weight of the active factors to be around 12 kDa. TCM from TNBC cell lines contained copious amounts of GRO chemokines and TGF-β cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-β, while the activity remained intact in the presence of either single receptor inhibitor. Together, our findings establish a direct link between the malignant potential of breast cancer cells and their ability to induce neutrophil migration. Our study also uncovers a novel coordinated function of TGF-β and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.

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Phytochemicals inhibit migration of triple negative breast cancer cells by targeting kinase signaling

BMC Cancer ◽

10.1186/s12885-019-6479-2 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 2

Author(s):

Pradip Shahi Thakuri ◽

Megha Gupta ◽

Sunil Singh ◽

Ramila Joshi ◽

Eric Glasgow ◽

...

Keyword(s):

Breast Cancer ◽

Cell Migration ◽

Cancer Cells ◽

Protein Kinases ◽

Cell Lines ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Migration And Invasion ◽

Tnbc Cell

Abstract Background Cell migration and invasion are essential processes for metastatic dissemination of cancer cells. Significant progress has been made in developing new therapies against oncogenic signaling to eliminate cancer cells and shrink tumors. However, inherent heterogeneity and treatment-induced adaptation to drugs commonly enable subsets of cancer cells to survive therapy. In addition to local recurrence, these cells escape a primary tumor and migrate through the stroma to access the circulation and metastasize to different organs, leading to an incurable disease. As such, therapeutics that block migration and invasion of cancer cells may inhibit or reduce metastasis and significantly improve cancer therapy. This is particularly more important for cancers, such as triple negative breast cancer, that currently lack targeted drugs. Methods We used cell migration, 3D invasion, zebrafish metastasis model, and phosphorylation analysis of 43 protein kinases in nine triple negative breast cancer (TNBC) cell lines to study effects of fisetin and quercetin on inhibition of TNBC cell migration, invasion, and metastasis. Results Fisetin and quercetin were highly effective against migration of all nine TNBC cell lines with up to 76 and 74% inhibitory effects, respectively. In addition, treatments significantly reduced 3D invasion of highly motile TNBC cells from spheroids into a collagen matrix and their metastasis in vivo. Fisetin and quercetin commonly targeted different components and substrates of the oncogenic PI3K/AKT pathway and significantly reduced their activities. Additionally, both compounds disrupted activities of several protein kinases in MAPK and STAT pathways. We used molecular inhibitors specific to these signaling proteins to establish the migration-inhibitory role of the two phytochemicals against TNBC cells. Conclusions We established that fisetin and quercetin potently inhibit migration of metastatic TNBC cells by interfering with activities of oncogenic protein kinases in multiple pathways.

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NLRP3 as Putative Marker of Ipilimumab-Induced Cardiotoxicity in the Presence of Hyperglycemia in Estrogen-Responsive and Triple-Negative Breast Cancer Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21207802 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7802 ◽

Cited By ~ 1

Author(s):

Vincenzo Quagliariello ◽

Michelino De Laurentiis ◽

Stefania Cocco ◽

Giuseppina Rea ◽

Annamaria Bonelli ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Triple Negative Breast Cancer ◽

High Glucose ◽

Breast Cancer Cells ◽

Triple Negative ◽

Anticancer Efficacy ◽

Low Glucose ◽

Mcf 7

Hyperglycemia, obesity and metabolic syndrome are negative prognostic factors in breast cancer patients. Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, achieving unprecedented efficacy in multiple malignancies. However, ICIs are associated with immune-related adverse events involving cardiotoxicity. We aimed to study if hyperglycemia could affect ipilimumab-induced anticancer efficacy and enhance its cardiotoxicity. Human cardiomyocytes and estrogen-responsive and triple-negative breast cancer cells (MCF-7 and MDA-MB-231 cell lines) were exposed to ipilimumab under high glucose (25 mM); low glucose (5.5 mM); high glucose and co-administration of SGLT-2 inhibitor (empagliflozin); shifting from high glucose to low glucose. Study of cell viability and the expression of new putative biomarkers of cardiotoxicity and resistance to ICIs (NLRP3, MyD88, cytokines) were quantified through ELISA (Cayman Chemical) methods. Hyperglycemia during treatment with ipilimumab increased cardiotoxicity and reduced mortality of breast cancer cells in a manner that is sensitive to NLRP3. Notably, treatment with ipilimumab and empagliflozin under high glucose or shifting from high glucose to low glucose reduced significantly the magnitude of the effects, increasing responsiveness to ipilimumab and reducing cardiotoxicity. To our knowledge, this is the first evidence that hyperglycemia exacerbates ipilimumab-induced cardiotoxicity and decreases its anticancer efficacy in MCF-7 and MDA-MB-231 cells. This study sets the stage for further tests on other breast cancer cell lines and primary cardiomyocytes and for preclinical trials in mice aimed to decrease glucose through nutritional interventions or administration of gliflozines during treatment with ipilimumab.

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DFF40-iRGD, As A Novel Chimeric Protein With Efficient Cytotoxic and Apoptotic Effects Against Triple-Negative Breast Cancer Cells

10.21203/rs.3.rs-618195/v1 ◽

2021 ◽

Author(s):

Raheleh Amrollahi-nia ◽

Vajihe Akbari ◽

Fatemeh Shafiee

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Triple Negative Breast Cancer ◽

Breast Cancer Cells ◽

Triple Negative ◽

Soluble Expression ◽

Soluble Form ◽

Apoptotic Effects ◽

Mcf 7

Abstract Purpose DNA fragmenting factor (DFF40), an endonuclease inducing irreversible apoptosis protein, is down-regulated in many types of tumor cells. iRGD is a tumor-penetrating peptide with high affinity to cancer cells overexpressing αVβ3 receptors. The aim of this study was to produce the recombinant DFF40-iRGD protein as a new molecule to selectively induce cytotoxicity in cancer cells and to evaluate its biological effects. Methods The three-dimensional structure of DFF40-iRGD was predicted using Modeller software and its interaction with αVβ3 receptor was evaluated by HADDOCK web-server. Recombinant DFF40 and DFF40-iRGD proteins were produced using intein fusion system in Escherichia coli BL21 (DE3). To improve the soluble expression, the inducer concentration, temperature and incubation time were optimized. After purification of DFF40 and DFF40-iRGD using chitin column, the cytotoxic and apoptotic effects of the proteins against MDA-MB-231 (αVβ3 positive) and MCF-7 (αVβ3 negative) cell lines were evaluated using cell viability assay and flow cytometric analysis. Results The results of molecular docking indicated the proper interaction of DFF40-iRGD with the integrin receptor comparable to iRGD. The optimum conditions of soluble expression of proteins were the induction with 0.5 mM and 0.1 mM of IPTG for DFF40 and DFF40-iRGD, respectively, at 7 ºC for 24 hours. After 48 hours of incubation, DFF40-iRGD exhibited significantly higher cytotoxic effect against MDA-MB-231 cells than MCF-7 cells as IC50 values of 0.77 and 1.64 µg/ml were found for MDA-MB-231 and MCF-7 cells, respectively. However, DFF40 cytotoxicity was not significantly different in two cell lines. Furthermore, Flow cytometry results showed that the fusion protein can induce remarkably apoptotic cell death in cancer cells. Conclusion in this study, DFF40-iRGD protein was produced in soluble form and its inhibitory effects on cancer cell survival and induction of apoptosis were demonstrated; therefore, it has the potential to be used as a drug candidate for targeted treatment of breast cancer, especially Triple Negative Breast Cancer Cells.

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R-Spondin 2 Promoted Proliferation, Invasion and Migration of Triple Negative Breast Cancer Cells Through Activating Wnt/β-Catenin Signaling Pathway After Axin2 Inhibition

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2020.2221 ◽

2020 ◽

Vol 10 (1) ◽

pp. 26-36

Author(s):

XiaoHu Sun ◽

Yue Yu ◽

Jie Ge ◽

Xin Wang ◽

XuChen Cao

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Signaling Pathway ◽

Triple Negative Breast Cancer ◽

Breast Cancer Cells ◽

Triple Negative ◽

Invasion And Migration ◽

Tnbc Cell ◽

And Migration ◽

Related Proteins

Protein R-spondin 2, which is known as roof plate-specific spondin 2, is an extracellular matrix secreted protein that participates in a wide range of biological processes. However, the expression of R-spondin 2 in triple negative breast cancer (TNBC) and its specific mechanism have not been reported. In this study, RT-qPCR and western blot were used to detect the expression of R-spondin 2 and Axin2 in cells. Cell transfection techniques were used to overexpress Axin2 and interfere with the expression of R-spondin 2. CCk-8 and clone formation assay were used to detect cell viability. Wound healing and Traswell techniques were used to test the rate of invasion and migration of TNBC cells. Western blot was used to detect the expression of related proteins. The results showed that the expression of R-spondin 2 in TNBC cell lines was significantly increased compared with normal breast cancer cells. After interfering with the expression of R-spondin 2 in TNBC cell lines, the rate of cell viability, invasion and migration were decreased. It was also found that the expressions of Axin2 and β-catenin and Cyclin-D1, which are wnt/β-catenin pathway related proteins, were significantly decreased. Subsequently, the overexpression of Axin2 can inhibit the proliferation, invasion and migration that were ever promoted by R-spondin 2 of TNBC cells. Moreover, the overexpression of Axin2 inhibited the activation of wnt/β-catenin signaling pathway, which was also activated by R-spondin 2 in TNBC cells. In a word, R-spondin 2 promoted proliferation, invasion and migration of triple negative breast cancer cells through activating wnt/β-catenin signaling pathway after Axin2 inhibition.

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Application of a Biphasic Mathematical Model of Cancer Cell Drug Response for Formulating Potent and Synergistic Targeted Drug Combinations to Triple Negative Breast Cancer Cells

Cancers ◽

10.3390/cancers12051087 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1087

Author(s):

Jinyan Shen ◽

Li Li ◽

Niall G. Howlett ◽

Paul S. Cohen ◽

Gongqin Sun

Keyword(s):

Breast Cancer ◽

Mathematical Model ◽

Targeted Therapy ◽

Cancer Cells ◽

Cell Lines ◽

Triple Negative Breast Cancer ◽

Breast Cancer Cells ◽

Triple Negative ◽

Drug Combinations ◽

Targeted Drug

Triple negative breast cancer is a collection of heterogeneous breast cancers that are immunohistochemically negative for estrogen receptor, progesterone receptor, and ErbB2 (due to deletion or lack of amplification). No dominant proliferative driver has been identified for this type of cancer, and effective targeted therapy is lacking. In this study, we hypothesized that triple negative breast cancer cells are multi-driver cancer cells, and evaluated a biphasic mathematical model for identifying potent and synergistic drug combinations for multi-driver cancer cells. The responses of two triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468, to a panel of targeted therapy drugs were determined over a broad range of concentrations. The analyses of the drug responses by the biphasic mathematical model revealed that both cell lines were indeed dependent on multiple drivers, and inhibitors of individual drivers caused a biphasic response: a target-specific partial inhibition at low nM concentrations, and an off-target toxicity at μM concentrations. We further demonstrated that combinations of drugs, targeting each driver, cause potent, synergistic, and cell-specific cell killing. Immunoblotting analysis of the effects of the individual drugs and drug combinations on the signaling pathways supports the above conclusion. These results support a multi-driver proliferation hypothesis for these triple negative breast cancer cells, and demonstrate the applicability of the biphasic mathematical model for identifying effective and synergistic targeted drug combinations for triple negative breast cancer cells.

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