Interaction of tumor cells and astrocytes promotes breast cancer brain metastases through TGF-β2/ANGPTL4 axes

Abstract Metastatic outcomes depend on the interactions of metastatic cells with a specific organ microenvironment. Our previous studies have shown that triple-negative breast cancer (TNBC) MDA-MB-231 cells passaged in astrocyte-conditioned medium (ACM) show proclivity to form brain metastases, but the underlying mechanism is unknown. The combination of microarray analysis, qPCR, and ELISA assay were carried out to demonstrate the ACM-induced expression of angiopoietin-like 4 (ANGPTL4) in TNBC cells. A stable ANGPTL4-knockdown MDA-MB-231 cell line was generated by ANGPTL4 short-hairpin RNA (shRNA) and inoculated into mice via left ventricular injection to evaluate the role of ANGPTL4 in brain metastasis formation. The approaches of siRNA, neutralizing antibodies, inhibitors, and immunoprecipitation were used to demonstrate the involved signaling molecules. We first found that ACM-conditioned TNBC cells upregulated the expression of ANGPTL4, a secreted glycoprotein whose effect on tumor progression is known to be tumor microenvironment- and tumor-type dependent. Knockdown of ANGPTL4 in TNBC MDA-MB-231 cells with shRNA decreased ACM-induced tumor cell metastatic growth in the brain and attributed to survival in a mouse model. Furthermore, we identified that astrocytes produced transforming growth factor-beta 2 (TGF-β2), which in part is responsible for upregulation of ANGPTL4 expression in TNBC through induction of SMAD signaling. Moreover, we identified that tumor cells communicate with astrocytes, where tumor cell-derived interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) increased the expression of TGF-β2 in astrocytes. Collectively, these findings indicate that the invading TNBC cells interact with astrocytes in the brain microenvironment that facilitates brain metastases of TNBC cells through a TGF-β2/ANGPTL4 axis. This provides groundwork to target ANGPTL4 as a treatment for breast cancer brain metastases.

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BSCI-11. STROMAL PLATELET DERIVED GROWTH FACTOR RECEPTOR-β (PDGFRβ) PROMOTES BREAST CANCER BRAIN METASTASIS

Neuro-Oncology Advances ◽

10.1093/noajnl/vdz014.009 ◽

2019 ◽

Vol 1 (Supplement_1) ◽

pp. i3-i3

Author(s):

Katie Thies ◽

Anisha Hammer ◽

Blake Hildreth ◽

Luke Russell ◽

Steven Sizemore ◽

...

Keyword(s):

Breast Cancer ◽

Growth Factor ◽

Brain Metastasis ◽

Brain Metastases ◽

Tumor Cells ◽

Mammary Tumor ◽

Growth Factor Receptor ◽

Platelet Derived Growth Factor ◽

Mammary Tumor Cells ◽

The Brain

Abstract Stromal platelet-derived growth factor receptor-beta (PDGFRβ) has emerged as an actionable mediator of breast tumor-stromal communication. As a receptor tyrosine kinase, PDGFRβ is activated by its ligand, PDGFB, which is released by neighboring tumor epithelium and endothelium. However, how PDGF signaling mediates breast cancer (BC) initiation, progression, and metastasis remains unclear. To evaluate PDGFRβ in this disease, we developed a mouse model of stromal-specific PDGFRβ activation using the Fsp-cre transgene previously published by our group. Mesenchymal-specific activation of PDGFRβ promotes preferential experimental brain metastasis of PDGFB-expressing mammary tumor cells when injected intravenously and accelerates intracranial tumor growth of these cells. Mammary tumor cells expressing low levels of PDGFB do not exhibit a similar increase in brain metastases in PDGFRβ mutant mice. To our knowledge, this is the first example where genetic manipulation of the stroma leads to an increased incidence of BCBM. Our pre-clinical data suggests that primary breast tumors that express high PDGFB could preferentially metastasize to the brain. To test this in patients, we analyzed PDGFB protein expression in a tissue microarray comprised of HER2-positive and triple negative BC primary tumors. While high PDGFB did not correlate with site-independent metastatic recurrence, it was prognostic of brain metastasis, mirroring our mouse data. Our findings suggest that high primary tumor PDGFB expression defines a subset of BC patients predisposed to brain metastases. These patients may benefit from therapeutic intervention of PDGFRβ signaling. To test this pre-clinically, we treated mice harboring intracranial tumors with the PDGFR-specific inhibitor, crenolanib. Excitingly, crenolanib treatment significantly inhibited the brain tumor burden in these mice. Combined, our findings (1) advocate that primary tumor expression of PDGFB is a novel prognostic biomarker for the development of BCBM and (2) support clinical trial evaluation of PDGFR inhibitors for the prevention and treatment of BCBM.

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A common goal to CARE: Cancer Advocates, Researchers, and Clinicians Explore current treatments and clinical trials for breast cancer brain metastases

npj Breast Cancer ◽

10.1038/s41523-021-00326-5 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Natalie S. Joe ◽

Christine Hodgdon ◽

Lianne Kraemer ◽

Kristin J. Redmond ◽

Vered Stearns ◽

...

Keyword(s):

Breast Cancer ◽

Clinical Trials ◽

Brain Metastases ◽

Detection Methods ◽

Future Research ◽

Breast Cancer Brain Metastasis ◽

Patient Advocates ◽

Breast Cancer Brain Metastases ◽

The Brain

AbstractBreast cancer is the most commonly diagnosed cancer in women worldwide. Approximately one-tenth of all patients with advanced breast cancer develop brain metastases resulting in an overall survival rate of fewer than 2 years. The challenges lie in developing new approaches to treat, monitor, and prevent breast cancer brain metastasis (BCBM). This review will provide an overview of BCBM from the integrated perspective of clinicians, researchers, and patient advocates. We will summarize the current management of BCBM, including diagnosis, treatment, and monitoring. We will highlight ongoing translational research for BCBM, including clinical trials and improved detection methods that can become the mainstay for BCBM treatment if they demonstrate efficacy. We will discuss preclinical BCBM research that focuses on the intrinsic properties of breast cancer cells and the influence of the brain microenvironment. Finally, we will spotlight emerging studies and future research needs to improve survival outcomes and preserve the quality of life for patients with BCBM.

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BSCI-04. TARGETING TRIPLE-NEGATIVE BREAST CANCER BRAIN METASTASES WITH A RE-ENGINEERED LUPUS AUTOANTIBODY

Neuro-Oncology Advances ◽

10.1093/noajnl/vdz014.003 ◽

2019 ◽

Vol 1 (Supplement_1) ◽

pp. i1-i2

Author(s):

Shenqi Zhang ◽

Christopher May ◽

Anupama Shirali ◽

Valentina Dubljevic ◽

James Campbell ◽

...

Keyword(s):

Breast Cancer ◽

Brain Metastases ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Metastatic Tumor ◽

Nucleoside Transporter ◽

Cell Nuclei ◽

Filter Model ◽

Breast Cancer Brain Metastases ◽

The Brain

Abstract An unusual lupus anti-DNA autoantibody, 3E10, has potential to be used against triple-negative breast cancer (TNBC) brain metastases. 3E10 penetrates live cell nuclei, inhibits DNA repair, and is selectively toxic to cancer cells with the PTEN and/or DNA-damage response (DDR)-deficiencies that are associated with brain metastases in TNBC. The ENT2 nucleoside transporter that 3E10 uses to cross cell membranes is highly expressed in tumors and in brain endothelial cells (BECs) at the blood-brain barrier (BBB), and 3E10 has previously delivered cargo proteins to ischemic brain in a rat stroke model. We have re-engineered 3E10 into an optimized fragment, called Deoxymab-1 (PAT-DX1), that has increased effect on PTEN/DDR-deficient tumor cells. In the present study we tested the ability of PAT-DX1 to cross the BBB and improve outcomes in a mouse model of TNBC brain metastases. PAT-DX1 crossed from apical to basolateral chambers in an hCMEC/D3 Transwell filter model of the BBB, and penetrated the nuclei of and was toxic to the brain-seeking 231-BR subclone of MDA-MB-231 TNBC cells, which harbors a loss of PTEN compared to parental cells. Brain metastases were generated in nude mice by intracardiac injection of 1.75x105 231-BR cells engineered for expression of luciferase, as confirmed by IVIS one week after injection. Mice with brain metastases were treated by tail vein injection of control (PBS, n=7) or DX1 (20 mg/kg, n=7) 3x/week for 4 weeks. Mice were observed for behavior and weights, and brain radiance efficiency was monitored by weekly IVIS to track metastatic tumor growth. PAT-DX1 significantly suppressed growth of brain metastases based on absolute and relative radiance efficiencies in the brain, increased the median survival of the mice from 38 to 52 days (P< 0.02), and was well tolerated. These results provide proof of concept for use of a re-engineered autoantibody against brain metastases.

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TMIC-10. THE ROLE OF THE BRAIN MICROENVIRONMENT IN THE MIGRATION OF BREAST CANCER CELLS IN BRAIN METASTASES

Neuro-Oncology ◽

10.1093/neuonc/noz175.1044 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi249-vi249

Author(s):

Cymon Kersch ◽

Leslie Muldoon ◽

DreeAnna Morris ◽

Edward Neuwelt

Keyword(s):

Breast Cancer ◽

Brain Metastases ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Αvβ3 Integrin ◽

Human Breast ◽

And Migration ◽

Breast Cancer Brain Metastases ◽

The Brain

Abstract BACKGROUND Breast cancer brain metastases have poor prognosis and few treatment options. When breast cancer cells invade the brain they interact with the vasculature and resident brain cells including microglia and astrocytes. We hypothesize that brain cells produce factors that enhance the growth and invasion of breast cancer cells in the brain. METHODS Human breast cancer cell lines (MDA-MB231BR-HER2 and HCC1954) were inoculated intracranially in athymic rats as xenograft models of brain metastasis. Brains were analyzed for pro-tumorigenic factor expression in the tumor microenvironment using fluorescent immunohistochemistry. In vitro assays assessed factors involved in breast cancer cell proliferation, morphology, and migration. RESULTS The tumor xenografts showed infiltration intothe perivascular space. Galectin-3 (Gal3), heparin-binding epidermal growth factor (HB-EGF), and Neuregulin (NRG1), factors known to interact with receptors expressed by breast cancer cells, co-localized with reactive microglia (Gal3) and astrocytes (HB-EGF and NRG1) in and around xenografts. In vitro, these factors individually increased HCC1954 cell proliferation and/or migration. In transwell co-culture assays, BV2 microglial cells increased the migration of HCC1954 cells >25 fold, which was prevented by cilengitide, an inhibitor of αvβ3-integrin cell adhesion protein. ELISA analysis demonstrated that BV2 microglia secrete Gal3 in the presence of HCC1954 cells. Gal3 is known to bind and induce clustering of αvβ3-integrin which is expressed on metastatic breast cancer cells. Immunohistochemistry of clinical specimens revealed that Gal3 is expressed in/around human breast cancer brain metastases. CONCLUSIONS These data suggest that factors produced in the tumor microenvironment promote the growth and migration of breast cancer cells in the brain. Gal3, produced and secreted by activated microglia in vitro and expressed in and around brain metastases, increases the invasive capability of breast cancer cells. The interactions of neoplastic cells with the brain environment may provide a target to improve therapy of brain metastases.

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The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation

Science Translational Medicine ◽

10.1126/scitranslmed.aal4682 ◽

2017 ◽

Vol 9 (391) ◽

pp. eaal4682 ◽

Cited By ~ 46

Author(s):

David P. Kodack ◽

Vasileios Askoxylakis ◽

Gino B. Ferraro ◽

Qing Sheng ◽

Mark Badeaux ◽

...

Keyword(s):

Breast Cancer ◽

Brain Metastases ◽

Treatment Strategies ◽

Brain Lesions ◽

Growth Delay ◽

Luminal Breast Cancer ◽

Pi3k Inhibition ◽

Mechanistic Basis ◽

Breast Cancer Brain Metastases ◽

The Brain

Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance ofHER2-amplified and/orPIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies forHER2-amplified and/orPIK3CA-mutant breast cancer brain metastases.

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Separation of cell survival, growth, migration, and mesenchymal transdifferentiation effects of fibroblast secretome on tumor cells of head and neck squamous cell carcinoma

Tumor Biology ◽

10.1177/1010428317705507 ◽

2017 ◽

Vol 39 (11) ◽

pp. 101042831770550 ◽

Cited By ~ 7

Author(s):

Veronika Maria Metzler ◽

Christian Pritz ◽

Anna Riml ◽

Angela Romani ◽

Raphaela Tuertscher ◽

...

Keyword(s):

Cell Proliferation ◽

Growth Factor ◽

Tumor Cell ◽

Cell Survival ◽

Tumor Cells ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Paracrine Factors ◽

Mesenchymal Transition ◽

Growth Factor Beta

Fibroblasts play a central role in tumor invasion, recurrence, and metastasis in head and neck squamous cell carcinoma. The aim of this study was to investigate the influence of tumor cell self-produced factors and paracrine fibroblast–secreted factors in comparison to indirect co-culture on cancer cell survival, growth, migration, and epithelial–mesenchymal transition using the cell lines SCC-25 and human gingival fibroblasts. Thereby, we particularly focused on the participation of the fibroblast-secreted transforming growth factor beta-1.Tumor cell self-produced factors were sufficient to ensure tumor cell survival and basic cell growth, but fibroblast-secreted paracrine factors significantly increased cell proliferation, migration, and epithelial–mesenchymal transition–related phenotype changes in tumor cells. Transforming growth factor beta-1 generated individually migrating disseminating tumor cell groups or single cells separated from the tumor cell nest, which were characterized by reduced E-cadherin expression. At the same time, transforming growth factor beta-1 inhibited tumor cell proliferation under serum-starved conditions. Neutralizing transforming growth factor beta antibody reduced the cell migration support of fibroblast-conditioned medium. Transforming growth factor beta-1 as a single factor was sufficient for generation of disseminating tumor cells from epithelial tumor cell nests, while other fibroblast paracrine factors supported tumor nest outgrowth. Different fibroblast-released factors might support tumor cell proliferation and invasion, as two separate effects.

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BSCI-02. tGLI1 IS A NOVEL, ACTIONABLE TARGET FOR THE TREATMENT OF BREAST CANCER BRAIN METASTASES

Neuro-Oncology Advances ◽

10.1093/noajnl/vdz014.001 ◽

2019 ◽

Vol 1 (Supplement_1) ◽

pp. i1-i1 ◽

Cited By ~ 1

Author(s):

Daniel Doheny ◽

Sherona Sirkisoon ◽

Tadas Rimkus ◽

Dongqin Zhu ◽

Noah Aguayo ◽

...

Keyword(s):

Breast Cancer ◽

Brain Metastases ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Unmet Need ◽

Current Standard ◽

Recurrent Gliomas ◽

Breast Cancer Brain Metastases ◽

The Brain ◽

Positive Breast Cancer

Abstract Despite improvements in early detection and intervention, breast cancer remains the second leading cause of cancer-related death in women and the second most common cancer to metastasize to the brain. Current standard of care options for breast cancer brain metastases (BCBM) include stereotactic radiosurgery, whole-brain radiotherapy, and surgical resection. Local and distant recurrences are common leading to significant morbidity; effective FDA-approved drugs for these patients remain a significant unmet need. Our laboratory discovered an alternative splice variant of glioma-associated oncogene homolog 1 (GLI1), termed truncated GLI1 (tGLI1) that is a tumor-specific gain-of-function transcription factor preferentially expressed in most BCBM samples and recurrent gliomas. Recent results established that tGLI1 promotes breast cancer stem cells (BrCSCs) and is associated with preferential metastasis to the brain and radioresistance, justifying tGLI1 as an ideal therapeutic target for BCBM patients. To identify tGLI1-targeting agents, we screened 1,520 compounds across three commercial drug libraries and found ketoconazole, an FDA-approved azole antifungal and component of previously studied anti-neoplastic regimens, selectively killed tGLI1-expressing breast cancer cells with heightened efficacy against the CSC subpopulation in vitro. tGLI1 knockdown abolished the ability of ketoconazole to target BrCSCs, indicating that ketoconazole effect is dependent on tGLI1. Intracardiac mouse studies showed ketoconazole selectively inhibited circulating tGLI1-positive breast cancer cells from developing into brain metastases and suppressed the progression of existing brain metastases. Mass spectrometry demonstrated ketoconazole effectively penetrated the blood-brain barrier (BBB) and blood-tumor barrier (BTB). Mechanistic studies suggest that ketoconazole-dependent cell kill is, in part, mediated through disruption of the tGLI1-STAT3 interaction. Collectively, our preclinical results demonstrate that ketoconazole is an effective inhibitor of BrCSCs and brain metastasis of tGLI1-positive breast cancer. Based on these promising preclinical data, we opened a window-of-opportunity study in patients with BCBM and recurrent gliomas to determine if ketoconazole treatment alters tGLI1 signaling in humans (NCT03796273).

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Screening and Identification of Novel Potential Biomarkers for Breast Cancer Brain Metastases

Frontiers in Oncology ◽

10.3389/fonc.2021.784096 ◽

2022 ◽

Vol 11 ◽

Author(s):

Lulu Wang ◽

Dan Zeng ◽

Qi Wang ◽

Li Liu ◽

Tao Lu ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Brain Metastases ◽

Candidate Genes ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Key Genes ◽

Breast Cancer Brain Metastases ◽

The Brain ◽

Potential Biomarkers

Brain metastases represent a major cause of mortality among patients with breast cancer, and few effective targeted treatment options are currently available. Development of new biomarkers and therapeutic targets for breast cancer brain metastases (BCBM) is therefore urgently needed. In this study, we compared the gene expression profiles of the brain metastatic cell line MDA-MB-231-BR (231-BR) and its parental MDA-MB-231, and identified a total of 84 genes in the primary screening through a series of bioinformatic analyses, including construction of protein-protein interaction (PPI) networks by STRING database, identification of hub genes by applying of MCODE and Cytohubba algorithms, identification of leading-edge subsets of Gene Set Enrichment Analysis (GSEA), and identification of most up-regulated genes. Eight genes were identified as candidate genes due to their elevated expression in brain metastatic 231-BR cells and prognostic values in patients with BCBM. Then we knocked down the eight individual candidate genes in 231-BR cells and evaluated their impact on cell migration through a wound-healing assay, and four of them (KRT19, FKBP10, GSK3B and SPANXB1) were finally identified as key genes. Furthermore, the expression of individual key genes showed a correlation with the infiltration of major immune cells in the brain tumor microenvironment (TME) as analyzed by Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA), suggesting possible roles of them in regulation of the tumor immune response in TME. Therefore, the present work may provide new potential biomarkers for BCBM. Additionally, using GSEA, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis, we determined the top enriched cellular functions or pathways in 231-BR cells, which may help better understand the biology governing the development and progression of BCBM.

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