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Commun Biol ; 4(1): 1091, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531530


During breast cancer metastasis, cancer cell invasion is driven by actin-rich protrusions called invadopodia, which mediate the extracellular matrix degradation required for the success of the invasive cascade. In this study, we demonstrate that TC10, a member of a Cdc42 subfamily of p21 small GTPases, regulates the membrane type 1 matrix metalloproteinase (MT1-MMP)-driven extracellular matrix degradation at invadopodia. We show that TC10 is required for the plasma membrane surface exposure of MT1-MMP at these structures. By utilizing our Förster resonance energy transfer (FRET) biosensor, we demonstrate the p190RhoGAP-dependent regulation of spatiotemporal TC10 activity at invadopodia. We identified a pathway that regulates invadopodia-associated TC10 activity and function through the activation of p190RhoGAP and the downstream interacting effector Exo70. Our findings reveal the role of a previously unknown regulator of vesicular fusion at invadopodia, TC10 GTPase, in breast cancer invasion and metastasis.

Clin Transl Radiat Oncol ; 28: 79-84, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33851037


Purpose/Objectives: Stereotactic radiosurgery (SRS) for metastatic disease to the brain is associated with higher in-brain failures compared to whole brain radiation therapy (WBRT). Here we investigated the relationship between low-dose fall off during SRS and location of new brain lesions. Materials and Methods: One hundred sixty-seven patients treated with single fraction or fractionated SRS for intact or resected brain metastases at our institution from January 2016 to June 2018 were reviewed. Patients with imaging findings of new brain metastases after the initial SRS were included. Patients with WBRT before SRS were excluded. MRI scans for repeat treatments were fused with initial SRS plan. New lesions were outlined on the initial SRS planning CT. The mean dose that the site of new lesions received from initial SRS was tabulated. Results: Thirty-eight patients met inclusion criteria. 165 new lesions were evaluated. There was a lower propensity to develop new brain lesions with increasing dose received by the regions from prior SRS, with 66%, 34%, 19%, 13%, 6%, 5%, 2% and 1% of new lesions appearing in regions that received less than 1 Gy, greater than or equal to 1, 2, 3, 4, 5, 6, and 7 Gy, respectively. Higher doses are received by smaller brain volumes during SRS. After accounting for volume, 14, 14, 11, 7, 2, 2, 1 and 1 new lesions appeared per 100 cm3 of brain in regions that received doses of less than 1 Gy, greater than or equal to 1, 2, 3, 4, 5, 6, and 7 Gy, respectively, from prior SRS. Conclusions: We identified low dose spillage during SRS to be associated with lower incidence of new brain metastases. Validation in larger dataset or prospective study of the combination of SRS with low dose WBRT would be crucial in order to establish causality of these findings.

Breast Cancer Res ; 20(1): 131, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30367629


BACKGROUND: Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, is not only essential for proper mammary ductal development, but also associated with breast cancer proliferation and growth. In the absence of AREG, mammary ductal growth is stunted and fails to expand. Furthermore, suppression of AREG expression in estrogen receptor-positive breast tumor cells inhibits in-vitro and in-vivo growth. METHODS: We crossed AREG-null (AREG-/-) mice with the murine luminal B breast cancer model, MMTV-PyMT (PyMT), to generate spontaneous breast tumors that lack AREG (AREG-/- PyMT). We evaluated tumor growth, cytokeratin-8 (K8)-positive luminal cells, cytokeratin-14 (K14)-positive myoepithelial cells, and expression of AREG, Ki67, and PyMT. Primary myoepithelial cells from nontumor-bearing AREG+/+ mice underwent fluorescence-activated cell sorting and were adapted to culture for in-vitro coculture studies with AT-3 cells, a cell line derived from C57Bl/6 PyMT mammary tumors. RESULTS: Intriguingly, PyMT-induced lesions progress more rapidly in AREG-/- mice than in AREG+/+ mice. Quantification of K8+ luminal and K14+ myoepithelial cells in non-PyMT AREG-/- mammary glands showed fewer K14+ cells and a thinner myoepithelial layer. Study of AT-3 cells indicated that coculture with myoepithelial cells or exposure to AREG, epidermal growth factor, or basic fibroblast growth factor can suppress PyMT expression. Late-stage AREG-/- PyMT tumors are significantly less solid in structure, with more areas of papillary and cystic growth. Papillary areas appear to be both less proliferative and less necrotic. In The Cancer Genome Atlas database, luminal-B invasive papillary carcinomas have lower AREG expression than luminal B invasive ductal carcinomas. CONCLUSIONS: Our study has revealed a previously unknown role of AREG in myoepithelial cell development and PyMT expression. AREG expression is essential for proper myoepithelial coverage of mammary ducts. Both AREG and myoepithelial cells can suppress PyMT expression. We find that lower AREG expression is associated with invasive papillary breast cancer in both the MMTV-PyMT model and human breast cancer.

Anfirregulina/metabolismo , Células Epiteliais/patologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/patologia , Anfirregulina/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/virologia , Feminino , Humanos , Glândulas Mamárias Animais/citologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/virologia , Vírus do Tumor Mamário do Camundongo/genética , Vírus do Tumor Mamário do Camundongo/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Invasividade Neoplásica/patologia , Polyomavirus/genética , Polyomavirus/imunologia
Breast Cancer Res ; 20(1): 24, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29636067


BACKGROUND: The interaction of breast cancer cells with other cells in the tumor microenvironment plays an important role in metastasis. Invasion and intravasation, two critical steps in the metastatic process, are influenced by these interactions. Macrophages are of particular interest when it comes to studying tumor cell invasiveness. Previous studies have shown that there is paracrine loop signaling between breast cancer cells and macrophages involving colony stimulating factor 1 (CSF-1) produced by tumor cells and epidermal growth factor (EGF) production by macrophages. In this paper, we identify a novel paracrine loop between tumor cells and macrophages involving neuregulin (NRG1) and notch signaling. METHODS: The aim of this study was to determine the role of NRG1, a ligand of the ErbB3 receptor, in macrophage stimulation of tumor cell transendothelial migration and intravasation. We used fluorescence-activated cell sorting (FACS) and western blot to determine ErbB3 and NRG1 expression, respectively. An in vitro transendothelial migration (iTEM) assay was used to examine the effects of short hairpin (sh)RNA targeting NRG1 in tumor cells and clustered regularly interspaced short palindromic repeats (CRISPR) knockout of jagged 1 (JAG1) in macrophages. Orthotopic xenograft injections in mice were used to confirm results in vivo. RESULTS: In our system, macrophages were the primary cells showing expression of ErbB3, and a blocking antibody against ErbB3 resulted in a significant decrease in macrophage-induced transendothelial migration of breast cancer cells. Stimulation of macrophages with NRG1 upregulated mRNA and protein expression of JAG1, a ligand of the Notch receptor, and JAG1 production by macrophages was important for transendothelial migration of tumor cells. CONCLUSIONS: This study demonstrates that stimulation of macrophages by tumor cell NRG1 can enhance transendothelial migration and intravasation. We also demonstrate that this effect is due to induction of macrophage JAG1, an important ligand of the Notch signaling pathway.

Neoplasias da Mama/genética , Proteína Jagged-1/genética , Neuregulina-1/genética , Migração Transendotelial e Transepitelial/genética , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Comunicação Parácrina/genética , Receptor ErbB-3/genética , Receptores Notch/genética , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
J Cell Biol ; 216(12): 4331-4349, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29061650


The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the matrix degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular matrix (ECM) with their ability to degrade the ECM in breast tumor cells. We identify two pathways at invadopodia important for integrin activation and delivery of matrix metalloproteinases: through the upstream recruiter CIB1 as well as the downstream effector GIT1. Rac3 activity, at and surrounding invadopodia, is controlled by Vav2 and ßPIX. These guanine nucleotide exchange factors regulate the spatiotemporal dynamics of Rac3 activity, impacting GIT1 localization. Moreover, the GTPase-activating function of GIT1 toward the vesicular trafficking regulator Arf6 GTPase is required for matrix degradation. Importantly, Rac3 regulates the ability of tumor cells to metastasize in vivo. The Rac3-dependent mechanisms we show in this study are critical for balancing proteolytic activity and adhesive activity to achieve a maximally invasive phenotype.

Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Integrina beta1/genética , Neoplasias Mamárias Animais/genética , Proteínas rac de Ligação ao GTP/genética , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Adesão Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Células HEK293 , Humanos , Integrina beta1/metabolismo , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/deficiência