RESUMO
Accurate modeling of angiogenesis in vitro is essential for guiding the preclinical development of novel anti-angiogenic agents and treatment strategies. The formation of new blood vessels is a multifactorial and multi-stage process dependent upon paracrine factors produced by stromal cells in the local microenvironment. Mesenchymal stem cells (MSCs) are multipotent cells in adults that can be recruited to sites of inflammation and tissue damage where they aid in wound healing through regenerative, trophic, and immunomodulatory properties. Primary stromal cultures derived from human bone marrow, normal prostate, or prostate cancer tissue are highly enriched in MSCs and stromal progenitors. Using conditioned media from these primary cultures, a robust pro-angiogenic response was observed in a physiologically-relevant three-dimensional fibrin matrix assay. To evaluate the utility of this assay, the allosteric HDAC4 inhibitor tasquinimod and the anti-VEGF monoclonal antibody bevacizumab were used as model compounds with distinct mechanisms of action. While both agents had a profound inhibitory effect on endothelial sprouting, only bevacizumab induced significant regression of established vessels. Additionally, the pro-angiogenic properties of MSCs derived from prostate cancer patients provides further evidence that selective targeting of this population may be of therapeutic benefit.
Assuntos
Células-Tronco Mesenquimais/fisiologia , Neovascularização Fisiológica/fisiologia , Próstata/citologia , Inibidores da Angiogênese/farmacologia , Bevacizumab/farmacologia , Células Cultivadas , Fibrina , Humanos , Masculino , Quinolonas/farmacologia , Nicho de Células-TroncoRESUMO
Tumor cell interactions with their microenvironment, and neighboring endothelial cells in particular, are critical for tumor cell survival and the metastatic process. Within the spectrum of tumors, melanomas are notorious for their ability to metastasize at a relatively early stage of development; however, little is known about the molecular pathways mediating this process. We recently performed a screen to assess critical mediators of melanoma metastasis by evaluating melanoma-endothelial cell communication. Neuropilin-2 (NRP2), a cell surface receptor involved in angiogenesis and axonal guidance, was found to be an important mediator of melanoma-endothelial cell cross-talk in these studies. Here we seek to further define the role of NRP2 in melanoma growth and progression. We use stable gene silencing of NRP2 in melanomas from varying stages of tumor progression to define the role of NRP2 in melanoma growth, migration, invasion, and metastasis. We found that NRP2 gene silencing in metastatic melanoma cell lines inhibited tumor cell growth in vitro; furthermore, knockdown of NRP2 expression in the metastatic melanoma cell line 1205Lu significantly inhibited in-vivo tumor growth and metastasis. We conclude that NRP2 plays an important role in mediating melanoma growth and metastasis and suggest that targeting this cell surface molecule may represent a significant therapeutic strategy for patients diagnosed with aggressive forms of melanoma.
Assuntos
Melanoma/genética , Melanoma/patologia , Neuropilina-2/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Metástase NeoplásicaRESUMO
Histone deacetylase inhibitors (HDACIs) are being tested in clinical trials for the treatment of solid tumors. While most studies have focused on the reexpression of silenced tumor suppressor genes, a number of genes/pathways are downregulated by HDACIs. This provides opportunities for combination therapy: agents that further disable these pathways through inhibition of residual gene function are speculated to enhance cell death in combination with HDACIs. A previous study from our group indicated that mitotic checkpoint kinases such as PLK1 and Aurora A are downregulated by HDACIs. We used in vitro and in vivo xenograft models of prostate cancer (PCA) to test whether combination of HDACIs with the pan-aurora kinase inhibitor AMG 900 can synergistically or additively kill PCA cells. AMG 900 and HDACIs synergistically decreased cell proliferation activity and clonogenic survival in DU-145, LNCaP, and PC3 PCA cell lines compared to single-agent treatment. Cellular senescence, polyploidy, and apoptosis was significantly increased in all cell lines after combination treatment. In vivo xenograft studies indicated decreased tumor growth and decreased aurora B kinase activity in mice treated with low-dose AMG 900 and vorinostat compared to either agent alone. Pharmacodynamics was assessed by scoring for phosphorylated histone H3 through immunofluorescence. Our results indicate that combination treatment with low doses of AMG 900 and HDACIs could be a promising therapy for future clinical trials against PCA.
Assuntos
Aurora Quinases/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ftalazinas/farmacologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores de Histona Desacetilases/administração & dosagem , Histonas/genética , Humanos , Masculino , Ftalazinas/administração & dosagem , Poliploidia , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Fuso Acromático/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Angiogenesis is required for tumor growth. WT1, a protein that affects both mRNA transcription and splicing, has recently been shown to regulate expression of vascular endothelial growth factor (VEGF), one of the major mediators of angiogenesis. In the present study, we tested the hypothesis that WT1 is a key regulator of tumor angiogenesis in Ewing sarcoma. We expressed exogenous WT1 in the WT1-null Ewing sarcoma cell line, SK-ES-1, and we suppressed WT1 expression using shRNA in the WT1-positive Ewing sarcoma cell line, MHH-ES. Suppression of WT1 in MHH-ES cells impairs angiogenesis, while expression of WT1 in SK-ES-1 cells causes increased angiogenesis. Different WT1 isoforms result in vessels with distinct morphologies, and this correlates with preferential upregulation of particular VEGF isoforms. WT1-expressing tumors show increased expression of pro-angiogenic molecules such as VEGF, MMP9, Ang-1, and Tie-2, supporting the hypothesis that WT1 is a global regulator of angiogenesis. We also demonstrate that WT1 regulates the expression of a panel of pro-angiogenic molecules in Ewing sarcoma cell lines. Finally, we found that WT1 expression is correlated with VEGF expression, MMP9 expression, and microvessel density in samples of primary Ewing sarcoma. Thus, our results demonstrate that WT1 expression directly regulates tumor angiogenesis by controlling the expression of a panel of pro-angiogenic genes.
Assuntos
Neoplasias Ósseas/irrigação sanguínea , Metaloproteinase 9 da Matriz/metabolismo , Neovascularização Patológica/metabolismo , Sarcoma de Ewing/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas WT1/metabolismo , Animais , Apoptose , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Movimento Celular , Proliferação de Células , Imunoprecipitação da Cromatina , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas WT1/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In cancer patients, the development of resistance to anti-angiogenic agents targeting the VEGF pathway is common. Increased pericyte coverage of the tumor vasculature undergoing VEGF targeted therapy has been suggested to play an important role in resistance. Therefore, reducing the pericytes coverage of the tumor vasculature has been suggested to be a therapeutic approach in breaking the resistance to and increasing the efficacy of anti-angiogenic therapies. To screen compound libraries, a simple in vitro assay of blood vessel maturation demonstrating endothelial cells and pericytes association while forming lumenized vascular structures is needed. Unfortunately, previously described 3-dimensional, matrix based assays are laborious and challenging from an image and data acquisition perspective. For these reasons they generally lack the scalability needed to perform in a high-throughput environment. With this work, we have developed a novel in vitro blood vessel maturation assay, in which lumenized, vascular structures form in one optical plane and mesenchymal progenitor cells (10T1/2) differentiate into pericyte-like cells, which associate with the endothelial vessels (HUVECs). The differentiation of the 10T1/2 cells into pericyte-like cells is visualized using a GFP reporter controlled by the alpha smooth muscle actin promoter (SMP-8). The organization of these vascular structures and their recruited mural cells in one optical plane allows for automated data capture and subsequent image analysis. The ability of this assay to screen for inhibitors of pericytes recruitment was validated. In summary, this novel assay of in vitro blood vessel maturation provides a valuable tool to screen for new agents with therapeutic potential.
Assuntos
Inibidores da Angiogênese/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Avaliação Pré-Clínica de Medicamentos/métodos , Actinas/metabolismo , Animais , Benzamidas/farmacologia , Linhagem Celular , Técnicas de Cocultura , Células Endoteliais/citologia , Fibroblastos/citologia , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Mesilato de Imatinib , Indóis/farmacologia , Lentivirus/metabolismo , Camundongos , Músculo Liso/metabolismo , Neovascularização Fisiológica , Pericitos/citologia , Piperazinas/farmacologia , Regiões Promotoras Genéticas , Pirimidinas/farmacologia , Pirróis/farmacologia , Células-Tronco/citologia , SunitinibeRESUMO
Tasquinimod is an orally active antiangiogenic drug that is currently in phase III clinical trials for the treatment of castration-resistant prostate cancer. However, the target of this drug has remained unclear. In this study, we applied diverse strategies to identify the histone deacetylase HDAC4 as a target for the antiangiogenic activity of tasquinimod. Our comprehensive analysis revealed allosteric binding (Kd 10-30 nmol/L) to the regulatory Zn(2+) binding domain of HDAC4 that locks the protein in a conformation preventing HDAC4/N-CoR/HDAC3 complex formation. This binding inhibited colocalization of N-CoR/HDAC3, thereby inhibiting deacetylation of histones and HDAC4 client transcription factors, such as HIF-1α, which are bound at promoter/enhancers where epigenetic reprogramming is required for cancer cell survival and angiogenic response. Through this mechanism, tasquinimod is effective as a monotherapeutic agent against human prostate, breast, bladder, and colon tumor xenografts, where its efficacy could be further enhanced in combination with a targeted thapsigargin prodrug (G202) that selectively kills tumor endothelial cells. Together, our findings define a mechanism of action of tasquinimod and offer a perspective on how its clinical activity might be leveraged in combination with other drugs that target the tumor microenvironment. Cancer Res; 73(4); 1386-99. ©2012 AACR.