RESUMO
Head and neck cancer etiology and architecture is quite diverse and complex, impeding the prediction whether a patient could respond to a particular cancer immunotherapy or combination treatment. A concomitantly arising caveat is obviously the translation from pre-clinical, cell based in vitro systems as well as syngeneic murine tumor models towards the heterogeneous architecture of the human tumor ecosystems. To bridge this gap, we have established and employed a patient-derived HNSCC (head and neck squamous cell carcinoma) slice culturing system to assess immunomodulatory effects as well as permissivity and oncolytic virus (OV) action. The heterogeneous contexture of the human tumor ecosystem including tumor cells, cancer-associated fibroblasts and immune cells was preserved in our HNSCC slice culturing approach. Importantly, the immune cell compartment remained to be functional and cytotoxic T-cells could be activated by immunostimulatory antibodies. In addition, we uncovered that a high proportion of the patient-derived HNSCC slice cultures were susceptible to the OV VSV-GP. More specifically, VSV-GP infects a broad spectrum of tumor-associated lineages including epithelial and stromal cells and can induce apoptosis. In sum, this human tumor ex vivo platform might complement pre-clinical studies to eventually propel cancer immune-related drug discovery and ease the translation to the clinics.
Assuntos
Neoplasias de Cabeça e Pescoço , Vírus Oncolíticos , Animais , Ecossistema , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Imunoterapia , Camundongos , Vírus Oncolíticos/fisiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapiaRESUMO
In vivo studies are the mainstay of translational immune-oncology and virotherapy research. In general oncology, bioluminescence imaging provides a convenient and reliable tool to visualize disseminated tumors and monitor growth kinetics or treatment effects. Unique aspects of this method in the field of oncolytic viruses are tracing the process of tumor-specific targeting, assessing potential off-target replication, and visualizing intratumoral spread. In addition, the longitudinal monitoring of virus activity kinetics over time is a very powerful feature supporting the subsequent, often elaborate, preclinical biodistribution and pharmtox program. Here we present a step-by-step standard imaging protocol used in our group for both tumor and virus monitoring, along with background information and general principles that should allow the reader to modify and adapt the protocol according to their needs.
Assuntos
Terapia Genética , Vetores Genéticos/genética , Imagem Molecular , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Animais , Análise de Dados , Modelos Animais de Doenças , Expressão Gênica , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Processamento de Imagem Assistida por Computador , Medições Luminescentes , Camundongos , Imagem Molecular/métodos , Neoplasias/diagnóstico , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Software , Distribuição Tecidual , Transgenes , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Oncolytic virotherapy is thought to result in direct virus-induced lytic tumour killing and simultaneous activation of innate and tumour-specific adaptive immune responses. Using a chimeric vesicular stomatitis virus variant VSV-GP, we addressed the direct oncolytic effects and the role of anti-tumour immune induction in the syngeneic mouse lung cancer model LLC1. METHODS: To study a tumour system with limited antiviral effects, we generated interferon receptor-deficient cells (LLC1-IFNAR1-/-). Therapeutic efficacy of VSV-GP was assessed in vivo in syngeneic C57BL/6 and athymic nude mice bearing subcutaneous tumours. VSV-GP treatment effects were analysed using bioluminescent imaging (BLI), immunohistochemistry, ELISpot, flow cytometry, multiplex ELISA and Nanostring® assays. RESULTS: Interferon insensitivity correlated with VSV-GP replication and therapeutic outcome. BLI revealed tumour-to-tumour spread of viral progeny in bilateral tumours. Histological and gene expression analysis confirmed widespread and rapid infection and cell killing within the tumour with activation of innate and adaptive immune-response markers. However, treatment outcome was increased in the absence of CD8+ T cells and surviving mice showed little protection from tumour re-challenge, indicating limited therapeutic contribution by the activated immune system. CONCLUSION: These studies present a case for a predominantly lytic treatment effect of VSV-GP in a syngeneic mouse lung cancer model.
Assuntos
Carcinoma Pulmonar de Lewis/terapia , Neoplasias Pulmonares/terapia , Terapia Viral Oncolítica/métodos , Vesiculovirus , Imunidade Adaptativa/imunologia , Animais , Antígenos Virais/genética , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Lewis/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Quimera , Citocinas/imunologia , Técnicas de Inativação de Genes , Imunidade Inata/imunologia , Técnicas In Vitro , Interferon Tipo I/imunologia , Interferon-alfa/imunologia , Interferon gama/imunologia , Neoplasias Pulmonares/genética , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Nus , Receptor de Interferon alfa e beta/genética , Vesiculovirus/genética , Vesiculovirus/imunologia , Proteínas do Envelope Viral/genética , Proteínas Virais/genéticaRESUMO
Oncolytic viruses, including the oncolytic rhabdovirus VSV-GP tested here, selectively infect and kill cancer cells and are a promising new therapeutic modality. Our aim was to study the efficacy of VSV-GP, a vesicular stomatitis virus carrying the glycoprotein of lymphocytic choriomeningitis virus, against prostate cancer, for which current treatment options still fail to cure metastatic disease. VSV-GP was found to infect 6 of 7 prostate cancer cell lines with great efficacy. However, susceptibility was reduced in one cell line with low virus receptor expression and in 3 cell lines after interferon alpha treatment. Four cell lines had developed resistance to interferon type I at different levels of the interferon signaling pathway, resulting in a deficient antiviral response. In prostate cancer mouse models, long-term remission was achieved upon intratumoral and, remarkably, also upon intravenous treatment of subcutaneous tumors and bone metastases. These promising efficacy data demonstrate that treatment of prostate cancer with VSV-GP is feasible and safe in preclinical models and encourage further preclinical and clinical development of VSV-GP for systemic treatment of metastatic prostate cancer.
Assuntos
Efeito Citopatogênico Viral , Modelos Animais de Doenças , Terapia Viral Oncolítica , Neoplasias da Próstata/terapia , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Apoptose , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias da Próstata/patologia , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
COPII vesicles mediate export of secretory cargo from the endoplasmic reticulum (ER). However, a standard COPII vesicle with a diameter of 60-90 nm is too small to export collagens that are composed of rigid triple helices of up to 400 nm in length. How do cells pack and secrete such bulky molecules? This issue is fundamentally important, as collagens constitute approximately 25% of our dry body weight and are essential for almost all cell-cell interactions. Recently, a potential mechanism for the biogenesis of mega-transport carriers was identified, involving packing collagens and increasing the size of COPII coats. Packing is mediated by TANGO1, which binds procollagen VII in the lumen and interacts with the COPII proteins Sec23/Sec24 on the cytoplasmic side of the ER. Cullin3, an E3 ligase, and its specific adaptor protein, KLHL12, ubiquitinate Sec31, which could increase the size of COPII coats. Recruitment of these proteins and their specific interactors into COPII-mediated vesicle biogenesis may be all that is needed for the export of bulky collagens from the ER. Nonetheless, we present an alternative pathway in which TANGO1 and COPII cooperate to export collagens without generating a mega-transport carrier.
Assuntos
Colágeno/metabolismo , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/metabolismoRESUMO
Collagens are secreted into the extracellular space where they assemble into a large complex protein network to form basement membrane and extracellular matrix. Collagens are therefore essential for cell attachment, tissue organization and the overall survival of all multicellular organisms. Collagens are synthesized in the endoplasmic reticulum (ER) but they are too big to fit into a conventional coat protein complex II (COPII) transport carrier of 60-90 nm average diameter. How are these molecules exported from the ER and then transported along the secretory pathway? We describe here the involvement of special packing machinery composed of hetero oligomers of transport and Golgi organization 1 (TANGO1) and cutaneous T-cell lymphoma-associated antigen 5 (cTAGE5) in the export of procollagen VII from the ER.
Assuntos
Retículo Endoplasmático/metabolismo , Pró-Colágeno/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Humanos , Fusão de Membrana , Transporte ProteicoRESUMO
TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18.DOI: http://dx.doi.org/10.7554/eLife.02784.001.
Assuntos
Retículo Endoplasmático/metabolismo , Pró-Colágeno/química , Proteínas Qa-SNARE/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Células Cultivadas , Clonagem Molecular , Células HeLa , Humanos , Fusão de Membrana , Microscopia de Fluorescência , Pró-Colágeno/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas SNARE/metabolismo , TransfecçãoRESUMO
COPII vesicles mediate the export of secretory cargo from endoplasmic reticulum (ER) exit sites. However, of 60-90 nm diameter COPII vesicles are too small to accommodate secreted molecules such as the collagens. The ER exit site-located proteins TANGO1 and cTAGE5 are required for the transport of collagens and therefore provide a means to understand the export of big cargo and the mechanism of COPII carrier size regulation commensurate with cargo dimensions.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Colágeno/metabolismo , Retículo Endoplasmático/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Complexo de Golgi/metabolismo , Humanos , Camundongos , Proteínas de Neoplasias/metabolismo , Transporte Proteico/fisiologiaRESUMO
Cutaneous T-cell lymphoma-associated antigen 5 (cTAGE5), an originally identified tumor antigen, is overexpressed in various cancer cell lines. The cDNA encodes an integral membrane protein containing two coiled-coil motifs and a proline-rich domain. We show that cTAGE5 specifically localizes to the endoplasmic reticulum (ER) exit sites. In addition, cTAGE5 forms a complex with TANGO1 (MIA3), a previously characterized cargo receptor for collagen VII, by the interaction of their coiled-coil motifs. Of interest, cTAGE5, as well as TANGO1, is capable of interacting with the inner-layer coatomer of COPII Sec23/24 complex through their C-terminal proline-rich domains and required for collagen VII secretion. We propose that cTAGE5 acts as a coreceptor of TANGO1 for collagen VII export from the ER.
Assuntos
Antígenos de Neoplasias/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Colágeno/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Neoplasias/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Transformada , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica , Transporte Proteico , Receptores de Superfície Celular/metabolismoRESUMO
Deleted in liver cancer 1 (DLC1) is a tumor suppressor protein that is frequently downregulated in various tumor types. DLC1 contains a Rho GTPase activating protein (GAP) domain that appears to be required for its tumor suppressive functions. Little is known about the molecular mechanisms that regulate DLC1. By mass spectrometry we have mapped a novel phosphorylation site within the DLC1 GAP domain on serine 807. Using a phospho-S807-specific antibody, our results identify protein kinase D (PKD) to phosphorylate this site in DLC1 in intact cells. Although phosphorylation on serine 807 did not directly impact on in vitro GAP activity, a DLC1 serine-to-alanine exchange mutant inhibited colony formation more potently than the wild type protein. Our results thus show that PKD-mediated phosphorylation of DLC1 on serine 807 negatively regulates DLC1 cellular function.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteína Quinase C/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Espectrometria de Massas , FosforilaçãoRESUMO
Deleted in Liver Cancer 1 (DLC1) is a GTPase-activating protein (GAP) with specificity for RhoA, RhoB, and RhoC that is frequently deleted in various tumor types. By inactivating these small GTPases, DLC1 controls actin cytoskeletal remodeling and biological processes such as cell migration and proliferation. Here we provide evidence that DLC1 binds to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) through a previously unrecognized polybasic region (PBR) adjacent to its RhoGAP domain. Importantly, PI(4,5)P(2)-containing membranes are shown to stimulate DLC1 GAP activity in vitro. In living cells, a DLC1 mutant lacking an intact PBR inactivated Rho signaling less efficiently and was severely compromised in suppressing cell spreading, directed migration, and proliferation. We therefore propose that PI(4,5)P(2) is an important cofactor in DLC1 regulation in vivo and that the PBR is essential for the cellular functions of the protein.
Assuntos
Lipídeos de Membrana/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Linhagem Celular , Sequência Conservada , Ensaio de Imunoadsorção Enzimática , Proteínas Ativadoras de GTPase , Guanosina Trifosfato/fisiologia , Humanos , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The phosphatase and tensin homolog (PTEN) gene is a tumor suppressor frequently deleted or mutated in sporadic tumors of the breast, prostate, endometrium and brain. The protein acts as a dual specificity phosphatase for lipids and proteins. PTEN loss confers a growth advantage to cells, protects from apoptosis and favors cell migration. The deleted in liver cancer 1 (DLC1) gene has emerged as a novel tumor suppressor downregulated in a variety of tumor types including those of the breast. DLC1 contains a Rho GTPase activating domain that is involved in the inhibition of cell proliferation, migration and invasion. To investigate how simultaneous loss of PTEN and DLC1 contributes to cell transformation, we downregulated both proteins by RNA interference in the non-invasive MCF7 breast carcinoma cell line. Joint depletion of PTEN and DLC1 resulted in enhanced cell migration in wounding and chemotactic transwell assays. Interestingly, both proteins were found to colocalize at the plasma membrane and interacted physically in biochemical pulldowns and coimmunoprecipitations. We therefore postulate that the concerted local inactivation of signaling pathways downstream of PTEN and DLC1, respectively, is required for the tight control of cell migration.
Assuntos
Neoplasias da Mama , Movimento Celular/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Proliferação de Células , Ativação Enzimática , Feminino , Quinase 1 de Adesão Focal/metabolismo , Forminas , Proteínas Ativadoras de GTPase , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica , PTEN Fosfo-Hidrolase/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genéticaRESUMO
Deleted in liver cancer 1 (DLC1) is a Rho-GTPase-activating protein (GAP) that is downregulated in various tumor types. In vitro, DLC1 specifically inactivates the small GTPases RhoA, RhoB and RhoC through its GAP domain and this appears to contribute to its tumor suppressor function in vivo. Molecular mechanisms that control DLC1 activity have not so far been investigated. Here, we show that phorbol-ester-induced activation of protein kinase C and protein kinase D stimulates association of DLC1 with the phosphoserine/phosphothreonine-binding 14-3-3 adaptor proteins via recognition motifs that involve Ser327 and Ser431. Association with 14-3-3 proteins inhibits DLC1 GAP activity and facilitates signaling by active Rho. We further show that treatment of cells with phorbol ester or coexpression of 14-3-3 proteins, blocks DLC1 nucleocytoplasmic shuttling, probably by masking a previously unrecognized nuclear localization sequence. The binding to 14-3-3 proteins is thus a newly discovered mechanism by which DLC1 activity is regulated and compartmentalized.
Assuntos
Proteínas 14-3-3/metabolismo , Núcleo Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas 14-3-3/genética , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Ativação Enzimática , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Humanos , Dados de Sequência Molecular , Ésteres de Forbol/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Proteína Quinase C/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Deleted in liver cancer (DLC) 1 and 2 are Rho GTPase-activating proteins that are frequently down-regulated in various types of cancer. Ectopic expression in carcinoma cell lines lacking these proteins has been shown to inhibit cell migration and invasion. However, whether the loss of DLC1 or DLC2 is the cause of aberrant Rho signaling in transformed cells has not been investigated. Here, we have down-regulated DLC1 and DLC2 expression in breast cancer cells using a RNA interference approach. Silencing of DLC1 led to the stabilization of stress fibers and focal adhesions and enhanced cell motility in wound-healing as well as chemotactic Transwell assays. We provide evidence that enhanced migration of cells lacking DLC1 is dependent on the Rho effector protein Dia1 but does not require the activity of Rho kinase. By contrast, DLC2 knockdown failed to affect the migratory behavior of cells, suggesting that the two proteins have distinct functions. This is most likely due to their differential subcellular localizations, with DLC1 found in focal adhesions and DLC2 being mainly cytosolic. Collectively, our data show that DLC1 is critically involved in the control of Rho signaling and actin cytoskeleton remodeling and that its cellular loss is sufficient for the acquisition of a more migratory phenotype of breast cancer cells.