RESUMEN
Precise vascular patterning is crucial for normal growth and development. The ERG transcription factor drives Delta-like ligand 4 (DLL4)/Notch signalling and is thought to act as a pivotal regulator of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC-specific focal adhesion kinase (FAK)-knockout (KO) and point-mutant FAK-knock-in mice, we show that loss of ECFAK, its kinase activity or phosphorylation at FAK-Y397, but not FAK-Y861, reduces ERG and DLL4 expression levels together with concomitant aberrations in vascular patterning. Rapid immunoprecipitation mass spectrometry of endogenous proteins identified that endothelial nuclear-FAK interacts with the deubiquitinase USP9x and the ubiquitin ligase TRIM25. Further in silico analysis confirms that ERG interacts with USP9x and TRIM25. Moreover, ERG levels are reduced in FAKKO ECs via a ubiquitin-mediated post-translational modification programme involving USP9x and TRIM25. Re-expression of ERG in vivo and in vitro rescues the aberrant vessel-sprouting defects observed in the absence of ECFAK. Our findings identify ECFAK as a regulator of retinal vascular patterning by controlling ERG protein degradation via TRIM25/USP9x.
Asunto(s)
Células Endoteliales , Factores de Transcripción , Animales , Células Endoteliales/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Ratones , Neovascularización Fisiológica/genética , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitinas/metabolismoRESUMEN
Tumor-targeting oncolytic viruses such as vaccinia virus (VV) are attractive cancer therapeutic agents that act through multiple mechanisms to provoke both tumor lysis and anti-tumor immune responses. However, delivery of these agents remains restricted to intra-tumoral administration, which prevents effective targeting of inaccessible and disseminated tumor cells. In the present study we have identified transient pharmacological inhibition of the leukocyte-enriched phosphoinositide 3-kinase δ (PI3Kδ) as a novel mechanism to potentiate intravenous delivery of oncolytic VV to tumors. Pre-treatment of immunocompetent mice with the PI3Kδ-selective inhibitor IC87114 or the clinically approved idelalisib (CAL-101), prior to intravenous delivery of a tumor-tropic VV, dramatically improved viral delivery to tumors. This occurred via an inhibition of viral attachment to, but not internalization by, systemic macrophages through perturbation of signaling pathways involving RhoA/ROCK, AKT, and Rac. Pre-treatment using PI3Kδ-selective inhibitors prior to intravenous delivery of VV resulted in enhanced anti-tumor efficacy and significantly prolonged survival compared to delivery without PI3Kδ inhibition. These results indicate that effective intravenous delivery of oncolytic VV may be clinically achievable and could be useful in improving anti-tumor efficacy of oncolytic virotherapy.
Asunto(s)
Adenina/análogos & derivados , Administración Intravenosa/métodos , Antineoplásicos/uso terapéutico , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Inmunoterapia/métodos , Viroterapia Oncolítica/métodos , Virus Oncolíticos/inmunología , Purinas/uso terapéutico , Quinazolinas/uso terapéutico , Quinazolinonas/uso terapéutico , Virus Vaccinia/inmunología , Adenina/farmacología , Adenina/uso terapéutico , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular , Terapia Combinada/métodos , Femenino , Ratones , Ratones Endogámicos BALB C , Purinas/farmacología , Quinazolinas/farmacología , Quinazolinonas/farmacología , Trasplante Homólogo , Resultado del Tratamiento , Carga TumoralRESUMEN
Oncolytic viruses based on adenovirus type 5 (Ad5) have been developed as a new class of therapeutic agents for cancers that are resistant to conventional therapies. Clinical experience shows that these agents are safe, but virotherapy alone has not achieved long-term cure in cancer patients. The vast majority of oncolytic adenoviruses used in clinical trials to date have deletion of the E3B genes. It has been demonstrated that the antitumor potency of the E3B-deleted mutant (dl309) is inferior to adenovirus with E3B genes intact. Tumors treated with dl309 show markedly greater macrophage infiltration than E3B-intact adenovirus. However, the functional mechanisms for this were not previously known. Here, we demonstrate that deletion of E3B genes increases production of chemokines by monocytes after adenovirus infection and increases monocyte migration. The E3B 14,700-Da protein (E3B-14.7K) inhibits STAT1 function by preventing its phosphorylation and nuclear translocation. The STAT1 inhibitor, fludarabine, rescues the effect of E3B-14.7K deletion by downregulating target chemokine expression in human and murine monocytes and results in an enhanced antitumor efficacy with dl309 in vivo. These findings have important implications for clinical use of E3B-deleted oncolytic adenovirus and other E3B-deleted adenovirus vector-based therapy.
Asunto(s)
Adenoviridae/fisiología , Proteínas E3 de Adenovirus/metabolismo , Monocitos/metabolismo , Virus Oncolíticos/fisiología , Factor de Transcripción STAT1/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Adenoviridae/metabolismo , Proteínas E3 de Adenovirus/genética , Análisis de Varianza , Animales , Western Blotting , Línea Celular , ADN Complementario/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Eliminación de Gen , Humanos , Inmunoprecipitación , Ratones , Microscopía Confocal , Virus Oncolíticos/metabolismo , Fosforilación/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción STAT1/antagonistas & inhibidores , Vidarabina/análogos & derivados , Vidarabina/farmacologíaRESUMEN
Vaccinia virus (VV) is an enveloped DNA virus from the poxvirus family and has played a crucial role in the eradication of smallpox. It continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer. However, the mechanisms of poxvirus entry, the host factors that affect viral virulence, and the reasons for its natural tropism for tumor cells are incompletely understood. By studying the effect of hypoxia on VV infection, we found that vascular endothelial growth factor A (VEGF-A) augments oncolytic VV cytotoxicity. VEGF derived from tumor cells acts to increase VV internalization, resulting in increased replication and cytotoxicity in an AKT-dependent manner in both tumor cells and normal respiratory epithelial cells. Overexpression of VEGF also enhances VV infection within tumor tissue in vivo after systemic delivery. These results highlight the importance of VEGF expression in VV infection and have potential implications for the design of new strategies to prevent poxvirus infection and the development of future generations of oncolytic VV in combination with conventional or biological therapies.
Asunto(s)
Proteínas Proto-Oncogénicas c-akt/metabolismo , Virus Vaccinia/patogenicidad , Factor A de Crecimiento Endotelial Vascular/metabolismo , Internalización del Virus , Animales , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/virología , Línea Celular Tumoral , Células Epiteliales/virología , Genes Reporteros , Humanos , Hipoxia , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Interferencia de ARN , ARN Interferente Pequeño , Mucosa Respiratoria/virología , Vaccinia/metabolismo , Vaccinia/virología , Virus Vaccinia/genética , Factor A de Crecimiento Endotelial Vascular/genética , Tropismo Viral , Replicación Viral/genéticaRESUMEN
Oncolytic adenoviruses based on serotype 5 (Ad5) have several shortcomings, including the downregulation of its receptor in cancer cells, high prevalence of neutralizing antibodies and hepatotoxicity. Another adenoviral serotype, Ad11, could overcome these obstacles. Here, we show that human cancer cell lines express higher levels of the Ad11 receptor CD46, resulting in much better infectivity than Ad5. Surprisingly, only 36% (9/25) of the cell lines were more sensitive to Ad11- than to Ad5-mediated cytotoxicity. Investigations revealed that it was the transcription of Ad11 E1A, not CD46 expression or virus infectivity, which determined the cell's sensitivity to Ad11 killing. Ad11 E1A mRNA levels have an effect on viral DNA replication, structural protein synthesis and infectious particle production. To test the hypothesis that increased E1A transcription would lead to improved Ad11 replication in Ad5-sensitive (but Ad11-less sensitive) cells, two Ad11 mutants (Ad11-Ad5-P and Ad11-Ad5-EP) were constructed where either the E1A promoter or enhancer-promoter, respectively, was replaced by that of Ad5. Ad11-Ad5-EP demonstrated increased E1A mRNA levels and replication, together with enhanced oncolytic potency in vitro and in vivo. This effect was found in both the Ad5-sensitive and Ad11-sensitive cancer cells, broadening the range of tumors that could be effectively killed by Ad11-Ad5-EP.
Asunto(s)
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Elementos de Facilitación Genéticos , Vectores Genéticos/genética , Virus Oncolíticos/genética , Regiones Promotoras Genéticas , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Efecto Citopatogénico Viral/genética , Desmogleína 2/genética , Vectores Genéticos/administración & dosificación , Humanos , Proteína Cofactora de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/genética , Neoplasias/mortalidad , Neoplasias/terapia , Viroterapia Oncolítica , Análisis de Supervivencia , Transcripción Genética , Replicación Viral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Pancreatic cancer remains one of the most lethal cancers and is refractory to immunotherapeutic interventions. Oncolytic viruses are a promising new treatment option, but current platforms demonstrate limited efficacy, especially for inaccessible and metastatic cancers that require systemically deliverable therapies. We recently described an oncolytic vaccinia virus (VV), VVLΔTKΔN1L, which has potent antitumor activity, and a regime to enhance intravenous delivery of VV by pharmacological inhibition of pharmacological inhibition of PI3 Kinase δ (PI3Kδ) to prevent virus uptake by macrophages. While these platforms improve the clinical prospects of VV, antitumor efficacy must be improved. METHODS: VVLΔTKΔN1L was modified to improve viral spread within and between tumors via viral B5R protein modification, which enhanced production of the extracellular enveloped virus form of VV. Antitumor immunity evoked by viral treatment was improved by arming the virus with interleukin-21, creating VVL-21. Efficacy, functional activity and synergy with α-programmed cell death protein 1 (α-PD1) were assessed after systemic delivery to murine and Syrian hamster models of pancreatic cancer. RESULTS: VVL-21 could reach tumors after systemic delivery and demonstrated antitumor efficacy in subcutaneous, orthotopic and disseminated models of pancreatic cancer. The incorporation of modified B5R improved intratumoural accumulation of VV. VVL-21 treatment increased the numbers of effector CD8+ T cells within the tumor, increased circulating natural killer cells and was able to polarize macrophages to an M1 phenotype in vivo and in vitro. Importantly, treatment with VVL-21 sensitized tumors to the immune checkpoint inhibitor α-PD1. CONCLUSIONS: Intravenously administered VVL-21 successfully remodeled the suppressive tumor-microenvironment to promote antitumor immune responses and improve long-term survival in animal models of pancreatic cancer. Importantly, treatment with VVL-21 sensitized tumors to the immune checkpoint inhibitor α-PD1. Combination of PI3Kδ inhibition, VVL-21 and α-PD1 creates an effective platform for treatment of pancreatic cancer.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Interleucina-12/genética , Glicoproteínas de Membrana/metabolismo , Neoplasias Pancreáticas/terapia , Inhibidores de Proteínas Quinasas/administración & dosificación , Virus Vaccinia/fisiología , Proteínas del Envoltorio Viral/metabolismo , Administración Intravenosa , Animales , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Terapia Combinada , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Interleucina-12/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Mesocricetus , Ratones , Viroterapia Oncolítica , Virus Oncolíticos/fisiología , Neoplasias Pancreáticas/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Microambiente Tumoral , Proteínas del Envoltorio Viral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The efficacy of oncolytic viruses depends on multiple actions including direct tumor lysis, modulation of tumor perfusion, and stimulation of tumor-directed immune responses. In this study, we investigated whether a sequential combination of immunologically distinct viruses might enhance antitumor efficacy through the induction of tumor-specific immunity and circumvention or mitigation of antiviral immune responses. EXPERIMENTAL DESIGN: The Syrian hamster as an immune-competent model that supports replication of both adenovirus and vaccinia virus was evaluated in vitro and in vivo. The antitumor efficacy of either virus alone or sequential combination of the two viruses was examined in pancreatic and kidney cancer models. The functional mechanism of the regimen developed here was investigated by histopathology, immunohistochemistry staining, CTL assay, and T-cell depletion. RESULTS: The Syrian hamster is a suitable model for assessment of oncolytic adenovirus and vaccinia virus. Three low doses of adenovirus followed by three low doses of vaccinia virus resulted in a superior antitumor efficacy to the reverse combination, or six doses of either virus alone, against pancreatic and kidney tumors in Syrian hamsters. A total of 62.5% of animals bearing either tumor type treated with the sequential combination became tumor-free, accompanied by the induction of effective tumor-specific immunity. This enhanced efficacy was ablated by CD3+ T-cell depletion but was not associated with humoral immunity against the viruses. CONCLUSION: These findings show that sequential treatment of tumors with oncolytic adenovirus and vaccinia virus is a promising approach for cancer therapy and that T-cell responses play a critical role.
Asunto(s)
Neoplasias Renales/terapia , Viroterapia Oncolítica , Neoplasias Pancreáticas/terapia , Adenoviridae/genética , Adenoviridae/inmunología , Adenoviridae/fisiología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Apoptosis , Células Cultivadas , Cricetinae , Femenino , Humanos , Inmunocompetencia , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Mesocricetus , Virus Oncolíticos/genética , Virus Oncolíticos/inmunología , Virus Oncolíticos/fisiología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virología , Carga Tumoral , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Virus Vaccinia/fisiología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC.
Asunto(s)
Angiostatinas/genética , Carcinoma de Células Escamosas/terapia , Endostatinas/genética , Vectores Genéticos/genética , Neoplasias de Cabeza y Cuello/terapia , Virus Oncolíticos/genética , Virus Vaccinia/genética , Adenoviridae/genética , Angiostatinas/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Efecto Citopatogénico Viral , Endostatinas/metabolismo , Femenino , Regulación Viral de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Viroterapia Oncolítica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello , Vacunas Virales , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The changes in cancer cell surface molecules and intracellular signaling pathways during tumorigenesis make delivery of adenovirus-based cancer therapies inefficient. Here we have identified carcinoembryonic antigen- related cell adhesion molecule 6 (CEACAM6) as a cellular protein that restricts the ability of adenoviral vectors to infect cancer cells. We have demonstrated that CEACAM6 can antagonize the Src signaling pathway, downregulate cancer cell cytoskeleton proteins, and block adenovirus trafficking to the nucleus of human pancreatic cancer cells. Similar to CEACAM6 overexpression, treatment with a Src-selective inhibitor significantly reduced adenovirus replication in these cancer cells and normal human epithelial cells. In a mouse xenograft tumor model, siRNA-mediated knockdown of CEACAM6 also significantly enhanced the antitumor effect of an oncolytic adenovirus. We propose that CEACAM6-associated signaling pathways could be potential targets for the development of biomarkers to predict the response of patients to adenovirus-based therapies, as well as for the development of more potent adenovirus-based therapeutics.
Asunto(s)
Adenoviridae/fisiología , Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Adenoviridae/ultraestructura , Antígenos CD/genética , Moléculas de Adhesión Celular/genética , Línea Celular , Neoplasias Colorrectales/genética , Citoesqueleto/metabolismo , Regulación hacia Abajo , Proteínas Ligadas a GPI , Regulación Neoplásica de la Expresión Génica , Humanos , Microscopía Electrónica de Transmisión , Neoplasias Pancreáticas/genética , ARN Interferente Pequeño/genética , Especificidad por Sustrato , Familia-src Quinasas/metabolismoRESUMEN
Pancreatic cancer is one of the most aggressive human tumors with a 5-year survival rate of only 3% and a striking resistance to chemotherapy and radiotherapy. The search for new therapeutic approaches includes strategies exploiting the deregulation of apoptotic pathways commonly found in cancer cells. The IAP proteins are inhibitors of apoptosis that have altered activity in numerous cancer types and are implicated in resistance to chemotherapy, and therefore are potentially interesting as therapeutic targets. We investigated alterations in the expression of IAPs and their inhibitors in pancreatic adenocarcinoma by using real-time PCR, in situ hybridization and immunohistochemistry. We found differential expression of various IAPs in this malignancy, and particularly we observed overexpression of cIAP-2, survivin, livin and XIAP. We also looked for correlations between the expression of IAPs and resistance to paclitaxel, doxorubicin, CDDP and 5-fluorouracil, and found that resistance to these drugs correlates most significantly with expression of cIAP-2. Using RNAi to downregulate these proteins we further confirmed that the levels of cIAP-2 and XIAP influence the response to the anti-cancer drugs, although only marginally for 5-FU. We conclude that anti-tumor strategies based on the inhibition of particular IAPs can be useful in targeting pancreatic adenocarcinoma.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/fisiopatología , Área Bajo la Curva , Secuencia de Bases , Cartilla de ADN , Resistencia a Antineoplásicos , Humanos , Inmunohistoquímica , Hibridación in Situ , Proteínas Inhibidoras de la Apoptosis/fisiología , Reacción en Cadena de la Polimerasa , Interferencia de ARNRESUMEN
S100P is a member of the S100 family of calcium-binding proteins and there have been several recent reports of its overexpression in pancreatic ductal adenocarcinoma (PDAC). We have used Far Western screening and in vitro interaction assays to identify and confirm a novel target protein for S100P. We have named this protein S100PBPR, and shown that its interaction with S100P is dependent on Ca(2+) or Mg(2+). S100PBPR was found to localize to cell nuclei where S100P is also present, and the two proteins co-immunoprecipitate. By in situ hybridization, S100PBPR transcript was found in islet cells but not duct cells of the healthy pancreas. Both S100P and S100PBPR were detected by quantitative real-time polymerase chain reaction in pancreatic intraepithelial neoplasia (PanIN) and PDAC samples, and in situ hybridization revealed the presence of S100PBPR transcript in malignant PDAC cells. These data suggest that an interaction between S100P and S100PBPR may be involved in early pancreatic cancer. S100P was further investigated in PanIN lesions and immunohistochemical analysis showed its expression to correlate significantly with increasing grade of PanINs, being found as early as PanIN-1 with more prevalent expression in PanIN-2 and -3. These data suggest that S100P can be added to the genetic progression model for PDAC.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas Portadoras/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Secuencia de Bases , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Clonación Molecular , Cartilla de ADN , Progresión de la Enfermedad , Humanos , Hibridación in Situ , Magnesio/metabolismo , Neoplasias Pancreáticas/patología , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , TransfecciónRESUMEN
BACKGROUND & AIMS: Markers to differentiate among pancreatic adenocarcinoma, chronic pancreatitis, and normal pancreas would be of significant clinical utility. This study was therefore designed to analyze the proteome of such specimens and identify new candidate proteins for differential diagnosis. METHODS: A PowerBlot analysis with more than 900 well-characterized antibodies was performed with tissue specimens from patients with chronic pancreatitis, pancreatic adenocarcinoma, and normal pancreas. Differential expression of selected proteins was confirmed on a larger scale by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry using tissue arrays. RESULTS: A total of 30 and 102 proteins showed significant deregulation between normal pancreas when compared with chronic pancreatitis and pancreatic adenocarcinoma, respectively, and although a substantial proportion were found similarly dysregulated in both chronic pancreatitis and pancreatic adenocarcinoma, several proteins were identified as potential disease-specific markers. CONCLUSIONS: A large number of proteins are differentially expressed in chronic pancreatitis and pancreatic adenocarcinoma compared with normal pancreas. Among these, expression analysis of UHRF1, ATP7A, and aldehyde oxidase 1 in combination could potentially provide a useful additional diagnostic tool for fine-needle aspirated or cytological specimens obtained during endoscopic investigations.
Asunto(s)
Adenocarcinoma/fisiopatología , Neoplasias Pancreáticas/fisiopatología , Pancreatitis Crónica/fisiopatología , Análisis por Matrices de Proteínas , Proteómica , Adenocarcinoma/genética , Biomarcadores de Tumor , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Pancreáticas/genética , Pancreatitis Crónica/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In order to expand our understanding of the molecular changes underlying the complex pathology of pancreatic malignancy, global gene expression profiling of pancreatic adenocarcinoma compared with normal pancreatic tissue was performed. Human cDNA arrays comprising 9932 elements were interrogated with fluorescence-labelled normal and adenocarcinoma samples (nine tumours, three normal pancreata, and three cell lines). The data were analysed for differential gene expression, which was confirmed by serial analysis of gene expression (SAGE), digital differential display (DDD) analysis, and immunohistochemistry for selected cases. The array data were filtered to produce lists of a total of 75 genes significantly up-regulated or down-regulated in pancreatic adenocarcinoma. Two of those showing the highest differential were members of the S100 family of Ca-binding proteins, namely S100P and S100A6, and therefore the S100 genes were studied in more detail. By immunohistochemical analysis of custom-built, pancreas-specific tissue arrays and commercially available, normal/cancer tissue arrays that included a wide variety of different tumour types, differential expression of S100P protein was found to be almost exclusive to pancreatic cancer. S100P could therefore represent a useful biomarker for pancreatic adenocarcinomas.