RESUMO
Insulinomas (INS) are the most common human and canine functioning pancreatic neuroendocrine tumours. The long-term prognosis for malignant INS is poor, because micrometastases are frequently missed during surgery. As human and canine malignant INS share clinical and histopathological features, dogs have been proposed as models for INS research. Using RNA-sequencing, we conducted a pilot study to better understand the underlying molecular mechanisms of canine INS. Normal canine pancreas and lymph node control tissues were compared with primary INS and INS-metastatic lymph nodes, revealing more than 3,000 genes differentially expressed in normal pancreas compared to primary INS. Only 164 genes were differentially expressed between primary INS and INS-metastatic lymph nodes. Hierarchical clustering analysis demonstrated similar genetic profiles in normal pancreas and early clinical stage primary INS, whereas late clinical stage primary INS resembled the genetic profile of INS-metastatic lymph nodes. These findings suggest that markers of malignant behaviour could be identified at the primary site of the disease. Finally, using the REACTOME pathways database, we revealed that an active collagen metabolism, extracellular matrix remodelling, beta-cell differentiation and non-beta-cell trans-differentiation might cause disease progression and hyperinsulinism in INS, identifying major pathways worthy of future research in this currently poorly controlled disease.
Assuntos
Doenças do Cão/genética , Insulinoma/genética , Proteínas de Neoplasias/genética , Transcriptoma/genética , Animais , Progressão da Doença , Doenças do Cão/patologia , Cães , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Insulinoma/patologia , Insulinoma/veterinária , Metástase Neoplásica , Análise de Sequência de RNARESUMO
OBJECTIVES: Oral malignant melanomas carry a poor-to-guarded prognosis because of their local invasiveness and high metastatic propensity. The Oncept melanoma vaccine is licensed to treat dogs with stage II or III locally-controlled oral malignant melanoma and this retrospective study aimed to assess survival of affected dogs treated with the vaccine in the UK. MATERIAL AND METHODS: Medical records of dogs with histopathologically-confirmed oral malignant melanoma that received the vaccine as part of their treatment were evaluated. Survival analyses for potential prognostic factors were performed. RESULTS: Sixty-nine dogs were included; 56 dogs, staged I to III, and with previous locoregional therapy, had a median survival time of 455 days (95% CI: 324 to 586 days). Based on Kaplan-Meier survival analysis with associated log-rank testing, no significant prognostic factors were identified for this population. Of the 13 patients with macroscopic disease treated with vaccine alone or in combination therapy, eight showed clinical response. Three patients with stage IV oral malignant melanoma survived 171, 178 and 288 days from diagnosis. CLINICAL SIGNIFICANCE: Patients treated with the melanoma vaccine in our study had survival times similar to their counterparts receiving the vaccine in the USA. There were observed responses in patients with macroscopic disease and so the vaccine could be considered as palliative treatment in dogs with stage IV disease.
Assuntos
Vacinas Anticâncer/uso terapêutico , Doenças do Cão/tratamento farmacológico , Melanoma/tratamento farmacológico , Neoplasias Bucais/veterinária , Neoplasias Cutâneas/tratamento farmacológico , Animais , Cães , Feminino , Masculino , Neoplasias Bucais/tratamento farmacológico , Prognóstico , Estudos Retrospectivos , Melanoma Maligno CutâneoRESUMO
Screening for expression of the high affinity receptor for IgE by reverse transcriptase PCR, revealed that almost all canine mast cell tumors expressed FcÉRIα mRNA, supporting the rationale for developing anti-neoplastic treatments based on molecules that could target this receptor. Use of cytotoxic cytokines to trigger an apoptotic signal is one strategy for inducing cell death in malignant mast cells. The coding sequences for canine IgE and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were identified through genome analyses. Selected regions of the coding sequences for these genes were cloned and compared to the predicted genome sequences. The Fc region of canine IgE, death domain of canine TRAIL and an IgE Fc: TRAIL fusion construct were generated and epitope-tagged proteins expressed, using a eukaryotic expression system. Specific binding of recombinant canine IgE Fc-containing proteins to recombinant human FcÉRIα and to a canine mast cell tumor line expressing FcÉRIα (C2), but not one failing to express FcÉRIα (MCLA), was demonstrated. Specific binding of the IgE: TRAIL fusion protein was not abrogated by the TRAIL moiety. These results are proof of principle that canine IgE targeting to FcÉRIα can be used as a platform for selective delivery of therapies to FcÉRIα-expressing cells, potentially enhancing their therapeutic index and efficacy.
Assuntos
Doenças do Cão/metabolismo , Mastócitos/metabolismo , Receptores de IgE/metabolismo , Neoplasias Cutâneas/veterinária , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose/fisiologia , Biópsia/veterinária , Western Blotting/veterinária , Linhagem Celular Tumoral , Clonagem Molecular , Doenças do Cão/imunologia , Doenças do Cão/patologia , Cães , Feminino , Citometria de Fluxo , Masculino , Mastócitos/imunologia , Mastócitos/patologia , RNA/química , RNA/genética , Receptores de IgE/genética , Receptores de IgE/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/imunologiaRESUMO
Canine mast cell tumours often express KIT mutations that result in constitutive activation of the c-kit receptor and which are associated with more aggressive disease. The aim of the current study was to determine whether small inhibitory RNA (SiRNA) molecules could specifically target canine KIT mRNA for knock-down. Canine beta-2 microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and KIT sequences were cloned into the psiCHECK™-2 vector. SiRNA molecules, designed to target gene-specific sequences, were co-transfected with plasmid DNA into Chinese hamster ovary (CHO) cells. Renilla and firefly luciferase activity was measured using the Dual-GLO(®) Luciferase Assay (Promega). Using this reporter system, canine housekeeping gene-specific SiRNA molecules demonstrated knockdown of their targets (72.0% knockdown for B2M and 94.5% knockdown for GAPDH). An SiRNA molecule targeting exon 2 of canine KIT successfully knocked-down reporter gene expression of a KIT(26-407) construct (90.8% knockdown). An SiRNA molecule targeting a 48 base-pair in-tandem duplication mutation in KIT exon 11 selectively knocked down expression of the KIT(1569-1966mutant) construct (93.1% knockdown) but had no effect on the KIT(1569-1918wild-type) construct. The results show that RNA interference can be used to inhibit canine KIT mRNA expression and has the potential to selectively target the mutant version of KIT that is expressed by some malignant mast cells.
Assuntos
Cães/genética , Técnicas de Silenciamento de Genes/veterinária , Proteínas Proto-Oncogênicas c-kit/genética , Interferência de RNA , Animais , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes/métodos , Lipase , RNA Interferente Pequeno/genética , Transfecção/veterináriaRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which preferentially induces apoptosis in cells that have undergone malignant transformation. In humans, non-neoplastic cells are normally protected from the effects of TRAIL by expressing decoy receptors, lacking death domains. In contrast, neoplastic cells tend to downregulate their decoy receptor expression, increasing their susceptibility to the pro-apoptotic effects of TRAIL, via the functional TRAIL receptors. The aim of the current study was to investigate the effect of TRAIL on the canine C2 mastocytoma cell line to determine whether this agent might be a suitable treatment for mast cell tumors in dogs. C2 and MDCK cells were cultured with recombinant human TRAIL. Apoptosis was assessed using a Caspase 3 and 7 chemiluminescence assay and flow cytometry following Annexin V:FITC labelling. Cell metabolism was assessed using a colorimetric MTT-based assay. C2 cells demonstrated greater sensitivity to TRAIL-induced apoptosis compared to MDCK cells by all assessment methods. The dog genome assembly was searched for orthologs of TRAIL and its receptors using published sequences from other species for reference. Although a canine ortholog for TRAIL was identified, only one TRAIL receptor ortholog (TNFRSF11B) could be found. C2, but not MDCK, cells expressed mRNA for TNFRSF11B, detected by RT-PCR. In other species, TNFRSF11B is a decoy receptor, as even though it has a death domain it is secreted due to its lack of a transmembrane domain. The effect of TRAIL on the C2 cell line suggests that this cytokine might be suitable for treatment of mast cell tumors in dogs.