Raffaella De Maria

met oncogene activation qualifies spontaneous canine osteosarcoma as a suitable pre-clinical model of human osteosarcoma†

The Met receptor tyrosine kinase (RTK) is aberrantly expressed in human osteosarcoma and is an attractive molecular target for cancer therapy. We studied spontaneous canine osteosarcoma (OSA) as a potential pre‐clinical model for evaluation of Met‐targeted therapies. The canine MET oncogene exhibits 90% homology compared with human MET, indicating that cross‐species functional studies are a viable strategy. Expression and activation of the canine Met receptor were studied utilizing immunohistochemical techniques in 39 samples of canine osteosarcoma, including 35 primary tumours and four metastases. Although the Met RTK is barely detectable in primary culture of canine osteoblasts, high expression of Met protein was observed in 80% of canine osteosarcoma samples acquired from various breeds. Met protein overexpression was also concordant with its activation as indicated by phosphorylation of critical tyrosine residues. In addition, Met was expressed and constitutively activated in canine osteosarcoma cell lines. OSA cells expressing high levels of Met demonstrated activation of downstream transducers, elevated spontaneous motility, and invasiveness which were impaired by both a small molecule inhibitor of Met catalytic activity (PHA‐665752) and met‐specific, stable RNA interference obtained by means of lentiviral vector. Similar to observations in human OSA, these data suggest that Met is commonly overexpressed and activated in canine OSA and that inhibition of Met impairs the invasive and motogenic properties of canine OSA cells. These data implicate Met as a potentially important factor for canine OSA progression and indicate that it represents a viable model to study Met‐targeted therapies. Copyright

CSPG4-Specific Immunity and Survival Prolongation in Dogs with Oral Malignant Melanoma Immunized with Human CSPG4 DNA

Purpose: Due to the many similarities with its human counterpart, canine malignant melanoma (cMM) is a valuable model in which to assess the efficacy of novel therapeutic strategies. The model is herein used to evaluate the immunogenicity, safety, and therapeutic efficacy of a human chondroitin sulfate proteoglycan-4 (hCSPG4) DNA-based vaccine. The fact that homology between hCSPG4 and cCSPG4 amino-acidic sequences stands at more than 80% provides the rationale for using an hCSPG4 DNA vaccine in the cMM model. Experimental Design: Dogs with stage II–III surgically resected CSPG4-positive oral MM were subjected to monthly intramuscular plasmid administration, which was followed immediately by electroporation (electrovaccination) for at least 6, and up to 20, months. The immunogenicity, safety, and therapeutic efficacy of the vaccine have been evaluated. Results: hCSPG4 electrovaccination caused no clinically relevant local or systemic side effects and resulted in significantly longer overall and disease-free survival times in 14 vaccinated dogs as compared with 13 nonvaccinated controls. All vaccinated dogs developed antibodies against both hCSPG4 and cCSPG4. Seven vaccinated dogs were also tested for a cCSPG4-specific T-cell response and only two gave a detectable interferon (IFN)γ response. Conclusion: Xenogeneic electrovaccination against CSPG4 is able to overcome host unresponsiveness to the “self” antigen and seems to be effective in treating cMM, laying the foundation for its translation to a human clinical setting. Clin Cancer Res; 20(14); 3753–62. ©2014 AACR.

Beta Galactosidase deficiency in a Korat cat: a new form of feline GM1 gangliosidosis

Abstract A 7-month-old Korat cat was referred for a slowly progressive neurological disease. Circulating monocytes and lymphocytes showed the presence of single or multiple empty vacuoles and blood leukocytes enzyme assay revealed a very low β-galactosidase activity level (4.7 nmol/mg per h) as compared to unaffected parents and relatives. Histologically, the cat, euthanized at the owner request at 21 months of age, presented diffuse vacuolization and enlargement of neurons throughout the brain, spinal cord and peripheral ganglia, severe cerebellar neuronal cell loss, and moderate astrocytosis. Stored material was stained with periodic acid-Schiff on frozen sections and with the lectins Ricinus communis agglutinin-I, concanavalin A and wheat germ agglutinin on paraffin-embedded sections. Ultrastructurally, neuronal vacuoles were filled with concentrically whorled lamellae and small membrane-bound vesicles. In the affected cat, β-galactosidase activity was markedly reduced in brain (18.9%) and liver (33.25%), while total β-hexosaminidase activity showed a remarkable increase. Quantitation of total gangliosides revealed a 3-fold increase in brain and 1.7-fold in liver of affected cat. High-performance thin layer chromatography (HPTLC) detected a striking increase of GM1-ganglioside. On densitometric analysis of HPTLC bands, the absorption of GM1-ganglioside band was 98.52% of all stained bands (GD1a ,GD1b, GT1b). Based on clinical onset, morphological and histochemical features, and biochemical findings, the Korat cat GM1-gangliosidosis is comparable with the human type II (juvenile) form. However, clinical progression, survival time and level of β-galactosidase deficiency do not completely fit with those of human type II GM1-gangliosidosis. The disease in the Korat cat is also different from other reported forms of feline GM1-gangliosidosis.

PDGFs and PDGFRs in canine osteosarcoma: new targets for innovative therapeutic strategies in comparative oncology.

Platelet derived growth factor receptor (PDGFR)α and PDGFRβ are tyrosine kinase receptors that are overexpressed in 70-80% of human osteosarcomas (OSAs) and may be suitable therapeutic targets for specific kinase inhibitors (TKIs). Canine OSA shows histopathological and clinical features similar to human OSA, and is considered an excellent model in comparative oncology. This study investigated PDGF-A, PDGF-B, PDGFRα and PDGFRβ expression in 33 canine OSA samples by immunohistochemistry and in seven primary canine OSA cell lines by Western blot and quantitative PCR analysis. Immunohistochemical data showed that PDGF-A and PDGF-B are expressed in 42% and 60% of the OSAs analysed, respectively, while PDGFRα and PDGFRβ were expressed in 78% and 81% of cases, respectively. Quantitative PCR data showed that all canine OSA cell lines overexpressed PDGFRα, while 6/7 overexpressed PDGFRβ and PDGF-A relative to a normal osteoblastic cell line. Moreover, in vitro treatment with a specific PDGFR inhibitor, AG1296, caused a dose- and time-dependent decrease in AKT phosphorylation. Collectively, these data show that PDGFRs/PDGFs are co-expressed in canine osteosarcomas, which suggests that an autocrine and/or paracrine loop is involved and that they play an important role in the aetiology of OSA. PDGFRs may be suitable targets for the treatment of canine OSA with a specific TKI.

Chondroitin sulfate proteoglycan-4: a biomarker and a potential immunotherapeutic target for canine malignant melanoma.

Chondroitin sulfate proteoglycan-4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA), is a membrane-bound chondroitin sulfate proteoglycan highly expressed by human melanoma cells. This phylogenetically conserved tumour antigen plays an important biological role in human melanoma, where it is used as a marker to diagnose forms with unusual characteristics, such as desmoplastic melanoma, and to detect melanoma cells in lymph nodes and peripheral blood, and as a target for immunotherapy because of its restricted distribution in normal tissues. To identify suitable targets to develop novel approaches of treating canine melanoma, CSPG4 was studies to see whether it is expressed in canine malignant melanomas. Immunohistochemical staining of 65 canine malignant melanomas with an anti-human CSPG4-specific antibody detected CSPG4 in 37 cases (56.9%). Positive staining was more frequent, albeit not significantly, in amelanotic compared to melanotic tumours and was statistically associated with tumours having both melanin and the epithelioid histotype. The frequency of CSPG4 expression was similar to that of other melanoma antigens used as diagnostic markers for canine malignant melanoma, such as Melan A and the protein recognized by the PNL2 monoclonal antibody. The results suggest that CSPG4 constitutes a new potential immunohistochemical marker of canine malignant melanoma and may represent an immunotherapeutic target as in humans.

Feline STK gene expression in mammary carcinomas

The human RON and its mouse homologue stk are members of the MET family of tyrosine kinase receptors. We have previously shown that the RON gene is over-expressed in human breast carcinomas. As cat mammary tumours have been proposed as a suitable model for aggressive human breast cancer, we identified the feline stk gene and studied its expression in cat mammary cancer. Feline stk sequences were found highly homologous to the stk and RON gene exons that encode the juxtamembrane and transmembrane domains of the stk and RON receptors. Feline stk-specific transcript was detected by RT–PCR in cat lung and in 7/8 feline mammary carcinomas and a synchronous skin metastasis examined. Western blot and immunohistochemical analyses were carried out with an antibody that recognized both the human RON and mouse stk receptors. This antibody specifically detected a 135 Kd feline protein and stained 10/34 mammary carcinoma archival samples. These data show that the pattern of expression and distribution of the stk protein in feline mammary cancer could be superimposed on that of the RON receptor in human breast cancer and suggest that these feline tumours are a suitable model to test innovative approaches to therapy of aggressive human breast carcinomas.

Immunohistochemical investigation of cell cycle and apoptosis regulators (Survivin, β-Catenin, P53, Caspase 3) in canine appendicular osteosarcoma

BackgroundOsteosarcoma (OSA) represents the most common canine primary bone tumour. Despite several pathways have been investigated so far, few molecules have been identified as prognostic tools or potential therapeutic targets, and there is still the need to find out molecular pathways with specific influence over OSA progression to facilitate earlier prognosis and treatment.Aims of the present study were to evaluate the immunohistochemical pattern and levels of expression of a panel of molecules (survivin, β-catenin, caspase 3 -inactive and active forms- and p53) involved in cell cycle and apoptosis regulation in canine OSA samples, known to be of interest in the study also of human OSA, and to detect specific relations among them and with histological tumour grade, disease free interval (DFI) and overall survival (OS).ResultsNuclear β-catenin immunostaining was detected in normal osteoblasts adjacent to the tumour, and in 47% of the cases. Cytoplasmic and/or membranous immunostaining were also observed. Nuclear survivin and p53 positive cells were found in all cases. Moderate/high cytoplasmic β-catenin expression (≥10% positive cells) was significantly associated with the development of metastasis (P = 0.014); moderate/high nuclear p53 expression (≥10% positive cells) was significantly associated with moderate/high histological grade (P = 0.017) and shorter OS (P = 0.049). Moderate/high nuclear survivin expression (≥15% positive cells) showed a tendency toward a longer OS (P = 0,088).ConclusionsThe present results confirmed p53 as negative prognostic marker, while suggested survivin as a potential positive prognostic indicator, rather than indicative of a poor prognosis. The detection of nuclear β-catenin immunostaining in normal osteoblasts and the absent/low expression in most of the OSAs, suggested that this pathway could not play a major role in oncogenic transformation of canine osteoblasts. Further studies are needed to confirm these hypotheses.

Corticosteroid Hormone Receptors and Prereceptors as New Biomarkers of the Illegal Use of Glucocorticoids in Meat Production

Despite the European ban on the use of growth promoters in cattle, veterinary surveillance reports indicate that the illicit use of corticosteroids persists both alone and in combination with anabolic hormones and β-agonists. Current control strategies should be informed by research into the effects of corticosteroids on bovine metabolism and improved through the development of specific, sensitive diagnostic methods that utilize potential molecular biomarkers of corticosteroid treatment. The actions of corticosteroids on target tissues are principally regulated by two receptors: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). The effects of these steroids are modulated by prereceptor enzyme-mediated metabolism: the two isoforms of the 11β-hydroxysteroid dehydrogenase (11β-HSDs) enzyme catalyze the interconversion between active glucocorticoids, such as cortisol, into inactive compounds, such as cortisone. This study aimed to determine whether the expression of the prereceptor system and of the corticosteroid receptors could be regulated in different target tissues by the administration of dexamethasone and prednisolone in cattle. It was observed that greater up-regulation of the GR and MR genes followed dexamethasone treatment in the muscle tissues than in the kidney, liver, and salivary glands; up-regulation of GR and MR expression following prednisolone treatment was higher in adipose tissue than in the other tissues. The thymus seemed to respond to dexamethasone treatment but not to prednisolone treatment. Both treatments significantly down-regulated 11β-HSD2 gene expression in the adrenal tissues, but only dexamethasone treatment down-regulated 11β-HSD2 expression in the bulbourethral and prostate glands. Together, these data indicate that the combination of GR, MR, and 11β-HSD2 could provide a useful biomarker system to detect the use of illicit glucocorticoid treatment in cattle.

Increased expression of insulin-like growth factor-1 receptor is correlated with worse survival in canine appendicular osteosarcoma.

Insulin-like growth factor 1 receptor (IGF-1R) is a cell membrane receptor widely expressed in tissues and involved in different cancers in humans. IGF-1R expression in human osteosarcoma has been associated with the development of tumour metastasis and with prognosis, and represents an attractive therapeutic target. The goal of this study was to investigate the expression of IGF-1R in canine osteosarcoma tissues and cell lines and assess its role and prognostic value. Samples from 34 dogs were examined by immunohistochemistry for IGF-1R expression. IGF-1R/AKT/MAPK signalling was evaluated by western blot and quantitative polymerase chain reaction in the cell lines. In addition, the in vitro inhibition of IGF-1R with pycropodophillin (PPP) was used to evaluate molecular and biological effects. Immunohistochemical data showed that IGF-1R was expressed in 71% of the analysed osteosarcoma samples and that dogs with higher levels of IGF-IR expression (47% of cases) had decreased survival (P < 0.05) when compared to dogs with lower IGF-IR expression. Molecular studies demonstrated that in canine osteosarcoma IGF-IR is activated by IGF-1 mostly in a paracrine or endocrine (rather than autocrine) manner, leading to activation of AKT/MAPK signalling. PPP caused p-IGF-1R dephosphorylation with partial blocking of p-MAPK and p-AKT, as well as apoptosis. It was concluded that IGF-1R is expressed and plays a role in canine osteosarcoma and that its expression is correlated with a poor prognosis. As in humans, IGF-1R may represent a good therapeutic target and a prognostic factor for canine osteosarcoma.

Inter-laboratory comparison of a yeast bioassay for the determination of estrogenic activity in biological samples.

An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expression of yeast enhanced green fluorescent protein (yEGFP), for the determination of estrogenic activity in extracts of calf urine samples. Urine samples were spiked with 1 and 5 ngmL(-1) 17beta-estradiol and 17alpha-ethynylestradiol, 10 and 50 ngmL(-1) mestranol, and 100 ngmL(-1) testosterone and progesterone. Sample extracts of blank and spiked urine samples were prepared at our laboratory and sent to seven laboratories together with a reagent blank, a DMSO blank, and eight 17beta-estradiol stock solutions in DMSO ranging in concentration from 0 to 545 ngmL(-1). Sample extracts and standards were coded and tested blindly. A decision limit (CCalpha) was determined based on the response of seven blank urine samples. Signals of the negative controls, e.g. urine samples spiked with 100 ngmL(-1) testosterone or progesterone, were all below the determined CCalpha and were thus screened as compliant. Positive controls, i.e. the urine samples spiked at two levels with 17beta-estradiol, 17alpha-ethynylestradiol and mestranol, were almost all screened as suspect, i.e. gave signals above the determined CCalpha. Determined EC(50) values calculated from the 17beta-estradiol dose-response curves obtained by the seven laboratories ranged from 0.59 to 0.95 nM.