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A úlcera de abomaso, classicamente definida como um processo de autodigestão da mucosa abomasal, é uma das abomasopatias mais comuns no gado leiteiro. É uma doença multifatorial em que o principal fator desencadeante nem sempre é identificado; estando na maioria das vezes, relacionado com fatores predisponentes. No interesse de fomentar o acervo de informações sobre a ocorrência de abomasopatias, este manuscrito teve como objetivo descrever os aspectos clínicos, epidemiológicos e patológicos dos casos de úlceras de abomaso em bovinos leiteiros diagnosticados na área coberta pela Universidade Federal de Fronteira Sul, Realeza, PR. Foram avaliados 98 casos de bovinos leiteiros do sudoeste do Paraná a partir dos dados de necropsias registrados entre abril de 2009 e dezembro de 2019, de forma retrospectiva, sendo essa lesão relatada em 87,50% dentre os animais que apresentavam algum tipo de enfermidade digestória ou metabólica. A mediana de vida foi a de 60 meses, sendo apenas 9,52% criados em sistema intensivo. A ocorrência de úlcera abomasal foi determinante para a morte de 54,17% dos animais necropsiados que apresentavam enfermidade digestória e/ou metabólica, evidenciando a importância de informação sobre a ocorrência dessa enfermidade, que acomete principalmente o gado leiteiro, podendo levar ao óbito do animal.

Made available in DSpace on 2019-10-06T16:06:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-02-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The mTOR/4E-BP1/eIF4E pathway plays important roles in the neoplastic transformation process and in tumour growth. In men, the mTOR/4E-BP1/eIF4E pathway was described as altered in different tumours, including prostate cancer (PC). Apart from humans, the dog is the only species that develops PC with high frequency and is considered a good model for comparative oncology initiatives. Due to limited information on this pathway in canine tumours, this study aimed to investigate mTOR, 4E-BP1 and eIF4E gene and protein expression in canine PC, as well as in metastatic and normal prostatic tissues, and to evaluate the correlations between gene/protein expression and Gleason score (GS) in PC. A total of 35 formalin-fixed paraffin-embedded (FFPE) samples, including 13 of normal prostatic tissue, 17 PC samples and 5 metastasis samples, were evaluated by immunohistochemistry and qPCR. mTOR gene mutation in the kinase domain was also investigated. We identified higher p-mTOR and eIF4E protein levels in canine PC with higher GS values (≥ 8) and a significant positive correlation in expression between these proteins. eIF4E overexpression was observed in metastasis relative to expression in normal samples. Our data suggest that p-mTOR and eIF4E expression is positively correlated with GS in canine PC, similar to the pattern in humans. More studies of the mTOR/4EBP1/eIF4E pathway should be performed to identify possible correlations of the proteins involved with clinical and pathologic findings in canine PC and the roles of these proteins as therapeutic targets for the treatment of canine PC. Department of Veterinary Pathology School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) Department of Veterinary Pathology School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) CNPq: 443884/2014-5

Made available in DSpace on 2022-04-29T08:31:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: Vascular endothelial growth factor-A (VEGF-A) and its receptor, VEGF receptor-2 (VEGFR-2), represent a complex family of angiogenic molecules consisting of different ligands and receptors. Due to the importance of VEGF-A/VEGFR-2 signaling in tumor proliferation and angiogenesis, this study aimed to evaluate the protein and gene expression levels of VEGF-A and VEGFR-2 in canine prostate cancer (PC). Methods: We analyzed VEGF-A and VEGFR-2 expression in 87 PC samples by immunohistochemistry and quantitative-polymerase chain reaction. PC samples were graded according to the Gleason score and the immunohistochemical staining for VEGF-A and VEGFR-2 was quantified using a selected threshold from the ImageJ Software. Microvascular density was assessed by cluster of differentiation 31 staining and counting the number of positive vessels. Additionally, the homology of VEGF-A and VEGFR-2 between humans and dogs was assessed, followed by the construction of a protein structure homology model to compare the tertiary structures of these proteins in both species. Results: Negative to weakly positive expression levels of VEGF-A and VEGFR-2 were observed in the epithelial cells of the normal prostate (NP) and prostatic hyperplasia samples. In contrast, the canine proliferative atrophy and PC samples exhibited higher VEGF-A (p

Made available in DSpace on 2022-04-28T19:41:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-06-01 HER2 is a prognostic and predictive marker widely used in breast cancer. Lapatinib is a tyrosine kinase inhibitor that works by blocking the phosphorylation of the receptor HER2. Its use is related to relatively good results in the treatment of women with HER2+ breast cancer. Thus, this study aimed to verify the effects of lapatinib on four canine primary mammary gland carcinoma cell cultures and two paired metastatic cell cultures. Cultures were treated with lapatinib at concentrations of 100, 500, 1000 and 3000 nM for 24 h and the 50% inhibitory concentration (IC50) for each cell culture was determined. In addition, a transwell assay was performed to assess the ability of lapatinib to inhibit cell migration. Furthermore, we verified HER2 expression by RT-qPCR analysis of cell cultures and formalin-fixed paraffin-embedded tissues from samples corresponding to those used in cell culture. Lapatinib was able to inhibit cell proliferation in all cell cultures, but it was not able to inhibit migration in all cell cultures. The higher the expression of HER2 in a culture, the more sensitive the culture was to treatment. This relationship may be an indication that the expression of HER2 may be a predictive factor and opens a new perspective for the treatment of primary and metastatic mammary gland cancer. Department of Veterinary Clinic Sao Paulo State University-UNESP Department of Veterinary Surgery and Animal Reproduction São Paulo State University-UNESP Institute of Health Sciences Paulista University-UNIP Department of Veterinary Clinic Sao Paulo State University-UNESP Department of Veterinary Surgery and Animal Reproduction São Paulo State University-UNESP

Simple Summary Prostatic diseases are important worldwide, being the prostate cancer (PC) the most common tumor in men. Among the factors associated with PC development, the preneoplastic lesions are well-recognized. Preneoplastic lesions are cellular morphological alterations, induced by different factors and present a potential to progression for PC. In this scenario, dogs are considered spontaneous models. Dogs naturally develops prostatic hyperplasia, preneoplastic lesions and PC. Among the preneoplastic lesions, the proliferative inflammatory atrophy (PIA) develops spontaneously in dogs. PIA is an epithelial lesion induced by prostatic chronic inflammation, leading to a proliferative atrophy of the prostate gland. Thus, this study aimed to perform a full PIA morphological, phenotypical and molecular characterization in dogs. After reviewing the archives of the veterinary pathology service, it was identified 171 dogs containing PIA in the prostate gland, and among the PC cases (N = 84), it was identified PIA lesions surrounding 60.7% of PC cases. Besides that, we identified loss of genes related to the maintenance of prostatic tissue and can predispose to malignant transformation. Moreover, mutations in androgen receptor gene were identified, demonstration alteration in DNA in PIA. Overall, these results support the hypothesis that PIA can be considered a preneoplastic lesion in canine prostate. Abstract Proliferative inflammatory atrophy (PIA) is an atrophic lesion of the prostate gland that occurs in men and dogs and is associated with a chronic inflammatory infiltrate. In this study, we retrospectively reviewed canine prostatic samples from intact dogs, identifying 50 normal prostates, 140 cases of prostatic hyperplasia, 171 cases of PIA, 84 with prostate cancer (PC), 14 with prostatic intraepithelial neoplasia (PIN) and 10 with bacterial prostatitis. PIA samples were then selected and classified according to the human classification. The presence of PIA lesions surrounding neoplastic areas was then evaluated to establish a morphological transition from normal to preneoplastic and neoplastic tissue. In addition, the expression of PTEN, P53, MDM2 and nuclear androgen receptor (AR) were analyzed in 20 normal samples and 20 PIA lesions by immunohistochemistry and qPCR. All PIA lesions showed variable degrees of mononuclear cell infiltration around the glands and simple atrophy was the most common histopathological feature. PIA was identified between normal glands and PC in 51 (61%) out of the 84 PC samples. PIA lesions were diffusely positive for molecular weight cytokeratin (HMWC). Decreased PTEN and AR gene and protein expression was found in PIA compared to normal samples. Overall, our results strongly suggest that PIA is a frequent lesion associated with PC. Additionally, this finding corroborates the hypothesis that in dogs, as is the case in humans, PIA is a pre neoplastic lesion that has the potential to progress into PC, indicating an alternative mechanism of prostate cancer development in dogs.

The giant anteater (Myrmecophaga tridactyla) is a vulnerable species from Central and South America, and is considered possibly extinct in Belize, Guatemala, El Salvador, and Uruguay. Due to the species’ conservation and reproductive importance, this research aimed to characterize the morphology, histochemical, immunohistochemical, and ultrastructural feature of the giant anteater prostate gland. For this, we collected 11 giant anteater prostate glands and performed macroscopic, morphological, histochemical, immunohistochemical, and ultrastructural analysis. Nine prostate glands from an adult subject and two from young subjects were studied. Grossly, the adult giant anteater prostate gland is divided in two distinct zones, the central zones (composed mainly of ducts) and the peripheral zones (of acini formed by secretory cells). The secretory cells showed positive periodic acid–Schiff staining. Furthermore, the immunohistochemical characterization revealed a similar human prostate pattern, with p63 staining basal cells, uroplakin III (UPIII) superficial cells of prostatic urethra, androgen receptor (AR) expressing nucleus of secretory and stromal cells, and prostatic specific antigen (PSA) staining prostatic epithelial cells. Overall, our research provided an in-depth morphological description of the giant anteater’s prostate gland, providing valuable information for futures studies focused on giant anteater conservation.

Made available in DSpace on 2021-06-25T10:55:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-25 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Rapamycin is an antifungal drug with antitumor activity and acts inhibiting the mTOR complex. Due to drug antitumor potential, the aim of this study was to evaluate its effect on a preclinical model of primary mammary gland tumors and their metastases from female dogs. Four cell lines from our cell bank, two from primary canine mammary tumors (UNESP-CM1, UNESP-CM60) and two metastases (UNESP-MM1, and UNESP-MM4) were cultured in vitro and investigated for rapamycin IC50. Then, cell lines were treated with rapamycin IC50 dose and mRNA and protein were extracted in treated and non-treated cells to perform AKT, mTOR, PTEN and 4EBP1 gene expression and global proteomics by mass spectrometry. MTT assay demonstrated rapamycin IC50 dose for all different tumor cells between 2 and 10 μM. RT-qPCR from cultured cells, control versus treated group and primary tumor cells versus metastatic tumor cells, did not shown statistical differences. In proteomics were found 273 proteins in all groups, and after data normalization 49 and 92 proteins were used for statistical analysis for comparisons between control versus rapamycin treatment groups, and metastasis versus primary tumor versus metastasis rapamycin versus primary tumor rapamycin, respectively. Considering the two statistical analysis, four proteins, phosphoglycerate mutase, malate dehydrogenase, l-lactate dehydrogenase and nucleolin were found in decreased abundance in the rapamycin group and they are related with cellular metabolic processes and enhanced tumor malignant behavior. Two proteins, dihydrolipoamide dehydrogenase and superoxide dismutase, also related with metabolic processes, were found in higher abundance in rapamycin group and are associated with apoptosis. The results suggested that rapamycin was able to inhibit cell growth of mammary gland tumor and metastatic tumors cells in vitro, however, concentrations needed to reach the IC50 were higher when compared to other studies. Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University-UNESP Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University-UNESP Institute of Health Sciences Universidade Paulista-UNIP Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University-UNESP Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University-UNESP CAPES: 001 FAPESP: 2019/24079-1 CNPq: 422139/2018-1

Made available in DSpace on 2019-10-06T16:22:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01. Added 1 bitstream(s) on 2019-10-09T18:35:19Z : No. of bitstreams: 1 S0100-736X2019000100040.pdf: 2621138 bytes, checksum: aa859149f83aebb38f1b4c7f4dd8d6b8 (MD5) Canine prostate gland is a hormonal dependent organ and its imbalance of estrogen and androgen receptor expressions are directly associated with the development of different diseases. Due to the lack of information regarding the behavior of the aforementioned receptors in canine prostate cancer (PC), this study aimed to identify estrogen receptor alpha (ERα), androgen receptor (AR), Ki67 and phosphatase and tensin homolog (PTEN) protein expressions in canine PC by immunohistochemistry. We found nuclear expression of ERα and AR in the epithelial cells of normal canine samples and a loss of protein expression in PC samples. Normal samples showed Ki67 expression in a few basal cells and the PC samples showed the highest mean of positive cells (253.1). Canine prostate cancer showed a high proliferative index, which was associated with independence of hormonal actuation. PTEN showed positive nuclear and cytoplasmic expression in normal canine samples and a loss in PC. Loss of ERα, AR and PTEN indicated that canine PC exhibits the same immunohistochemical phenotype as in human patients with PC resistant to hormonal therapy. Therefore, canine PC should be considered as a model to study human PC resistant to hormonal therapy. Departamento de Clínica Veterinária Faculdade de Medicina Veterinária e Zootecnia (FMVZ) Universidade Estadual Paulista (Unesp), Rua Prof. Dr. Walter Mauricio Correra s/n, Rubião Junior Departamento de Anestesiologia Faculdade de Medicina de Botucatu, Av. Prof. Montenegro s/n Instituto de Ciências Biomédicas Abel Salazar (ICBAS) Universidade do Porto (UP), Rua Jorge de Viterbo Ferreira 228 Departamento de Clínica Veterinária Faculdade de Medicina Veterinária e Zootecnia (FMVZ) Universidade Estadual Paulista (Unesp), Rua Prof. Dr. Walter Mauricio Correra s/n, Rubião Junior Departamento de Anestesiologia Faculdade de Medicina de Botucatu, Av. Prof. Montenegro s/n

A espécie canina apresenta semelhanças com a espécie humana com relação às lesõesprostáticas, sendo a única espécie a apresentar tumores espontâneos e com frequência, dessemodo representa um importante modelo para estudo comparativo de afecções desta glândula.O desenvolvimento das neoplasias prostáticas é multifatorial e dentre eles, alteraçõesgenéticas e epigenéticas estão envolvidas, portanto o cão pode ser um modelo natural eespontâneo para o estudo das lesões pré neoplásicas e neoplásicas da próstata, com futuro altopotencial de ser utilizado como modelo em testes pré-clínicos de medicamentos. Uma das viasimportantes para o desenvolvimento dos carcinomas prostáticos é a WNT canônica,dependente da proteína β-catenina. Nesta revisão, abordaremos o papel desta via e suaparticipação na carcinogênese prostática, importante em humanos e cães, além de algumasproteínas envolvidas na regulação da mesma

Mammary neoplasms affect a population of uncastrated and elderly female dogs and most of these neoplasms are malignant. In order to study this disease cell culture presents itself as a promising preclinical model, creating the opportunity to deposit cell lines at a cell bank, allowing a great repetition of the assays and making the validation of the results more reliable. Including, in vitro experiments for vasculogenic mimicry (VM) evaluation. VM is related to cancer cells capable of generate vascular-like structures without endothelial cells, mimicking the vasculogenic process. The aim of this study was to establish and characterize ten cell lines from canine mammary gland tumour according to immunophenotype and tumorigenicity, and with its ability to form vasculogenic mimicry-like structures in vitro. Fifteen samples from canine mammary gland carcinoma were collected and cultured in vitro and ten cell lines were established and characterized. Cells were evaluated for morphology, phenotype, vascular mimicry and tumorigenicity. All cell lines presented spindle shape morphology and expressed concomitant pan-cytokeratin and CK8/18. Four cell lines had vasculogenic mimicry ability and two cell showed in vivo tumorigenic potential. Cell characterization of those lines will help to create a database for more knowledge of mammary gland carcinomas in dogs, including studies of tumor behavior and new therapeutic targets.

Made available in DSpace on 2019-10-06T16:41:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) This study aimed to investigate collagen (Coll-I, III, IV) and elastin in canine normal prostate and prostate cancer (PC) using Picrosirius red (PSR) and Immunohistochemical (IHC) analysis. Eight normal prostates and 10 PC from formalin-fixed, paraffin-embedded samples were used. Collagen fibers area was analyzed with ImageJ software. The distribution of Coll-I and Coll-III was approximately 80% around prostatic ducts and acini, 15% among smooth muscle, and 5% surrounding blood vessels, in both normal prostate and PC. There was a higher median area of Coll-III in PC when compared to normal prostatic tissue (p = 0.001 for PSR and p = 0.05 for IHC). Immunostaining for Coll-IV was observed in the basal membrane of prostate acini, smooth muscle, blood vessels, and nerve fibers of normal and PC samples. Although there was no difference in Coll-IV area between normal tissue and PC, tumors with Gleason score 10 showed absence of Coll-IV, when compared to scores 6 and 8 (p = 0.0095). Elastic fibers were found in the septa dividing the lobules and around the prostatic acini of normal samples and were statistically higher in PC compared to normal tissue (p = 0.00229). Investigation of ECM components brings new information and should be correlated with prognosis in future studies. Department of Veterinary Pathology School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) Department of Veterinary Surgery and Anesthesiology School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) Department of Veterinary Pathology School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) Department of Veterinary Surgery and Anesthesiology School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) CNPq: 422139/2018-1 CNPq: 443884/2014-5

Made available in DSpace on 2019-10-06T16:13:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The cancer cell population is heterogeneous, and cancer stem cells (CSCs) are important for tumor growth and maintenance. The CSC population is associated with different neoplastic characteristics, such as cell migration, resistance to apoptosis, radiation therapy and chemotherapy. To increase the knowledge of CSCs in canine prostate cancer (PC), we characterized CSC markers in canine PC tissues and tumorspheres. We performed immunohistochemistry of OCT3/4, Nestin, NANOG, CD44 and CD24 in 10 normal canine prostatic tissue samples, 10 prostatic hyperplastic (PH) tissue samples and 28 PC tissue samples. Then, we established two canine prostate cancer cell cultures and characterized the CSC profile of tumorspheres grown from these cultures. Normal and PH tissues were positive for Nestin, NANOG, CD44 and CD24 only in the basal cell layer. OCT3/4 was expressed in the luminal cells of normal and PH tissues. There was no significant difference in Nestin expression among the prostatic tissues. However, we found higher expression of NANOG and CD44 in canine PC tissues than that in normal and PH tissues. Tumorspheres from canine prostate cancer cells express OCT3/4, Nestin, NANOG and CD44, indicating that these markers may be potential cancer stem cell markers in canine PC. The results obtained can be useful to better characterize the stem cell population in canine prostatic cancer and to guide future studies in comparative oncology. São Paulo State University – UNESP Department of Veterinary Surgery and Anaesthesiology School of Veterinary Medicine and Animal Science São Paulo State University – UNESP Department of Veterinary Clinic School of Veterinary Medicine and Animal Science School of Veterinary Science The University of Queensland, Gatton Campus São Paulo State University – UNESP Department of Veterinary Surgery and Anaesthesiology School of Veterinary Medicine and Animal Science São Paulo State University – UNESP Department of Veterinary Clinic School of Veterinary Medicine and Animal Science FAPESP: 2015/25400-7 FAPESP: 2017/25822-4

Made available in DSpace on 2018-12-11T17:01:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-06-01 The PTEN, AR, MDM2 and p53 protein network plays a central role in the development of many human cancers, thus eliciting the development of targeted cancer therapeutics. Dogs spontaneously develop tumours, and they are considered a good model for comparative oncology initiatives. Due to the limited information on these proteins in canine tumours, this study aimed to investigate gene and protein alterations in PTEN, AR, MDM2 and p53 in canine prostate cancer (PC). Protein expression was evaluated by immunohistochemistry (15 normal, 22 proliferative inflammatory atrophy (PIA) and 19 PC samples) and Western blotting (2 normal prostate tissue, 2 BPH, 2 PIA samples and 2 PC samples) and gene expression by RT-qPCR (10 normal, 10 PIA and 15 PC samples) of formalin-fixed tissue. We identified nuclear and cytoplasmic expression of PTEN and p53 in all samples, with only nuclear staining found for MDM2 and AR. Our results revealed high expression of MDM2 in PC and PIA samples compared to normal samples, whereas PTEN, P53 and AR expression was down-regulated in PC compared to normal tissue. All tumour samples (n = 19) showed loss of nuclear PTEN expression, and all cancer mimickers showed positive nuclear staining. Therefore, nuclear PTEN staining could be a good diagnostic marker for differentiating between malignant lesions and mimickers. Canine prostate carcinogenesis involves increased expression of MDM2 in association with decreased expression of PTEN, p53 and AR, such as occurs in hormone refractory PC in men. Thus, dogs may be an important model for studying advanced stage PC. Faculdade de Ciências Agronômicas Univ. Estadual Paulista - UNESP Department of Veterinary Clinic School of Veterinary Medicine and Animal Science Univ. Estadual Paulista - UNESP Department of Urology Botucatu Medical School - FMB Univ. Estadual Paulista - UNESP Faculdade de Ciências Agronômicas Univ. Estadual Paulista - UNESP Department of Veterinary Clinic School of Veterinary Medicine and Animal Science Univ. Estadual Paulista - UNESP Department of Urology Botucatu Medical School - FMB Univ. Estadual Paulista - UNESP

Collagen (Coll) is the most common protein in the extracellular matrix, responsible for providing tissue structure and support. In some types of cancer, including prostate cancer (PC) abundant collagen was identified and related to tumor progression and metastasis. This study aimed to investigate Coll-I, III, IV and elastin in normal canine prostatic tissue and PC, using the Picrosirius red (PSR) and Immunohistochemical (IHC) analysis. Eight normal prostates and 10 PC from formalin-fixed, paraffin-embedded samples were used. Collagen fibers area was analyzed with ImageJ software. The distribution of Coll-I and Col-III was approximately 80% around prostatic ducts and acini, 15% among smooth muscle and 5% around in blood vessels, in both normal prostate and PC. Immunostaining for type IV collagen was observed in the basal membrane of prostate acini, smooth muscle, blood vessels, and never fibers of normal and PC samples. Elastic fibers were found in the septa dividing the lobules and around the prostatic acini of normal samples. A high amount of elastic fibers was observed around the ducts and the urethra in normal and canine PC. The distribution and area percentage of staining for collagen are similar in normal and neoplastic canine prostate when analyzed with PSR and IHC.

Made available in DSpace on 2018-12-11T17:18:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Prostate cancer is a heterogeneous disease with high levels of clinical and gene heterogeneity, consequently offering several targets for therapy. Dogs with naturally occurring prostate cancer are useful models for molecular investigations and studying new treatment efficacy. Three genes and proteins associated with the WNT pathway (β-catenin, APC and E-cadherin) and Caveolin-1 (CAV-1) were evaluated in canine pre-neoplastic proliferative inflammatory atrophy (PIA), prostate cancer and metastatic disease. The APC gene methylation status was also investigated. As in human prostate cancer, cytoplasmic and nuclear β-catenin, which are fundamental for activating the canonical WNT pathway, were found in canine prostate cancer and metastasis. Membranous E-cadherin was also lost in these lesions, allowing cellular migration to the stroma and nuclear localization of β-catenin. In contrast to human prostate tumours, no APC downregulation or hypermethylation was found in canine prostate cancer. The CAV-1 gene and protein overexpression were found in canine prostate cancer, and as in humans, the highest levels were found in Gleason scores ≥8. In conclusion, as with human prostate cancer, β-catenin and E-cadherin in the WNT pathway, as well as Caveolin-1, are molecular drivers in canine prostate cancer. These findings provide additional evidence that dogs are useful models for studying new therapeutic targets in prostate cancer. São Paulo State University (UNESP) Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Department of Veterinary Pathology School of Agricultural and Veterinarian Sciences International Center for Research (CIPE) AC Camargo Hospital, Liberdade Department of Clinical Genetics Vejle Hospital and Institute of Regional Health University of Southern Denmark São Paulo State University (UNESP) Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Department of Veterinary Pathology School of Agricultural and Veterinarian Sciences CNPq: 131323/2014-8 FAPESP: 2012/16068-0 CNPq: 443884/2014-5

Made available in DSpace on 2018-12-11T17:20:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-01 An unusual variant of prostate adenocarcinoma (PC) expressing nuclear p63 in secretory cells instead of the typical basal expression has been reported in men. Nevertheless, the biological behavior and clinical significance of this phenomenon is unknown. In dogs, this unusual PC subtype has not been described. In this study, p63 immunoexpression was investigated in 90 canine PCs and 20 normal prostate tissues (NT). The p63 expression pattern in luminal or basal cells was confirmed in a selected group of 26 PCs and 20 NT by immunohistochemistry and/or Western blotting assays. Eleven canine PC samples aberrantly expressing p63 (p63+) in secretory cells were compared with 15 p63 negative (p63-) cases in the context of several molecular markers (high molecular weight cytokeratin- HMWC, CK8/18, CK5, AR, PSA, chromogranin, NKX3.1, PTEN, AKT and C-MYC). P63+ samples were positive for CK5, HMWC and CK8/18 and negative for PSA, NKX3.1, PTEN and chromogranin. Five p63+ PCs were negative for AR, and the remaining six samples had low AR expression. In contrast, p63- PC showed AR and PSA positive expression in all 15 samples. Only five p63- PCs were positive for CK5. Both p63+ and p63- PC samples showed higher cytoplasmic AKT expression and nuclear C-MYC staining in comparison with normal tissues. Metastatic (N = 12) and non-metastatic (N = 14) PCs showed similar immunoexpression for all markers tested. In contrast to human PC, canine PC aberrantly expressing p63 showed higher expression levels of HMWC and CK5 and lower levels of NKX3.1. Canine p63+ PC is a very rare PC group showing a distinct phenotype compared to typical canine PC, including AR and PSA negative expression. Although in a limited number of cases, p63 expression was not associated with metastasis in canine PC, and cytoplasmic p63 expression was observed in animals with shorter survival time, similar to human PC cases. Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University-UNESP Department of Morphology Instituto de Biociências São Paulo State University-UNESP Department of Clinical Genetics Vejle Hospital and Institute of Regional Health Research University of Southern Denmark Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University-UNESP Department of Morphology Instituto de Biociências São Paulo State University-UNESP

The epithelial-mesenchymal transition (EMT) is a fundamental event responsible for the invasiveness and metastasis of epithelial tumours. The EMT has been described in many human cancers, but there are few reports of this phenomenon in veterinary oncology. Due to the importance of this process, the current study evaluated mesenchymal and epithelial marker protein expression in prostate lesions from dogs. Our results indicate both a loss of E-cadherin and translocation of β-catenin from the membrane to the cytoplasm and nucleus in the tumour group. Vimentin expression in the tumour group was higher than in normal tissue. All of the metastases were positive for prostate-specific antigen, pan-cytokeratin and E-cadherin, although fewer positive cells were present than in the primary tumours. The immunohistochemical results showed a loss of epithelial markers and a gain of a mesenchymal marker among metastatic cells, suggesting that the EMT occurs during the metastatic process of canine prostate carcinoma.

Made available in DSpace on 2018-12-11T17:19:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) NKX3.1/C-MYC cross-regulation has been reported in the normal human prostate, and loss of NKX3.1 and gain of C-MYC seem to be important events in prostate cancer development and progression. The dog can be an interesting model for human prostatic disease, and yet only one previous research study has shown deregulation of NKX3.1 and MYC in the canine prostate. To address the expression of NKX3.1 and C-MYC in different canine prostatic lesions, this study verified the gene and protein expression of NKX3.1 and C-MYC in normal canine prostatic tissues. We identified a 26 kDa band that corresponded to the NKX3.1 protein, while C-MYC showed a 50 kDa band on Western blotting analysis of all prostatic tissues. We observed that NKX3.1 protein and transcript were down-regulated in prostate cancer (PC) samples compared with non-neoplastic samples. We also observed that C-MYC protein was overexpressed in PC samples compared with normal (P =.001) and proliferative inflammatory atrophy (PIA) samples (P =.003). We found a positive correlation between NKX3.1 and C-MYC protein expression in normal and PIA samples. Interestingly, a negative correlation (NKX3.1 downregulation and MYC overexpression) was observed between NKX3.1 and MYC transcripts in PC. Thus, samples with higher C-MYC expression also exhibited higher NKX3.1 expression, which indicates the regulation of C-MYC by NKX3.1 protein. As in humans, these two genes and proteins were found to be related to canine prostate cancer. However, in contrast from what is observed in humans, in canine PC samples, the downregulation of NKX3.1 cannot be explained by DNA hypermethylation. Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University – UNESP Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University – UNESP FAPESP: CNPq 306055/2011-2 FAPESP: FAPESP 2010/13774-6 FAPESP: FAPESP 2012/16068-0 FAPESP: FAPESP 2012/18426-1

Made available in DSpace on 2018-12-11T17:21:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 Saúde Animal Veterinary Hospital Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University - UNESP College of Veterinary Medicine Western University of Health Sciences Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University - UNESP