RESUMO
Canine mammary tumours (CMT) have histological, clinicopathological and molecular resemblances to human breast cancer (HBC), positioning them as viable models for studying the human disease. CMT initiation and progression occur spontaneously in immune-competent animals, which challenge the suggested limitations of genetically modified mice, also enabling the evaluation of immunotherapies in canine patients. Dogs have shorter life expectancy compared to humans, and cancer advances more rapidly in this species. This makes it possible to perform studies about the clinical efficacy of new therapeutic modalities in a much shorter time than in human patients. The identification of biomarkers for tumour subtypes, progression and treatment response paves the way for the development of novel therapeutic and diagnostic approaches. This review addresses the similarities between CMT and HBC and the molecular signatures identified in CMT samples that have been explored to date. We proposed a detailed molecular exploration of the CMT stroma using state-of-the-art methods in transcriptomics and proteomics. Using CMT as an analog for HBC not only helps to understand the complexities of the disease, but also to advance comparative oncology to the next level to prove the claim of dogs as a valid translational model.
Assuntos
Modelos Animais de Doenças , Doenças do Cão , Neoplasias Mamárias Animais , Cães , Animais , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/genética , Doenças do Cão/patologia , Doenças do Cão/genética , Feminino , Humanos , Neoplasias da Mama/veterinária , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Pesquisa Translacional BiomédicaRESUMO
Mammary tumours (MT) are one of the most prevalent malignancies in female dogs and women. Currently, molecular analyzes have shown that each tumour type presents its own genetic signature. In this context, liquid biopsy allows a comprehensive genetic characterisation of the tumour, enabling early diagnosis and personalised treatment of patients. In women, deleterious mutations inherited in BRCA2 gene are associated with an increased risk of breast cancer, resistance to therapies and worse prognosis. In female dogs, there are many divergent data on the involvement of BRCA2 gene with mammary carcinogenesis and what its pathogenic potential is. Therefore, the objective was to identify BRCA2 gene variants in 20 plasma DNA samples, from 10 newly diagnosed dogs with mammary cancer (RD), five control (CTR) and five mastectomized patients. Eleven single nucleotide polymorphisms (SNPs) were detected, most of them in the exon 11 and two indels (deletion/insertion) in the BRCA2 gene. However, there was no statistically significant difference in the SNPs/indels detected between the groups. In addition, only one SNP (p.T1425P) and one deletion (p.L2307del) were considered deleterious using in silico computational models. Interestingly, most common variants were present in the plasma of all groups, except for the Ile2614Thr, Ile2614Val, Thr1425Pro and p.L2307del variants. Thus, we observed that SNPs are common in the BRCA2 gene of female dogs with MT, with a similar condition identified in women with breast cancer. Liquid biopsy approach in dogs with MT is useful for genetic and therapeutic proposals.
Assuntos
Neoplasias da Mama , Doenças do Cão , Genes BRCA2 , Neoplasias Mamárias Animais , Animais , Neoplasias da Mama/veterinária , Doenças do Cão/diagnóstico , Doenças do Cão/genética , Cães , Feminino , Predisposição Genética para Doença , Biópsia Líquida/veterinária , Neoplasias Mamárias Animais/diagnóstico , Neoplasias Mamárias Animais/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
LIN28 is a RNA-binding protein including two highly conserved homologous, LIN28A and LIN28B. Proto-oncogenes such as LIN28A and LIN28B are generally targeted by the let-7 miRNAs in different types of human cancers. Here, we determined the expression of LIN28A in canine mammary tumor samples and the LIN28/let-7 pathway in canine mammary cell lines. In those cell lines, we identified a functional LIN28/let-7 pathway which exhibited high expression of let-7 members and low expression of its targets, including LIN28A and LIN28B. However, the mammary carcinoma tissue samples showed a frequent expression of LIN28A being expressed mainly in the epithelial cells. No association was observed between LIN28A expression and histopathological classification and grade, TNM and survival time. Our results suggested a possible role of the LIN28A protein in the development of canine mammary carcinomas due to the high frequency observed in the tumor samples (28 of 32). The in vitro experiments suggested that the LIN28/let-7 pathway is active in the tumor cells evaluated. However, more studies are necessary to elucidate the exact role of LIN28/let-7 pathway in canine mammary carcinomas.
LIN28 é uma proteína de ligação ao RNA, com duas formas homólogas altamente conservadas, LIN28A e LIN28B. Os proto-oncogenes LIN28A e LIN28B são regulados pela família de miRNAs let-7 em diferentes tipos de cânceres em humanos. No presente trabalho, o objetivo foi determinar a expressão de LIN28A em amostras de tumor mamário de cadelas e a via LIN28/let-7 em linhagens celulares mamaÌrias caninas. Nestas linhagens, atraveÌs das teÌcnicas de qPCR e RNAseq, foi identificado que a via LIN28/let-7 apresenta-se funcional, com alta expressaÌo dos membros da famiÌlia let-7 e baixa expressaÌo de seus alvos, entre eles LIN28A e LIN28B. No entanto, as amostras de tecidos de carcinomas mamaÌrios caninos demonstraram expressaÌo frequente de LIN28A, sendo observada principalmente em ceÌlulas epiteliais. NaÌo foram observadas associaçoÌes entre expressaÌo de LIN28A com classificaçaÌo e gradaçaÌo histopatoloÌgicas, TNM e tempo de sobrevida. Nossos resultados sugerem uma possível relação da proteína LIN28A no desenvolvimento de carcinomas mamários caninos devido à alta frequência observada nas amostras tumorais (28 de 32). Os experimentos in vitro sugerem que a via LIN28/let-7 é ativa nas linhagens celulares caninas avaliadas. Entretanto, estudos funcionais ainda são necessários para elucidar a função exata da via LIN28/let-7 nos carcinomas mamários caninos.
Assuntos
Animais , Feminino , Cães , Neoplasias Mamárias Animais/genética , Proteínas de Ligação a RNA/análise , MicroRNAs/análise , Reação em Cadeia da PolimeraseRESUMO
TP53 and PGAM1 genes play a key role in glycolysis which is an essential metabolic pathway of cancer cells for obtaining energy. The purpose of this work was to evaluate PGAM1 and TP53 mRNA expressions in canine mammary carcinomas (CMC) and to correlate them with animal data and tumour histological features. None of the nine samples analysed revealed PGAM1 DNA sequence variations. PGAM1 and TP53 RNA expressions from 21 CMC were analysed using a one-step reverse transcription-PCR kit and its platform system. Most CMC samples had low levels of PGAM1 mRNA (71.5%) and normal expression of TP53 mRNA (95.2%). Our results suggest a different feature of the Warburg effect on canine mammary cancer cells compared to human cells.
Assuntos
Doenças do Cão , Neoplasias Mamárias Animais/genética , Fosfoglicerato Mutase/genética , Proteína Supressora de Tumor p53/genética , Animais , Doenças do Cão/genética , Cães , Glicólise , Neoplasias Mamárias Animais/metabolismo , Fosfoglicerato Mutase/metabolismo , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mammary tumours are the first and third most incident neoplasm in women and cats, respectively. Approximately 85% of feline mammary gland tumours are malignant and aggressive, especially the triple-negative and HER-2+ molecular subtypes. Triple-negative basal-like feline mammary carcinomas (FMCs) are considered suitable models due to the clinical and morphological similarities with human basal-like triple-negative breast cancer (TNBC). In women, TNBC has a poor prognosis and is often associated with mutations in the tumour suppressor genes BRCA1 and BRCA2. In light of this, the aim of the present investigation was to screen somatic and germline variants of BRCA1 and BRCA2 in nine female cats bearing FMCs. Matched whole blood and FMC samples were obtained for genetic analysis. Additional tumour samples were obtained for histopathological and immunohistochemical evaluation. Genomic DNA was isolated and 27 exonic regions of BRCA1 and BRCA2 genes were amplified and screened by next-generation sequencing. A somatic variant with high functional impact was found in exon 11 of BRCA2 at a frequency of 4.34% in one FMC-bearing cat. Four germline variants with moderate impact were detected in three of the nine FMC-bearing cats and were restricted to exon 9 of BRCA1. It is concluded that the germline genetic variants found in one-third of FMC-bearing animals might be associated with a higher risk of hereditary mammary carcinogenesis.
Assuntos
Neoplasias da Mama , Carcinoma , Doenças do Gato , Neoplasias Mamárias Animais , Neoplasias de Mama Triplo Negativas , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Neoplasias da Mama/veterinária , Carcinoma/veterinária , Doenças do Gato/genética , Gatos , Feminino , Genes BRCA2 , Predisposição Genética para Doença , Humanos , Neoplasias Mamárias Animais/genética , Mutação , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/veterináriaRESUMO
Mammary tumors (MTs) in bitches are similar to breast cancers in women. Thus, they can be used as a model for human breast cancer and findings can be extrapolated for use in human medicine. BRCA1 is a tumor suppressor gene. When the gene has a mutation, it cannot repair damaged DNA, which causes genetic instability and tumorigenesis. Therefore, we aimed to study the frequency of single nucleotide polymorphisms (SNPs) in the BRCA1 gene that are associated with distinct histological types of malignant MT in bitches. The study population consisted of 91 bitches, including a control group of 6 animals with healthy mammary glands and 85 animals with MTs. All animals underwent a presurgery evaluation consisting of a questionnaire administered to the person responsible for the animal, a physical examination, collection of peripheral blood for hematological and serum biochemistry evaluations, an electrocardiogram, and a preanesthesia evaluation. In addition, distant metastasis was studied via chest radiography and abdominal ultrasound. After evaluations were complete, the animals that could undergo surgery were administered general anesthesia and underwent a mastectomy or mammary gland sample collection. Histopathological examination and molecular analysis were performed to identify mutations in the BRCA1 gene. Histopathological examinations found 10 different types of malignant tumors in 36 sick animals. Tumor samples plus samples from the 6 control animals were subjected to DNA extraction, polymerase chain reaction (PCR) analysis, and genetic sequencing. The tumor with the highest incidence (33.33%) was a complex carcinoma, followed by carcinoma in mixed tumor (13.88), tubular carcinoma (13.88) and carcinosarcoma (13.88). Molecular analysis revealed 3 different SNP points in 5 samples (4006G>A, 3619A>G, and 3761C>T). The allelic variant 4006G>A (1/36) resulted in the alteration of the amino acid valine by isoleucine (V1336 I). The mutation 3619A>G (2/36) inserted the amino acid alanine instead of threonine (T1207 A). The mutation 3761C>T (2/36) led to the alteration of the amino acid serine by phenylalanine (S1254 F), a mutation for which there are no published reports. The histological types that showed BRCA1 mutations were complex carcinoma (1/5), carcinoma in mixed tumor (1/5), papillary carcinoma (1/5) and tubular carcinoma (2/5). Software analysis identified the new SNP (nucleotide 3761) in BRCA1 and 2 point mutations in nucleotides 4006 and 3619 and responsible for genetic instability. The development of breast cancer is caused by many endogenous and exogenous factors. The results of our study show that these factors have a greater presence in female, mixed breed, uncastrated, and older dogs, confirming the data in the veterinary literature. In the present study, we found different histological types of malignant breast tumors with mutations in the BRCA1 gene, as other authors have reported. However, we also found the mutation 3761C>T, which, to the best of our knowledge, has not been reported in the literature. This shows the need for studies in veterinary medicine that assess mutations in the BRCA1 gene and the most common histological types. In conclusion, SNPs in the BRCA1 gene cause genetic instability, resulting in additional mutations that lead to the development of breast tumors. They are point mutations that affect transcription, resulting in truncated proteins. These proteins may have a loss of function, leading to carcinogenesis.(AU)
Assuntos
Animais , Feminino , Cães , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/diagnóstico por imagem , Genes BRCA1 , Polimorfismo de Nucleotídeo Único/genética , Doenças do Cão/genética , CãesRESUMO
Triple-negative tumors are characterized immunohistochemically by the absence of positivity to sex hormone receptors and to human epidermal growth factor receptor 2. Additionally, they are differentiated into basal-like and non-basal (or null) subtypes, based on the presence of basal cytokeratin expression (CK5/6, 14, and17). Triple-negative subtypes are yet to be characterized in male dogs, to our knowledge. We report herein the clinical and pathologic findings and molecular characterization of carcinoma in the mammary glands of 2 male dogs. Case 1 was diagnosed as a grade II tubulopapillary carcinoma; case 2 was diagnosed as a grade II carcinoma in a mixed tumor. The tumors were characterized phenotypically as triple-negative basal and triple-negative non-basal, respectively.
Assuntos
Carcinoma/veterinária , Neoplasias Mamárias Animais/genética , Receptores de Estrogênio/genética , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma/genética , Carcinoma/patologia , Cães , Regulação Neoplásica da Expressão Gênica , Imuno-Histoquímica , Masculino , Neoplasias Mamárias Animais/patologia , Receptores de Progesterona/metabolismoRESUMO
Mammary gland tumors are a heterogeneous group of neoplastic diseases. Genetic studies make it possible to determine genetic profiles and identify new molecular markers. The aim of the study was to evaluate the gene expression profile of canine mammary carcinomas and identify potential prognostic markers. Twelve mammary cancer samples from bitches were collected for the evaluation of global gene expression. Microarray assays were performed using commercial kits. Statistical analysis of the microarray was done using moderate t-statistic and adjusted using the Benjamini and Hochberg procedure. Differential connectivity analysis was also performed. Enrichment analyses were conducted using WebGestalt. P-values were calculated using hypergeometric statistics and adjusted using the Benjamini and Hochberg procedure. The HYAL-1 gene was validated using quantitative PCR (qPCR). There were 878 upregulated genes and 821 downregulated genes in the neoplasms studied. Enrichment analysis (individual analysis) identified the HYAL-1 gene as a potential marker of tumorigenesis and tumor recurrence. Differential connectivity analysis demonstrated 262 differentially connected genes.
Assuntos
Doenças do Cão/genética , Neoplasias Mamárias Animais/genética , Animais , Biomarcadores Tumorais/metabolismo , DNA de Neoplasias , Doenças do Cão/enzimologia , Cães , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/metabolismo , Neoplasias Mamárias Animais/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
Targeting self-renewal and tumorigenicity has been proposed as a potential strategy against cancer stem cells (CSCs). Epigenetic proteins are key modulators of gene expression and cancer development contributing to regulation and maintenance of self-renewal and tumorigenicity. Here, we have screened a small-molecule epigenetic inhibitor library using 3D in vitro models in order to determine potential epigenetic targets associated with self-renewal and tumorigenicity in Canine Mammary Cancer (CMC) cells. We identified inhibition of BET proteins as a promising strategy to inhibit CMC colonies and tumorspheres formation. Low doses of (+)-JQ1 were able to downregulate important genes associated to self-renewal pathways such as WNT, NOTCH, Hedgehog, PI3K/AKT/mTOR, EGF receptor and FGF receptor in CMC tumorspheres. In addition, we observed downregulation of ZEB2, a transcription factor important for the maintenance of self-renewal in canine mammary cancer cells. Furthermore, low doses of (+)-JQ1 were not cytotoxic in CMC cells cultured in 2D in vitro models but induced G2/M cell cycle arrest accompanied by upregulation of G2/M checkpoint-associated genes including BTG2 and CCNG2. Our work indicates the BET inhibition as a new strategy for canine mammary cancers by modulating the self-renewal phenotype in tumorigenic cells such as CSCs.
Assuntos
Carcinogênese/genética , Proliferação de Células/genética , Doenças do Cão/genética , Epigênese Genética , Neoplasias Mamárias Animais/genética , Fatores de Transcrição/genética , Animais , Antineoplásicos/farmacologia , Azepinas/farmacologia , Biomarcadores Tumorais/genética , Carcinogênese/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doenças do Cão/patologia , Cães , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Testes Genéticos/métodos , Indazóis/farmacologia , Neoplasias Mamárias Animais/patologia , Família Multigênica/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/fisiologia , Piridonas/farmacologia , Triazóis/farmacologiaRESUMO
The E-cadherin loss has frequently been associated with transcriptional repression mediated by transcription factors, such as the Zinc Finger E-Box Binding Homeobox-2 (ZEB2). Invasive micropapillary carcinomas (IMPCs) of the breast are aggressive neoplasms frequently related to lymph node metastasis and poor overall survival. In the canine mammary gland, IMPCs has just been reported and, based on its behavioral similarity with the human IMPCs, appears to be a good spontaneous model to this human entity. This study aimed to evaluate the relationship between E-cadherin and ZEB2 in a spontaneous canine model of invasive micropapillary carcinoma of the mammary gland. The correlation among gene expression (ZEB2 and CDH1) and clinicopathological findings was also explored. Nineteen cases of IMPC of the canine mammary gland were obtained, protein and mRNA expression were investigated through immunohistochemistry and RNA In Situ Hybridization, respectively. To better understand the relationship between E-cadherin and ZEB2, immunofluorescence was performed in canine IMPCs. Immunohistochemically, most of IMPCs showed 1+ (14/19, 73.7%) for E-cadherin; and positivity for ZEB2 was diagnosed in 47.4% of the IMPCs. Regarding the RNA In Situ Hybridization (ISH), most of IMPCs showed 4+ and 0+ for E-cadherin (CDH1) and ZEB2 respectively. Through immunofluorescence, the first and second more frequent combinatorial group were E-cadherin+ZEB2- and E-cadherin+ZEB2+; neoplastic cells showing concomitantly weak expression for E-cadherin and positivity for ZEB2 were frequently observed. A negative correlation was observed between E-cadherin and progesterone receptor expression in IMPCs. Based on these results, canine mammary IMPCs show E-cadherin lost and, at times reveals nuclear positivity for the transcription factor ZEB2 that seems to exert transcriptional repression of the CDH1.