Expression and significance of p53, rb,p21/waf-1, p16/ink-4a,and PTEN tumor suppressors in canine melanoma

The role of tumor suppressor genes in the pathogenesis of canine melanoma is incompletely understood. The genes encoding the tumor suppressors p53, Rb, p21 (waf-1), p16 (ink-4a), and PTEN have been postulated to contribute to the pathogenesis of melanoma in humans and experimental animal models. To assess whether inactivation of these genes similarly contributes to the origin and progression of canine melanoma, we examined their expression in seven distinct canine melanoma cell lines and in 31 retrospective samples (representing 29 dogs) of spontaneous canine melanoma. Various patterns suggestive of loss of tumor suppressor function emerged in these cell lines. The most frequently observed abnormality was loss or significant reduction of p16 expression in six of seven cell lines and in 21 of 26 tumor samples. Loss or significant reduction of PTEN expression was seen in four of seven cell lines and in 13 of 27 tumor samples. Although p53 was detectable in all the cell lines and in 24 of 30 tumors, exclusion of p53 from the nuclear compartment was observed in each of the cell lines and in 18 of 25 tumor samples. These results indicate that loss of function of these tumor suppressor proteins is a common occurrence that may contribute to the origin of canine melanoma. In our sample population, abnormalities in the expression or localization of one or more tumor suppressor proteins occurred with similar frequency in malignant and benign tumors; thus, additional work is necessary to determine how these proteins may impact disease progression and response to therapy.     

Inactivation of p53 and retinoblastoma family pathways in canine osteosarcoma cell lines

Canine osteosarcoma (OS) has been used as a model system for the study of cancer biology and treatment despite the lack of information regarding its pathogenesis. Expression of tumor suppressor genes known to participate in malignant transformation were studied in five different OS cell lines. Each of the cell lines exhibited properties of transformed cells, and those that were tested grew in soft agarose and formed osteoid-containing tumors when injected subcutaneously into nude mice. p53 function was determined to be defective in each cell line as indicated by the lack of induction of p53-responsive genes, p21 and mdm2, following treatment with 5-fluorouracil. p53 mRNA and protein levels were elevated in three cell lines and were extremely low in two cell lines. p53 protein overexpression correlated with the presence of mutations within the DNA binding domain. Four cell lines appeared to contain normal retinoblastoma (Rb) mRNA and Rb protein and no detectable p16 mRNA or protein. In contrast, the remaining cell line contained high levels of p16 mRNA and protein and significantly reduced levels of Rb, p107, and p130 proteins. These results underscore the importance of inactivating p53 and Rb family pathways in canine OS and suggest that unlike human OS, cells derived from canine OS contain mutations that simultaneously inactivate all three Rb family members.     

PTEN 基因在犬乳腺肿瘤组织中的表达及临床意义

有关酪氨酸磷酸酶基因（Phosphatase and tensin homolog deleted on chromosometen,PTEN）在乳腺肿瘤中的检测在人医早有报道。为了研究PTEN基因在犬乳腺肿瘤组织中的表达情况,笔者运用实时荧光PCR定量检测了38例不同的犬乳腺肿瘤组织（包括15例良性乳腺肿瘤和23例恶性乳腺肿瘤）、4例正常犬乳腺组织。结果发现：PTEN基因在犬恶性乳腺肿瘤组织中表达量明显低于其在良性乳腺肿瘤和正常乳腺组织中的表达量,两者差异极显著（P〈0.001）;PTEN在良性乳腺肿瘤组织中的表达与正常犬乳腺组织相比,差异不显著（P〉0.05）;发生了淋巴结转移的乳腺癌PTEN基因的表达量与未发生转移的乳腺癌组织的表达量间差异亦不显著（P〉0.05）,且PTEN的表达量与肿瘤组织的大小和发病动物年龄无关。结论：PTEN蛋白表达异常可能与乳腺肿瘤发生、发展相关,可考虑作为判断犬乳腺肿瘤生物学行为和预测的指标。     

TP53 gene mutations in canine osteosarcoma

Objective— To investigate mutations of the TP53 gene in canine osteosarcoma (OS).
Study Design— Clinical historic cohort study.
Animals— Client-owned dogs.
Methods— OS (n=59) were screened for mutations of the complete TP53 gene using polymerase chain reaction and the mutation was analyzed by single-strand conformational polymorphism. Clinical outcome of dogs with TP53-mutated OS were compared with dogs with OS without a mutation after complete surgical excision of the primary tumor.
Results— TP53 gene mutations were observed in 24 of 59 (40.7%) OS; 3 mutated OS had 2 mutations. The alterations consisted mainly of point mutations (74%). Dogs with mutated OS had a significantly shorter survival time (ST) after surgery than dogs with normal tumor TP53 gene expression ( P =.03). Other significant prognosticators for ST and disease-free interval included elevated serum alkaline phosphatase ( P <.01) and tumor grade ( P =.01).
Conclusion— TP53 genetic mutations are common in canine OS and may have a prognostic value.
Clinical Relevance— Mutations of the TP53 gene may influence survival and should be considered when evaluating canine OS.     

Anti‐Tumor Effect of Adenovirus‐Mediated P53 Gene Therapy on the Growth of Canine Osteosarcoma Transplanted into Nude Mice

Introduction:  It has been reported that 40–50% of canine osteosarcoma cases have p53 mutations. The p53 tumor supressor gene plays a central role in cell cycle regulation and induction of apoptosis. We previously showed that adenoviral vector expressing canine P53 (AxCA‐cp53) inhibited growth of cultured canine osteosarcoma cell lines. Here, we evaluated anti‐tumor effect of adenovirus‐mediated p53 gene therapy on the growth of canine osteosarcomas transplanted into nude mice.
Methods:  Nine nude mice were subcutaneously injected with cells of a canine osteosarcoma cell line (POS) having p53 gene mutation. The transplanted tumors formed into nude mice were injected with AxCA‐cp53, AxCA‐LacZ (adenovirus vector expressing LacZ) or PBS (3 mice each) 7 times during 15 days. Tumor sizes were measured every 3 days for 27 days after injection with the adenovirus vectors. Expression efficiency of the adenovirus‐mediated gene transfer was examined by X‐gal staining and P53 immunostaining. Effects of the P53 expression on cell cycle control were examined by RT‐PCR for expression of p21 gene downstream of P53.
Results:  Significant differences in the tumor size was observed between the transplanted osteosarcoma tissues injected with AxCA‐cp53 and those injected with AxCA‐LacZ or PBS. Expressions of LacZ and P53 were confirmed at the injection sites of the tumors. Moreover, p21 mRNA expression was shown to be induced in the AxCA‐cp53‐injected tumors, indicating the funciton of P53 to induce cell cycle arrest.
Conclusions:  Adenoviral vector expressing canine P53 inhibited the growth of canine ostersarcoma transplanted into nude mice.     

Comparative study of anti-oncogenic microRNA-145 in canine and human malignant melanoma

MicroRNA-145 (miRNA-145; miR-145) is aberrantly expressed in most of human cancers and plays a significant role in carcinogenesis and cancer progression. In the current study, we focused on how miR-145 plays a role in canine and human malignant melanomas. MiR-145 was significantly downregulated in canine malignant melanoma tissues and canine melanoma cell lines, as well as human melanoma cell lines tested. The ectopic expression of miR-145 showed a significant growth inhibition in both canine and human melanoma cells tested, and the effect was achieved partly by suppressing c-MYC in canine melanoma LMeC and in human melanoma A2058 and Mewo cells. At the same time, a suppressive tendency on cell migration in canine melanoma KMeC cells and significant suppression of cell migration in human melanoma A2058 cells by suppressing FASCIN1 were also found. These findings suggest that miR-145 acts as a tumor suppressor in both canine and human malignant melanomas.     

Morphologic manifestations of gene-specific molecular alterations ("genetic addictions") in mouse models of disease

Neoplasia in both animals and humans results in part from lasting activation of tumor-promoting genes ("oncogenes") or diminished function of genes responsible for preventing neoplastic induction ("tumor suppressor genes"). The concept of "genetic addiction" has emerged to indicate that neoplastic cells cannot maintain a malignant phenotype without sustained genotypic abnormalities related to aberrant activity of oncogene(s) and/or inactivity of tumor suppressor gene(s). Interestingly, some genetic abnormalities reliably produce distinct morphologic patterns that can be used as structural signatures indicating the presence of a specific molecular alteration. Examples of such consistent genetic/microanatomic pairings have been identified for mutated oncogenes, such as rising mucin-producing capacity with RAS overexpression, and mutated tumor suppressor genes-including PTEN eliciting cell hypertrophy, RB1 dictating neuroendocrine differentiation, and TRP53 encouraging sarcomatous transformation. Familiarity with the concept of genetic addiction, as well as the ability to recognize such regular genomic-phenotypic relationships, are of paramount importance for comparative pathologists who are engaged in phenotyping genetically engineered mice to help unravel genomic intricacies in both health and disease.     

Immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors

Immunohistochemical and histochemical stains are useful adjunct techniques in the diagnosis of canine cutaneous round cell tumors, which can appear histologically similar. We applied a panel of monoclonal antibodies (recognizing tryptase, chymase, serotonin for mast cells; CD1a, CD18, MHC class II for histiocytes; CD3 for T lymphocytes; CD79a for B lymphocytes and plasma cells) and one histochemical stain (naphthol AS-D chloroacetate for chymase activity) to formalin-fixed, paraffin-embedded sections of canine cutaneous mast cell tumors, histiocytomas, lymphosarcomas, plasmacytomas, and unidentified round cell tumors. Of 21 tumors with a histologic diagnosis of mast cell tumor, 7/7 (100%) grade I, 6/7 (85.7%) grade II, and 3/7 (42.9%) grade III tumors were diagnosed as mast cell tumors based on positive staining for tryptase antigen and chymase activity. Mast cells were positive for both tryptase antigen and chymase activity, indicating equal efficacy of tryptase immunohistochemistry and chymase histochemistry. Chymase was detected immunohistochemically in both tumor and nontumor cells, while serotonin was not detected in most mast cell tumors, and thus, neither was useful in the diagnosis of mast cell tumors. Immunohistochemistry to detect CD18 and MHC class II was equally effective in staining histiocytomas, although lymphosarcoma must be ruled out through the use of CD3 and CD79a immunohistochemistry. Immunohistochemistry using three different monoclonal antibodies to human CD1a showed no cross-reactivity in canine histiocytomas and was not useful. A final diagnosis was obtained for 4/5 (80%) of the unidentified tumors, indicating the usefulness of multiple stains in poorly differentiated round cell tumors.     

Characterization, Expression and Function of c‐MET in Canine Spontaneous Cancers

Introduction:  Aberrant expression of the proto‐oncogene c‐Met has been noted in a variety of human cancers. In dogs, inappropriate Met expression has been identified in canine osteosarcoma (OSA) tumor samples. To better define the potential role of Met dysregulation in canine cancer, we cloned canine Met, HGF, and HGF activator and evaluated their expression patterns in a variety of canine tumor cell lines.
Methods:  Canine Met, HGF, and HGF activator were cloned from normal canine liver and canine OSA cell lines using primers based on regions of homology between mouse and human sequences as well as 5' and 3' RACE.
Results:  Inappropriate expression of Met was found in canine cell lines derived from OSAs, mast cell tumors, histiocytic sarcomas, hemangiosarcoma, and melanomas. Both HGF and HGF activator were found to be expressed in several of these tumor cell lines, providing evidence of a possible autocrine loop of Met stimulation. Incubation of canine tumor cell lines with rhHGF resulted in Met autophosphorylation and activation of the downstream signaling elements Gab1, Akt and Erk1/2. Scattering of tumor cells in response to HGF occurred under conditions of cell stress, such as serum starvation. Lastly, the Met inhibitor PHA‐665752 blocked HGF induced phosphorylation of canine Met and Gab1.
Conclusions:  These studies provide evidence that similar to the case in human tumors, aberrant Met expression may play an important role in the biology of canine cancer. As such, inhibition of Met function may represent a potentially useful novel therapeutic approach.     

Expression of a tumor-associated antigen, RCAS1, in canine mammary tumors

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1), one of novel cancer cell-surface antigens, is strongly expressed in invasive cancers. RCAS1 inhibits the in vitro growth of lymphocytes such as T cells and natural killer (NK) cells, and induces apoptotic cell death. We investigated the expression of RCAS1 in canine mammary tumor cell lines and tumor cells by immunohistochemistry, and also in situ deoxyribonucleic acid (DNA) fragmentation in tumor-infiltrating lymphocytes (TILs) by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. All canine mammary tumor cell lines expressed RCAS1 at both the messenger ribonucleic acid (mRNA) and protein level. Immunohistochemically, RCAS1 was negative in 100% of normal mammary glands, but was expressed in 100% of malignant tumors examined. In most malignant mammary tumors, RCAS1 was localized in the cytoplasm with no polarity of expression. In benign mammary tumors, it was detected on the luminal surface of the tumor cell. RCAS1 expression or localization was significantly correlated with malignancy. In situ DNA fragmentation of CD3-positive TILs was observed in RCAS1-expressing tumors. RCAS1-expressing tumors, indicating a possible induction of apoptotic cell death in TILs through RCAS1 expression. These observations suggest that RCAS1 probably plays an important role in tumor progression and escape from immune surveillance in canine mammary tumors.     

Immunohistochemical analysis of cyclin A, cyclin D1 and P53 in mammary tumors, squamous cell carcinomas and basal cell tumors of dogs and cats

The involvement of cyclin A, cyclin D1 and p53 proteins in canine and feline tumorigenesis was analyzed immunohistochemically. In the present study, a total of 176 cases were examined, among which there were 108 canine cases (75 mammary lesions, 16 squamous cell carcinomas and 17 basal cell tumors) and 68 feline cases (43 mammary lesions, 20 squamous cell carcinomas and 5 basal cell tumors). Speckled nuclear staining for cyclin A was observed in 19/38 (50%) canine malignant mammary tumors and 18/37 (48.6%) feline mammary carcinomas, while this was not seen in benign mammary tumors of either dogs or cats. Marked intense nuclear cyclin A staining was seen in 7/16 (43.8%) canine squamous cell carcinomas and 18/20 (90.0%) feline squamous cell carcinomas. Only 3/17 (17.6%) canine basal cell tumors showed slight and scattered staining for cyclin A. Expression of cyclin D1 was very rare in both canine and feline tumors. Nuclear staining of p53 was found in 7/37 (18.9%) feline mammary carcinomas. Intense immunoreactivity for p53 was found in 6/16 (37.5%) canine squamous cell carcinomas and 8/20 (40%) feline squamous cell carcinomas. These results suggest that cyclin A may have a role in the proliferation of canine malignant mammary tumors, feline mammary carcinomas and squamous cell carcinomas of dogs and cats, and p53 may associate with the tumorigenesis of feline mammary carcinomas and squamous cell carcinomas of dogs and cats.     

5-Lipoxygenase expression in benign and malignant canine prostate tissues

5-Lipoxygenase (5-LO) is overexpressed in human prostate carcinomas (PCs), and its inhibition decreases proliferation and induces apoptosis in prostate cancer cell lines. We hypothesized that 5-LO would be overexpressed in canine PC compared with benign prostate tissue and may be important in the pathogenesis of the disease. Immunoblot analysis of canine PC and benign prostatic hyperplasia (BPH) tissues demonstrated 5-LO expression in both. 5-LO immunohistochemical staining was not significantly different within the stromal or epithelial components of canine primary PC, BPH or suppurative prostatitis, suggesting that differential expression of this enzyme does not occur in these conditions. The percentage of tumour cells expressing 5-LO was significantly lower in metastatic PC lesions compared with primary PC (P < 0.0001). This decreased expression may indicate down-regulation or altered expression of the enzyme with progression of canine PC to a metastatic phenotype.     

Immunohistochemical characterization of canine hyperplastic hepatic lesions and hepatocellular and biliary neoplasms with monoclonal antibody hepatocyte paraffin 1 and a monoclonal antibody to cytokeratin 7.

Immunostaining with monoclonal antibody (MoAb) hepatocyte paraffin 1 (Hep Par 1) and an MoAb to cytokeratin 7 (CK7) was performed on 105 formalin-fixed, paraffin-embedded canine hyperplastic and neoplastic hepatic lesions. Hep Par 1 was detected in 12/12 hyperplastic nodules, 17/17 hepatocellular adenomas, and 37/40 hepatocellular carcinomas. The staining was disseminated, granular, and cytoplasmic. This antibody did not react with normal or neoplastic biliary epithelium. Other hepatic tumors or tumors metastatic to the liver did not bind Hep Par 1 except one metastatic intestinal carcinoma. MoAb to CK 7 stained all hyperplastic biliary epithelium and benign cholangiocellular tumors (5/5) and 14/18 cholangiocellular carcinomas. One hepatocellular carcinoma had cells positive for both Hep Par 1 and CK 7. Liver was the only normal tissue tested that reacted with MoAb Hep Par 1. Only five nonhepatic tumors (one adrenocortical carcinoma, one interstitial cell tumor of the testis, one melanoma, and two salivary adenocarcinomas) of 277 tumors tested had focal/multifocal staining for Hep Par 1. Prolonged fixation did not alter the staining with Hep Par 1. We conclude that Hep Par 1 is a specific and sensitive marker for canine hepatocellular tumors and allows distinction between hepatocellular and biliary neoplasms.     

Cloning and expression analysis of prohibitin mRNA in canine mammary tumors

Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3′-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors.     

The 5‐lipoxygenase inhibitor tepoxalin induces oxidative damage and altered PTEN status prior to apoptosis in canine osteosarcoma cell lines

The 5‐lipoxygenase (5‐LOX) inhibitor tepoxalin has been shown to slow canine osteosarcoma (OSA) tumour xenografts growth, yet the mechanisms are poorly elucidated. Further examination of tepoxalin in canine OSA cell lines shows that tepoxalin treated cells undergo apoptosis through caspase‐3 activation and annexin staining. Interestingly, apoptosis is superseded by an increase in reactive oxygen species (ROS), as measured by activation of dihydrorhodamine 123 and mitosox. This increase in ROS appears to be related to the 5‐LOX inhibitor regardless of cellular 5‐LOX status, and was not observed after treatment with the tepoxalin metabolite RWJ20142. Additionally, 5‐LOX inhibition by tepoxalin appears to increase phosphatase and tensin (PTEN) homolog activity by preventing its alkylation or oxidation. PTEN modification or inhibition allows phosphoinositide‐3 (PI3) kinase activity thereby heightening activation of protein kinase B (AKT) phosphorylation. Our data suggest that off target oxidation and LOX inhibition play roles in the apoptotic response.     

Mutations of phosphatase and tensin homolog deleted from chromosome 10 in canine hemangiosarcoma

We examined the presence of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) abnormalities that could contribute to the origin or progression of naturally occurring canine endothelial tumors (hemangiosarcoma). Our results document somatic point mutations or deletions encompassing the PTEN C-terminal domain in canine hemangiosarcoma that might provide cells a survival advantage within their microenvironment. This represents the first characterization of a naturally occurring, highly metastatic tumor with biologically significant mutations of PTEN in the C-terminal domain.     

犬常见肿瘤流行病学调查及组织病理学诊断

为探讨犬常见肿瘤的发病规律，试验收集哈尔滨地区2016-2017年宠物医院部分犬肿瘤病例71例，采用组织病理学方法对犬肿瘤进行病理学诊断，并对患病动物的品种、性别、年龄、发生部位和饮食情况与肿瘤发生关系进行统计分析。结果显示，71例确诊肿瘤病例中，恶性肿瘤28例，包括鳞状上皮细胞癌3例，基底细胞癌5例，乳腺癌14例，淋巴瘤2例，肾母细胞瘤、精原细胞癌、生殖细胞癌、侵袭性血管黏液瘤各1例；良性肿瘤43例，包括良性乳腺肿瘤15例，纤维瘤10例，平滑肌瘤5例，乳头状瘤和精原细胞瘤各3例，造釉细胞瘤、耵聍腺瘤、脂肪瘤、结节样增生、性索间质细胞瘤、颗粒细胞瘤、肛周腺瘤各1例。上述病例中，乳腺和皮肤是犬肿瘤的高发部位，分别占全部患犬的40.8%和34.0%；患病犬年龄1~16岁不等，平均发病年龄为9.1岁，7岁以后是肿瘤的高发期，中老年犬仍为主要发病群体，但低龄犬发病数量上升；各品种犬均可发生肿瘤，其中杂种犬发病率最高，其次是贵宾犬和京巴犬，且主要为乳腺肿瘤；饮食习惯主要以剩饭剩菜类和肉拌饭类为主，分别占患犬总数的39%和30%；部分肿瘤类型与犬的性别、是否绝育有关，如乳腺肿瘤多发生于未绝育的老年母犬。本研究结果为犬肿瘤的流行病学及诊断提供了参考依据，对犬肿瘤的预防和诊断有一定的借鉴意义。