Effects of transplantation sites on tumour growth,pulmonary metastasis and ezrin expression of canine osteosarcoma cell lines in nude mice

To determine the influence of the transplantation site of canine osteosarcoma (OS) cell lines on tumour growth and pulmonary metastasis, three OS cell lines were transplanted into nude mice via subcutaneous (SC), intratibial (IT) or intravenous (IV) injection. IT‐xenografts exhibited greater potential for developing primary masses and pulmonary metastasis than SC‐xenografts. In IT and IV xenografts, lung micrometastases along with phosphorylated ezrin–radixin–moesin (p‐ERM) overexpression were found in mice xenografted with HMPOS and OOS cells after 1 week and metastasis was found with decreased p‐ERM expression at later time points. The expression of ezrin and p‐ERM in the primary tumours of IT‐xenografted mice was higher than those in SC‐xenografted mice with HMPOS and OOS cells. The results suggest that the orthotopic transplantation site plays an important role in the spontaneous metastasis of canine OS and that ezrin phosphorylation may be involved in the early metastatic mechanism of canine OS cells.     

Establishment of four pairs of canine mammary tumour cell lines derived from primary and metastatic origin and their E-cadherin expression

Four new pairs of canine mammary carcinoma cell lines derived from both primary and metastatic lesions were established. The cells were cultured in RPMI‐1640 with 10% fetal bovine serum and they showed stable growth for more than 120 passages. Using these cell lines, the expression of E‐cadherin was measured by flow cytometry and the function of E‐cadherin was evaluated by cell aggregation assay and results from the primary and metastatic lesions were compared statistically. E‐cadherin was strongly expressed in all of the cell lines, without a notable difference between cells of primary and metastatic origin. In the cell aggregation assay, the function of E‐cadherin was significantly weaker in the cells of primary origin (p < 0.05), as compared with cells of metastatic origin. The present results suggest that a reduction in E‐cadherin function may be implicated in the invasive and metastatic potential of canine mammary tumour cells; however, further study will be needed to clarify E‐cadherin function in the context of the metastasis of canine mammary carcinoma.     

Serum vascular endothelial growth factor concentrations and postsurgical outcome in dogs with osteosarcoma

Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor, playing putative roles in both tumour growth and metastasis. The purpose of this study was to correlate pretreatment serum concentrations of VEGF in dogs with osteosarcoma (OSA) with disease‐free interval (DFI) and overall survival (OS). Additionally, the effect of serum from dogs with OSA on ex vivo canine endothelial cell (EC) growth was determined. Pretreatment platelet‐corrected serum VEGF levels correlated significantly with DFI. No other examined variable predicted outcome. The ability of sera from dogs with OSA to stimulate canine EC proliferation did not correlate with VEGF concentration or outcome. These data support a role for VEGF in the development or progression of metastatic disease in dogs with OSA. The VEGF concentration in tested sera was not a major determinant of ex vivo canine EC proliferation in this study.     

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.     

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.     

The adrenergic receptor antagonists propranolol and carvedilol decrease bone sarcoma cell viability and sustained carvedilol reduces clonogenic survival and increases radiosensitivity in canine osteosarcoma cells

Adrenergic receptor (AR) expression has been demonstrated at several sites of primary and metastatic tumour growth and may influence proliferation, survival, metastasis and angiogenesis. AR antagonists like propranolol and carvedilol inhibit proliferation, induce apoptosis and synergize with chemotherapy agents in some cancers. Radiation resistance is mediated in many cells by upregulation of pro‐survival pathways, which may be influenced by ARs. Studies evaluating AR antagonists combined with radiation are limited. The purpose of this study was to determine the effect of propranolol and carvedilol on viability and radiosensitivity in sarcoma cell lines. The hypothesis was that propranolol and carvedilol would increase radiosensitivity in four primary bone sarcoma cell lines. Single agent propranolol or carvedilol inhibited cell viability in all cell lines in a concentration‐dependent manner. The mean inhibitory concentrations (IC₅₀) for carvedilol were approximately 4‐fold lower than propranolol and may be clinically relevant in vivo. Immunoblot analysis confirmed AR expression in both human and canine sarcoma cell lines; however, there was no correlation between baseline AR protein expression and radiosensitivity. Short duration treatment with carvedilol and propranolol did not significantly affect clonogenic survival. Prolonged exposure to propranolol and carvedilol significantly decreased the surviving fraction of canine osteosarcoma cells after 3Gy radiation. Based on our results and possible in vivo activity in dogs, further studies investigating the effects of carvedilol on sarcoma are warranted.     

Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma

The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer‐related signalling pathways despite differences in activating mutations.     

Prognostic significance of a new histologic grading system for canine osteosarcoma

Histologic grade is an important determinant in clinical outcome of human osteosarcoma (OS). In this study, the histologic characteristics of primary and metastatic canine OS were evaluated using a new classification system. Histologic characteristics were classified in 166 primary and 34 metastatic canine OS. Prognostic variables for clinical outcome were determined using multivariate analysis. Most OS were histologically characterized by severe to extreme cellular pleomorphism, a variable number of mitoses, small to moderate amounts of matrix, a high percentage of tumor cells, and minimal to moderate amounts of necrosis. Tumor invasion into vessels was present in 117/152 (71%) tumors, and 12/50 (24%) of the regional lymph nodes had evidence of metastasis. Classification of the 166 tumors resulted in seven (4%) grade 1, 34 (21%) grade II, and 125 (75%) grade III OS. In the multivariate analysis, histologic grade III OS and elevated pretreatment plasma alkaline phosphatase (AP) levels were independent predictors of clinical outcome. Dogs with high-grade tumors and elevated AP should be carefully evaluated for the presence of metastatic disease before starting adjunctive therapy protocols.     

Immunophysiological studies of interleukin-2 and canine lymphocytes.

Interleukin-2-dependent pathways of lymphocyte activation were investigated in canine peripheral blood lymphocytes (PBL) following stimulation with T-cell mitogens including phytohemagglutinin, phorbol ester (TPA), calcium ionophore (ionomycin), and human recombinant interleukin-2 (hrIL-2). The ability of the stimulated cells to produce interleukin-2 (IL-2) was determined using murine indicator cell lines. IL-2 receptor expression by mitogen-stimulated canine PBL was confirmed by the binding of hrIL-2 with high affinity, and with characteristics comparable to those of the human and murine IL-2 receptor. Examination of serum and PBL from two dogs that were treated with hrIL-2 and human recombinant tumor necrosis factor for systemic mast cell tumors showed that in one dog, IL-2 could be measured in the serum. Concurrently, the in vitro mitogenic response of this dog's PBL to hrIL-2 occurred earlier, possibly reflecting an increase in the relative number of IL-2-responsive cells within the PBL population.     

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.     

MicroRNAs as tumour suppressors in canine and human melanoma cells and as a prognostic factor in canine melanomas

Malignant melanoma (MM) is one of the most aggressive cancers in dogs and in humans. However, the molecular mechanisms of its development and progression remain unclear. Presently, we examined the expression profile of microRNAs (miRs) in canine oral MM tissues and paired normal oral mucosa tissues by using the microRNA‐microarray assay and quantitative RT‐PCR. Importantly, a decreased expression of miR‐203 was significantly associated with a shorter survival time. Also, miR‐203 and ‐205 were markedly down‐regulated in canine and human MM cell lines tested. Furthermore, the ectopic expression of miR‐205 had a significant inhibitory effect on the cell growth of canine and human melanoma cells tested by targeting erbb3. Our data suggest that miR‐203 is a new prognostic factor in canine oral MMs and that miR‐205 functions as a tumour suppressor by targeting erbb3 in both canine and human MM cells.     

The therapeutic effects of SET/I2PP2A inhibitors on canine melanoma

Canine melanoma is one of the most important diseases in small animal medicine. Protein phosphatase 2A (PP2A), a well conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. SET/I2PP2A is an endogenous inhibitor for PP2A, which directly binds to PP2A and suppresses its phosphatase activity. Elevated SET protein levels have been reported to exacerbate human tumor progression. The role of SET in canine melanoma, however, has not been understood. Here, we investigated the potential therapeutic role for SET inhibitors in canine melanoma. The expression of SET protein was observed in 6 canine melanoma cell lines. We used CMeC-1 cells (primary origin) and CMeC-2 cells (metastatic origin) to generate cell lines stably expressing SET-targeting shRNAs. Knockdown of SET expression in CMeC-2, but not in CMeC-1, leads to decreased cell proliferation, invasion and colony formation. Phosphorylation level of p70 S6 kinase was decreased by SET knockdown in CMeC-2, suggesting the involvement of mTOR (mammalian target of rapamycin)/p70 S6 kinase signaling. The SET inhibitors, OP449 and FTY720, more effectively killed CMeC-2 than CMeC-1. We observed PP2A activation in CMeC-2 treated with OP449 and FTY720. These results demonstrated the potential therapeutic application of SET inhibitors for canine melanoma.     

Tumor suppressor PTEN is mutated in canine osteosarcoma cell lines and tumors

Canine osteosarcoma (OS) cell lines contain mutations that directly or indirectly inactivate the tumor suppressor genes p53 and retinoblastoma. Another important tumor suppressor, PTEN, is mutated in many human cancers. To determine whether inactivation of PTEN plays a role in the pathogenesis of canine OS, we studied its expression in canine OS cell lines and tumors. Four of five canine OS cell lines (CO2, C03, CO5, and CO7) constitutively express high levels of the phosphorylated form of Akt, an indirect indicator of aberrant PTEN expression. PTEN protein is essentially absent from three of these cell lines (CO2, CO5, and CO7), whereas C03 contains a potentially inactivating amino acid substitution in PTEN at codon 340. Genomic hybridization experiments indicate that CO2, CO5, and CO7 contain large deletions within the PTEN gene. Ten of 15 OS tumors exhibit variable or negative PTEN staining. Evaluation of a PTEN-negative staining tumor by Southern blotting indicates that the PTEN gene is deleted in this tumor. These results indicate that PTEN is mutated or downregulated in a high percentage of canine OS cell lines and tumors and likely plays an important role in the pathogenesis of the disease.     

Establishment of a pair of novel cloned tumour cell lines with or without metastatic potential from canine mammary adenocarcinoma

We produced 23 cloned cell lines from parental CHMp, which was previously established from a canine mammary adenocarcinoma patient in our laboratory. Two representative cloned cell lines, namely, CHMp-5b and -13a, were selected and characterized for cellular morphology, growth potential and expression of some tumour-related proteins. Subsequently, we transplanted the 2 tumour cell lines orthotopically into female nude mice to examine their tumorigenicity and metastatic potential. Interestingly, despite sharing the same origin, only CHMp-5b cells metastasized to the lung. Our results indicate that a comparison between these 2 cell lines at the molecular level will help us understand mechanisms of tumour progression, especially in the context of distant metastases originating from canine mammary gland tumours.     

Cyclopedic protein expression analysis of cultured canine mammary gland adenocarcinoma cells from six tumours

We characterised cultured canine mammary gland adenocarcinoma cells by exhaustive step protein expression analysis to identify factors associated with tumour progression or metastasis of canine mammary gland tumour. Cultured adenocarcinoma cells derived from a total of 3 primary and 3 metastatic lesions from 3 dogs (CHMp/m, CIPp/m and CNMp/m, where CHM, CIP, and CNM indicate the 3 animals) were used in this study. The expression of 24 proteins reported to be related to tumourigenesis or malignancy of human breast cancers were examined by Western blot analysis using 24 antibodies. The expression of sialyl Lewis X [sLe(x)] was only observed in CHMm cells, which were derived from pleural effusion. This expression was further confirmed by immunohistochemistry. The levels of some factors, such as 14-3-3sigma, cyclinD1 and Rb, differed among cells or between the primary and metastatic cells in the pair. Though the difference in their expression was not consistent within the cells from primary and metastatic origin, this characterisation should provide useful information for further molecular analysis of these cultured cells. Since some of the factors, such as sLe(x), 14-3-3sigma, cyclinD1 and Rb, showed different levels of expression in the pair, these cultured cells might be meaningful tools for clarification of distant metastasis in canine mammary gland tumours.