Overexpression of human epidermal growth factor receptor 2 in canine primary lung cancer.

Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various human cancers. HER2-targeted therapies showed clinical responses in humans with HER2-positive tumors. The incidence of canine primary lung cancer (cPLC) is increasing, but there are no effective systemic therapies for dogs with late-stage cPLC. HER2-targeted therapy could be an option for cPLC, but HER2 expression in cPLC remains unknown. We evaluated HER2 expression in cPLC. Immunohistochemical analysis revealed that 3 samples (19%) scored 3+; 8 (50%), 2+; 5 (31%); and 1+ and 0 (0%), 0. Of the cPLC tissues, 69% were HER2 positive (scored ≥2+). These data would lead to further evaluation of the role of HER2 in cPLC as a mechanism of malignancy and therapeutic target.

Aberrantly expressed snoRNA, snRNA, piRNA and tRFs in canine melanoma.

Among small non-coding RNAs (sncRNAs/sRNAs), the functional regulation of microRNAs (miRNAs) has been studied in canine oral melanoma (COM). However, the expression level of other sncRNAs, like small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), transfer RNA-derived fragments (tRFs) and PIWI-interacting RNAs (piRNAs), in COM is unknown. The aim of this study was to investigate sncRNAs other than miRNAs in COM from our small RNA sequencing project (PRJNA516252). We found that several snRNAs and piRNAs were upregulated, whereas tRFs and snoRNAs were downregulated in COM. Upregulation of U1 snRNA and piR-972, and downregulation of tRNA-ser (1) and snoRA24 was confirmed in dog melanoma tissue and cell lines by quantitative reverse transcription PCR. Consistently, the expression of tRNA-ser (1) and snoRA24 in plasma of COM cases was also decreased. Finally, we found a similar expression trend of U1 and snoRA24 in the human cutaneous melanoma cell line, MEWO, compared with human epidermal melanocyte cells (HEMa-Lp). In our study, snRNA, snoRNA, tRFs and piRNA were dysregulated during melanoma progression. Moreover, the melanoma-associated expression of U1 and snoRA24 was similar in human and dog melanoma.

Transcriptome analysis of dog oral melanoma and its oncogenic analogy with human melanoma.

Dogs have been considered as an excellent immunocompetent model for human melanoma due to the same tumor location and the common clinical and pathological features with human melanoma. However, the differences in the melanoma transcriptome between the two species have not been yet fully determined. Considering the role of oncogenes in melanoma development, in this study, we first characterized the transcriptome in canine oral melanoma and then compared the transcriptome with that of human melanoma. The global transcriptome from 8 canine oral melanoma samples and 3 healthy oral tissues were compared by RNA­Seq followed by RT­qPCR validation. The results revealed 2,555 annotated differentially expressed genes, as well as 364 novel differentially expressed genes. Dog chromosomes 1 and 9 were enriched with downregulated and upregulated genes, respectively. Along with 10 significant transcription site binding motifs; the NF­κB and ATF1 binding motifs were the most significant and 4 significant unknown motifs were indentified among the upregulated differentially expressed genes. Moreover, it was found that canine oral melanoma shared >80% significant oncogenes (upregulated genes) with human melanoma, and JAK­STAT was the most common significant pathway between the species. The results identified a 429 gene signature in melanoma, which was up­regulated in both species; these genes may be good candidates for therapeutic development. Furthermore, this study demonstrates that as regards oncogene expression, human melanoma contains an oncogene group that bears similarities with dog oral melanoma, which supports the use of dogs as a model for the development of novel therapeutics and experimental trials before human application.

Micro RNA Transcriptome Profile in Canine Oral Melanoma.

MicroRNAs (miRNAs) dysregulation contribute the cancer pathogenesis. However, the miRNA profile of canine oral melanoma (COM), one of the frequent malignant melanoma in dogs is still unrevealed. The aim of this study is to reveal the miRNA profile in canine oral melanoma. MiRNAs profile of oral tissues from normal healthy dogs and COM patients were compared by next-generation sequencing. Along with tumour suppressor miRNAs, we report 30 oncogenic miRNAs in COM. The expressions of miRNAs were further confirmed by quantitative real-time PCR (qPCR). Pathway analysis showed that deregulated miRNAs impact on cancer and signalling pathways. Three oncogenic miRNAs targets (miR-450b, 301a, and 223) from human study also were down-regulated in COM and had a significant negative correlation with their respective miRNA. Furthermore, we found that miR-450b expression is higher in metastatic cells and regulated MMP9 expression through a PAX9-BMP4-MMP9 axis. In silico analysis indicated that miR-126, miR-20b, and miR-106a regulated the highest numbers of differentially expressed transcription factors with respect to human melanoma. Chromosomal enrichment analysis revealed the X chromosome was enriched with oncogenic miRNAs. We comprehensively analyzed the miRNA's profile in COM which will be a useful resource for developing therapeutic interventions in both species.

Immunohistochemical evaluation of HER2 expression in canine thyroid carcinoma.

The human epidermal growth factor receptor 2 (HER2) is expressed in various human cancers including thyroid cancers (TC) and is used as a diagnostic marker and therapeutic target. Canine TC (cTC), the most common endocrine malignancy in dogs, shows a high metastasis rate, and HER2-targeted therapy could be a candidate for treatment. Here, we immunohistochemically evaluated HER2 expression in 21 paraffin-embedded cTC tissues and scored the degree of expression based on intensity and positivity (score: 0-3+). Four samples (19%) scored 3+; 6 (29%), 2+; 7 (33%), 1+; and 4 (19%), 0. Therefore, 48% of the cTC tissues were HER2 positive (scored ≥2+). These data may lead to further evaluation of the role of HER2 in cTC as a mechanism of malignancy and a therapeutic target.

Anti-tumor effects of the histone deacetylase inhibitor vorinostat on canine urothelial carcinoma cells.

Canine urothelial carcinoma (cUC) is the most common tumor of the lower urinary tract in dogs. Although chemotherapy and radical surgery have improved the overall survival, most dogs with cUC succumb to metastasis or recurrence. Therefore, the development of an effective systematic therapy is warranted. In this study, a comprehensive drug screening test using a cUC cell line was performed and the anti-tumor effect of a histone deacetylase (HDAC) inhibitor was evaluated. Comprehensive drug screening was performed on cUC cells. Based on this screening, the anti-proliferation effect of vorinostat, an HDAC inhibitor clinically applied in humans, was evaluated using several cUC cell lines in sulforhodamine B and flow cytometry assays. Western blot analysis was also performed to evaluate the degree of acetylation of histone H3 as well as the expression and phosphorylation of cell cycle-related molecules. The anti-tumor effect of vorinostat in vivo was evaluated using a xenograft model. Finally, immunohistochemistry was performed on acetyl-histone H3 in cUC and the relationship between the degree of acetylation and prognosis was examined using Kaplan-Meier survival analysis. Drug screening revealed that HDAC inhibitors consistently inhibited the growth of cUC cells. Vorinostat inhibited the growth of 6 cUC cell lines in a dose-dependent manner and induced G0/G1 cell cycle arrest. Western blot analysis showed that vorinostat mediated the acetylation of histone H3, the dephosphorylation of p-Rb, and the upregulation of p21 upon exposure to vorinostat. Furthermore, inhibition of tumor growth was observed in the xenograft model. In clinical cUC cases, neoplastic urothelium showed significant deacetylation of histones compared to the normal control, where lower histone acetylation levels were associated with a poor prognosis. In conclusion, the therapeutic potential of vorinostat was demonstrated in cUC. Histone deacetylation may be related to cUC tumor progression.

Detection of human epidermal growth factor receptor 2 overexpression in canine anal sac gland carcinoma.

Canine anal sac gland carcinoma (ASGC) frequently occurs in the apocrine glands of the canine anal sac and shows aggressive biological behavior. The expression of human epidermal growth factor receptor 2 (HER2) has been reported in various human and canine tumors. HER2 is a promising therapeutic target of these tumors, and HER2-targeted drugs, such as trastuzumab and lapatinib, have improved the outcome of these patients. In this study, HER2 expression in ASGC was evaluated to investigate its potential as a therapeutic target for canine ASGC. HER2 mRNA expression in surgically resected ASGC tissues was significantly higher than that in normal anal sac tissue. To evaluate the expression of HER2 protein, paraffin-embedded ASGC tissues were immunohistochemically evaluated. Strong and broad staining of HER2 was detected in ASGC tissues, while HER2 was weakly to moderately stained in normal anal sac apocrine glands and squamous epithelia. The degree of HER2 expression in ASGC tissues was scored based on its intensity and positivity (score: 0-3+). Scoring of HER2 expression revealed 6 samples (24%) scored 3+, 14 (56%) scored 2+, and 5 (20%) scored 1+, with no samples scoring 0. In all, 80% of canine ASGC tissues were positive for HER2 (scored ≥2+). Furthermore, putative HER2-overexpressed cells in ASGC were detected with trastuzumab by flow cytometry. These preliminary data may lead to further evaluation of the role of HER2 in canine ASGC as a mechanism of malignancy and as a therapeutic target for HER2-targeted therapy.

The role of SET/I2PP2A in canine mammary tumors.

Canine mammary tumor is the most common neoplasm in female dogs, and it has generated considerable attention as a translational model for human breast cancer. Ser/Thr protein phosphatase 2A (PP2A) plays a critical role as a tumor suppressor, and SET/I2PP2A, the endogenous inhibitory protein of PP2A, binds directly to PP2A and suppresses its phosphatase activity. Here, we investigated the role of SET in the tumorigenic growth in canine mammary tumor as well as in the sensitivity of tumors to existing therapeutics. Elevated protein levels of SET were observed in advanced-stage of canine mammary tumor tissues of dogs compared with paired normal tissues. Knockdown of SET expression in a canine mammary tumor cell line CIP-m led to increased PP2A activity and decreased cell proliferation, colony formation, and in vivo tumor growth. We observed suppression of mTOR, ß-catenin, and NFκB signaling by SET knockdown. The sensitivity of CIP-m cells to doxorubicin was decreased by SET knockdown, while SET knockdown in CIP-m cells did not affect sensitivity to 4-OH-tamoxifen, carboplatin, bortezomib, and X-ray radiation. These data suggest that SET plays important roles in the tumor progression of a subset of canine mammary tumor by suppressing PP2A activity and enhancing mTOR, ß-catenin, and NFκB signaling.

Circulating microRNA-214 and -126 as potential biomarkers for canine neoplastic disease.

Circulating microRNAs in the blood may provide diagnostic and prognostic information about canine neoplastic diseases, and their profiles may be conserved between human and canine species. We performed RT-qPCR to obtain the profiles of circulating plasma microRNA-214 and -126 in total 181 cases of canine neoplastic diseases and healthy controls. MicroRNA-214 levels were high in 2 epithelial tumours (thyroid and mammary carcinomas) and 4 non-epithelial tumours (osteosarcoma, histiocytic sarcoma, chondrosarcoma, and hemangiosarcoma). In contrast, microRNA-126 levels were high in 6 epithelial tumours (mammary, hepatocellular, squamous cell, thyroid, transitional cell carcinomas, and adenocarcinoma) and 4 non-epithelial tumours (osteosarcoma, mast cell tumour, melanoma, and hemangiosarcoma). The diagnostic potential of microRNA-214 was relatively high in sarcomas, whereas that of microR-126 was high in most types of the tumours. MicroRNA-214 and -126 were prognostic predictors in 2 groups (adenocarcinoma and non-epithelial tumours except for osteosarcoma) and 3 groups (epithelial tumours, adenocarcinoma, and melanoma), respectively. Additionally, the microRNA levels did not show a strong correlation with the other clinical parameters. In conclusion, circulating microRNA-214 and -126 have the potential to be diagnostic and prognostic biomarkers for canine neoplastic diseases. Furthermore, their profiles may be key references as well for exploring novel biomarkers for human cancers.

Anti-neoplastic effects of topoisomerase inhibitors in canine mammary carcinoma, melanoma, and osteosarcoma cell lines.

Numerous topoisomerase inhibitors with proven efficacy have been used extensively to treat various human neoplasms. However, among these, only doxorubicin has been used and studied extensively in veterinary oncology. The current study was performed to evaluate the responsiveness of canine osteosarcoma (cOSA), mammary gland tumour (cMGT), and malignant melanoma (cMM) cell lines to several topoisomerase inhibitors. In addition, the correlation between the sensitivity to treatment and multi-drug resistant (MDR) factors was investigated. cOSA cell lines exhibited higher sensitivity than cMGT and cMM cell lines to all the topoisomerase inhibitors tested in vitro; this was associated with the levels of multi-drug resistance protein 1 (MDR1) gene expression in the cOSA cell lines. Treatment of cOSA (HMPOS) and cMGT cell line (CHMp) xenograft mouse models with etoposide markedly delayed tumour progression in HMPOS xenografts, but failed to elicit lasting anti-tumour effects on CHMp xenograft mice. The present findings suggest that MDR1 represents a molecular signature for prediction of treatment efficacy of topoisomerase inhibitors, especially that of etoposide, which may be a clinically useful anti-tumour agent for cOSA; however, further study is necessary to refine the treatment protocol.

MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases.

Malignant endothelial proliferative diseases including human angiosarcoma (AS) and canine hemangiosarcoma (HSA) are serious diseases with a grave prognosis. Establishing liquid biopsy-based biomarkers for screening has definite clinical utility; however, plasma miRNAs up- or down-regulated in these sarcomas have been unclear. For identifying possible diagnostic plasma miRNAs for these sarcomas, we investigated whether plasma miR-214 and miR-126, which miRNAs play important roles in angiogenesis and tumorigenesis, were elevated in malignant endothelial proliferative diseases. For this investigation, human angiosarcoma and canine hemangiosarcoma cell lines and clinical plasma samples of canine hemangiosarcoma were examined by performing miRNA qRT-PCR. We report here that human angiosarcoma and canine hemangiosarcoma cell lines over-secreted miR-214 and miR-126 via microvesicles; in addition, their levels in the plasma samples from canines with hemangiosarcoma were increased. Moreover, the surgical resection of primary tumors decreased the levels of plasma miR-214 and miR-126. Our findings suggest that these malignant endothelial proliferative diseases over-secreted miR-214 and miR-126, thus suggesting that these miRNAs have potential as diagnostic biomarkers for malignant endothelial proliferative diseases in canine and possible in human angiosarcoma.

Skin photoprotection and consumption of coffee and polyphenols in healthy middle-aged Japanese females.

BACKGROUND: Reactive oxygen species are known to mediate skin photoaging, which results in the formation of pigmented spots and wrinkles. Coffee is the largest source of polyphenols, which supplies a large number of antioxidants in one's daily life. However, little is known about how much coffee and polyphenol consumption influences skin health. In this study, a cross-sectional survey of the diet, environmental factors, and skin conditions was conducted in healthy Japanese females to explore the influence of coffee and polyphenol consumption on skin conditions. MATERIALS AND METHODS: Non-smoking, healthy female subjects with moderate sun exposure in their daily lives were recruited for this study (n = 131, age range: 30-60 years old) and recorded their food and beverage intake and life circumstances using questionnaires. The skin water content, transepidermal water loss, and elasticity were measured on the cheek of each subject using non-invasive methods: a Corneometer, a Tewameter, and a Cutometer, respectively. Wrinkles and pigmented spots were evaluated using digital photograph images. RESULTS: Consumption of coffee and total polyphenols from all sources and from coffee showed a statistically significant correlation towards a decrease in pigmented spot scores (P < 0.05). Subjects with high total polyphenol consumption from coffee or chlorogenic acids (the third tertile group) showed the lowest score of ultraviolet pigmented spots (P < 0.05). CONCLUSION: Coffee and polyphenol consumption was associated with low facial pigmented spots in Japanese middle-aged females. We speculated that coffee helps protect human skin from photoaging, and polyphenols, including chlorogenic acids, may contribute to the decreased hyperpigmentation of pigmented spots.

Inflammatory cytokines induce a reduction in E-cadherin expression and morphological changes in MDCK cells.

Epithelial-mesenchymal transition (EMT) is a fundamental phenomenon in organisms that occurs during gastrulation, wound healing, and cancer metastasis. Various cytokines induce EMT processes through complex mechanisms. Inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), induce EMT in human cell lines. However, whether inflammatory cytokines can affect EMT processes in canine cell lines remains unclear. In this study, we investigated the role of transforming growth factor beta (TGF-ß), TNF-α, and IL-6 in Madin-Darby canine kidney (MDCK) cells. We found that the localization of E-cadherin, a cell adhesion molecule, was shifted and that its expression was decreased. We also observed morphological changes in MDCK cells under persistent stimulation of inflammatory cytokines. Morphological changes in cells may occur during late stages of EMT processes; inflammatory cytokines may be important in these changes.