Jong-Il Shin

Analysis of a new histological and molecular-based classification of canine mammary neoplasia.

Canine mammary tumors (CMTs) are morphologically and biologically heterogeneous, prompting several attempts to classify such tumors on the basis of their histopathological characteristics. Recently, molecular-based analysis methods borrowed from human breast cancer research have also been applied to the classification of CMTs. In this study, canine mammary neoplasms (n = 648) occurring in Korea from 2008 to 2011 were analyzed according to the histological classification and grading system proposed by Goldschmidt et al. Furthermore, randomly selected mammary carcinomas (n = 159) were classified according to the molecular subtype using immunohistochemical characteristics. Canine mammary neoplasia accounted for 52.6% (648/1250) of the tumors in female dogs, and 51.7% (340/648) of these were malignant. All of the carcinoma-anaplastic subtypes were grade III tumors (5/5, 100%), while most of the carcinoma-tubular subtypes (15/18, 83.3%) and carcinoma arising in a complex adenoma/mixed-tumor subtype (115/135, 85.2%) were grade I tumors. Tumor cell invasion into lymphatic vessels was most common in the comedocarcinoma, carcinoma-anaplastic, and inflammatory carcinoma subtypes. The most frequently occurring molecular subtype (70/159, 44%) was luminal A. However, the basal-like subtype was the most malignant and was frequently associated with grade III tumors and lymphatic invasion. The carcinoma-solid subtypes were also often of the basal-like subtype. Reclassification of CMTs using the newly proposed histopathological classification system and molecular subtyping could aid in determining the prognosis and the most suitable anticancer treatment for each case.

Salubrinal-Mediated Upregulation of eIF2α Phosphorylation Increases Doxorubicin Sensitivity in MCF-7/ADR Cells.

Eukaryotic translation initiation factor 2 alpha (eIF2α), which is a component of the eukaryotic translation initiation complex, functions in cell death and survival under various stress conditions. In this study, we investigated the roles of eIF2α phosphorylation in cell death using the breast cancer cell lines MCF-7 and MCF-7/ADR. MCF-7/ADR cells are MCF-7-driven cells that have acquired resistance to doxorubicin (ADR). Treatment of doxorubicin reduced the viability and induced apoptosis in both cell lines, although susceptibility to the drug was very different. Treatment with doxorubicin induced phosphorylation of eIF2α in MCF-7 cells but not in MCF-7/ADR cells. Basal expression levels of Growth Arrest and DNA Damage 34 (GADD34), a regulator of eIF2α, were higher in MCF-7/ADR cells compared to MCF-7 cells. Indeed, treatment with salubrinal, an inhibitor of GADD34, resulted in the upregulation of eIF2α phosphorylation and enhanced doxorubicin-mediated apoptosis in MCF-7/ADR cells. However, MCF-7 cells did not show such synergic effects. These results suggest that dephosphorylation of eIF2α by GADD34 plays an important role in doxorubicin resistance in MCF-7/ADR cells.

Effects of Obesity and Obesity-Related Molecules on Canine Mammary Gland Tumors

Obesity can affect the clinical course of a number of diseases, including breast cancer in women and mammary gland tumors in female dogs, via the secretion of various cytokines and hormones. The objective of this study was to examine the expression patterns of obesity-related molecules such as aromatase, leptin, and insulin-like growth factor 1 receptor (IGF-1 R) in canine mammary carcinomas (CMCs) on the basis of the body condition score (BCS). Comparative analyses of the expression of these molecules, together with prognostic factors for CMCs, including hormone receptors (HRs; estrogen and progesterone receptors), lymphatic invasion, central necrosis of the tumor, and histologic grade, were performed on 56 CMCs. The mean age of CMC onset was lower in the overweight or obese group (8.7 ± 1.9 years) than in the lean or ideal body weight group (10.4 ± 2.7 years). The proportion of poorly differentiated (grade III) tumors was significantly higher in the overweight or obese female dogs. Aromatase expression was significantly higher in the overweight or obese group and was correlated with the expression of HRs (P = .025). These findings suggest that overweight or obese status might affect the development and behavior of CMCs by tumor-adipocyte interactions and increased HR-related tumor growth.

Obesity, expression of adipocytokines, and macrophage infiltration in canine mammary tumors

Obesity influences the development, progression and prognosis of human breast cancer and canine mammary cancer (MC) but the precise underlying mechanism is not well-documented in the fields of either human or veterinary oncology. In the present study, the expression of major adipocytokines, including leptin, adiponectin, and leptin receptor (ObR) in benign (n = 28) and malignant (n = 70) canine mammary tumors was investigated by immunohistochemistry and on the basis of the subjects body condition score (BCS). To evaluate the relationship between obesity and chronic inflammation of the mammary gland, macrophages infiltrating within and around tumoral areas were counted. The mean age of MC development was lower in overweight or obese dogs (9.0 ± 1.8 years) than in lean dogs or optimal bodyweight (10.2 ± 2.9 years), and the evidence of lymphatic invasion of carcinoma cells was found more frequently in overweight or obese group than in lean or optimal groups. Decreased adiponectin expression and increased macrophage numbers in overweight or obese subjects were significantly correlated with factors related to a poor prognosis, such as high histological grade and lymphatic invasion. Leptin expression was correlated with progesterone receptor status, and ObR expression was correlated with estrogen receptor status of MCs, regardless of BCS. Macrophage infiltration within and around the tumor may play an important role in tumor progression and metastasis in obese female dogs and may represent a prognostic factor for canine MCs.

CD44+/CD24– Cancer Stem Cells Are Associated With Higher Grade of Canine Mammary Carcinomas

The CD44+/CD24– phenotype identifies cancer stem cell (CSC) properties in canine mammary carcinoma (MC); however, the histopathological features associated with this phenotype remain to be elucidated. Here, we determined whether the CD44+/CD24– phenotype was associated with hormonal receptor (HR; estrogen receptor [ER] and/or progesterone receptor [PR]) status and/or triple (ER, PR, and human epithelial growth factor receptor 2)–negative (TN) subtype; conventional histological evaluation was also performed. We found that, as single markers, both CD44+ and CD24+ were associated with less aggressive histological types, low grade, and a non-TN subtype; both markers were associated with HR positivity. On the other hand, a CD44+/CD24– phenotype was associated with higher grade of carcinoma. Therefore, our results suggest that immunohistochemical phenotyping for CD44/CD24 is useful for the evaluation of tumor behavior as well as CSC-like properties in canine MCs.

Efficacy of clade 2.3.2 H5 commercial vaccines in protecting chickens from clade 2.3.4.4 H5N8 highly pathogenic avian influenza infection

Emerging clade 2.3.4.4 of the highly pathogenic avian influenza (HPAI) virus strain H5N8, which had been detected sporadically in domestic poultry in China, started to affect wild birds and poultry in South Korea in 2014. The virus was spread to Germany, Italy, the Netherlands, United Kingdom, and even United States by migratory birds. Here, we tested currently used commercial clade 2.3.2 H5 vaccines to evaluate mortality, clinical signs, virus shedding, and histological damage after experimental infection of chickens with the clade 2.3.4.4 HPAI H5N8 virus. Although the vaccination protected chickens from death, it failed to prevent chickens from shedding the virus and from tissue damage according to histological examination. These results suggest that the use of appropriate vaccines that match the currently epidemic HPAI virus is recommended, and continuous HPAI surveillance and testing of currently used commercial vaccines should be performed.

Analysis of Hypoxia-Inducible Factor-1α Expression Relative to Other Key Factors in Malignant Canine Mammary Tumours.

Hypoxia-inducible factor (HIF)-1α plays important physiological roles, but is also of significance in carcinogenesis in man and animals. This study aimed to identify HIF-1α expression in malignant canine mammary tumours (CMTs) and to find correlations with other key factors by using immunohistochemistry (IHC). The histological classification, grading and evaluation of lymphatic invasion were achieved by examining sections stained by haematoxylin and eosin. Determination of molecular subtype, expression of HIF-1α, oestrogen receptor (OR), progesterone receptor (PR), human epidermal growth factor receptor (HER)-2, Ki67, vascular endothelial growth factor (VEGF) and E-cadherin was evaluated by IHC in 87 samples of malignant CMTs. HIF-1α expression correlated significantly with histological type, grade of cancer, negativity for OR and expression of Ki67 and VEGF. Lymphatic invasion, molecular subtype, PR, HER-2 and E-cadherin levels did not significantly correlate with HIF-1α expression. The results of this study imply that HIF-1α may potentially play a role in increased malignancy of CMTs, as it does in human breast cancer.

Evaluation of Clinicopathological Characteristics and Oestrogen Receptor Gene Expression in Oestrogen Receptor-negative, Progesterone Receptor-positive Canine Mammary Carcinomas

The existence of the oestrogen receptor-negative (OR(-))/progesterone receptor-positive (PR(+)) phenotype in canine mammary carcinomas (CMCs) is not well understood, although this phenotype was reported consistently in previous studies. In the present study, immunohistochemistry (IHC) was performed to categorize CMCs with the OR(-)/PR(+) phenotype and compare their clinicopathological features with OR(+)/PR(+) tumours. Of a total of 305 CMCs, 36 (11.8%) were categorized as OR(-)/PR(+) and showed intermediate characteristics between those of OR(+)/PR(+) and OR(-)/PR(-) cases. OR mRNA levels were measured in formalin-fixed, paraffin wax-embedded samples using a novel branched-chain DNA assay method. Similar to the IHC result, one-way analysis of variance showed that the mean normalized OR mRNA level of OR(-)/PR(+) tumours (11.4 ± 16.34) was between that of the OR(-)/PR(-) (mean 4.7 ± 6.35) and OR(+)/PR(+) (mean 15.8 ± 11.95) (P = 0.033) tumours. Only three of the 36 OR(-)/PR(+) tumours completely lacked OR mRNA expression. The OR(-)/PR(+) tumours were not categorized as an independent group nor were they included in the other groups on post-hoc analysis. OR(-)/PR(+) tumours were associated with factors related to poor prognosis compared with OR(+)/PR(+) tumours, but OR(-)/PR(-) tumours were associated with the worst prognostic indicators. Further studies are required in order to determine the clinical significance of the OR(-)/PR(+) phenotype.

Differential and correlated expressions of p16/p21/p27/p38 in mammary gland tumors of aged dogs

The inhibitory effect of neutering on mammary gland tumor development in dogs has been well described. However, we observed that the effect of neutering on tumor malignancy may be altered by aging. Therefore, we characterized mammary tumors in aged dogs by analyzing the expression of cellular senescence markers. Expressions of p16, p38, p21, and p27 antibodies, which are senescence-associated markers, were assessed in canine mammary tumors of aged dogs via immunohistochemical analysis. In addition, correlations between those expressions were analyzed. Expression of p16 was negatively associated with strong nuclear p27 expression. Expression of p38 was observed in most of the mammary tumors examined, and negative p38 expression was related to positive p21 expression. Moreover, p21 expression was associated with p27 expression; negative p21 expression was associated with negative p27 expression, while positive p21 expression was associated with positive p27 expression. The results confirm that the p21- and p27-encoding genes have similar expression patterns in the mammary tumors of aged dogs. In the present study, we characterized the expression of cellular senescence markers in these tumors and elucidated the relationships among their expression patterns.

A novel synthetic chalcone derivative promotes caspase-dependent apoptosis through ROS generation and activation of the UPR in MH7A cells

Chalcones, one class of polyphenolic compounds synthesized as secondary metabolites during flavonoid biosynthesis in plants, have anti-inflammatory and antitumorigenic effects. In this study, we investigated the roles of a novel chalcone derivative, (E)-3-(3,5-dimethoxyphenyl)-1-(1-hydroxynaphthalen-2-yl)prop-2-en-1-one (DK-59), on rheumatoid arthritis (RA) fibroblast-like synoviocyte MH7A cells. DK-59 treatment reduced cell viability and induced caspase-mediated apoptosis in MH7A cells; activation of caspase-7 and caspase-3, as well as fragmentation of poly (ADP-ribose) polymerase (PARP-1), occurred in a time-dependent manner, and treatment with a pan-caspase inhibitor, Z-VAD, significantly reduced DK-59-mediated PARP-1 cleavage. DK-59-mediated apoptosis was caused by the production of intracellular reactive oxygen species (ROS); DK-59 treatment induced ROS production in a time-dependent manner, and treatment with the antioxidant N-acetyl-l-cysteine significantly inhibited DK-59-mediated fragmentation of PARP-1 and recovered cell viability. Many genes that function in the unfolded protein response (UPR), including inositol-requiring protein 1, X-box binding protein 1, and C/EBP homologous protein (CHOP), were upregulated by DK-59 treatment. CHOP knockdown by lentivirus infection reduced DK-59-mediated PARP-1 cleavage, suggesting that CHOP plays a proapoptotic role in DK-59-mediated apoptosis in MH7A cells. Taken together, the results suggest that DK-59-mediated induction of ROS and activation of the UPR can be used as a target for the therapeutic treatment of RA.