BACKGROUND
breast cancer is expected to be the most common malignancy among women in the united states in  <dig>  <cit> .
although several prognostic markers in human breast cancer have been investigated, only a small number of markers are in clinical use, possibly because of a poor correlation between the findings of animal studies and clinical trials.
therefore, there is a need for more appropriate therapeutic targets and additional models to improve the understanding and biological characterization of human breast cancer and therapeutic development.
ezh <dig> is a catalytic subunit of the epigenetic regulator polycomb repressive complex  <dig> .
prc <dig>  which includes ezh <dig>  suppressor of zeste  <dig> , and embryonic ectoderm development , trimethylates histone  <dig> lysine residue  <dig>  and leads to silencing of genes involved in processes such as stem cell maintenance and tumor progression without dna sequence modification .
recently, overexpression or mutation of ezh <dig> have been found in a wide range of human tumors, including breast, prostate, urinary bladder, ovarian, lung, gastric cancer, renal cell carcinoma, and glioblastoma .
the evidence indicates that ezh <dig> plays a role in the initiation, development, progression, and metastasis of cancer and drug resistance  <cit> .
in particular, ezh <dig> has been connected to the aggressiveness of breast cancer  <cit> .
in addition, ezh <dig> has been reported to be an adverse prognostic marker for breast cancer and an index of an unfavorable tamoxifen outcome .
a correlation between loss of differentiation and deregulated expression of ezh <dig> has also been proposed in human breast cancer  <cit> .
recent evidence implicates ezh <dig> in transcriptional activation, but the mechanisms are not clearly defined  <cit> .
in order to investigate the feasibility of an additional animal model to improve our understanding of ezh <dig>  we collected and investigated naturally occurring canine mammary tumors .
mammary tumors are the most commonly diagnosed neoplasms in female dogs and nearly 50 % are malignant  <cit> .
one study has found an annual incidence of mammary tumors of  <dig> – <dig>  %  and  <dig>  %   <cit> .
this incidence is greater than that of human breast cancers.
dog and human lineages are similar in terms of both nucleotide divergence and rearrangements  <cit>  and dogs have been suggested as additional tumor models  <cit> .
although histomorphological features may differ between human breast cancer and cmts, they share many similarities in terms of age of onset, risk factors, molecular marker expression, behavior, and prognosis .
in addition, the incidence of cmts is sufficiently high to secure proper number of subjects in clinical trials and the size of dogs makes multimodality protocols feasible  <cit> .
therefore, it is expected that the study of cancer using mammary tumors of domesticated dog might provide new insights into cancer understanding and therapy development.
the purpose of our present study was to investigate ezh <dig> in cmts.
we found that ezh <dig> is overexpressed in clinical samples of canine mammary carcinomas.
RESULTS
histological evaluation
the clinical and morphologic features of the  <dig> mammary gland cases were identified .
there were five non-neoplastic lesions including lobular hyperplasia and duct ectasia.
sixty-nine cmt cases showed benign morphological features  and malignant features  such as carcinoma with simple tubular or tubulopapillary type, complex type, mixed type, solid type, anaplastic and inflammatory carcinoma, mucinous carcinoma, lipid-rich carcinoma, and comedocarcinoma .
the most common type of carcinoma identified was the complex type , followed by mixed type  and simple tubular type .
canine mammary carcinomas exhibited a malignancy ranging from 1– <dig> as follows:  <dig> ,  <dig> , and  <dig> .table  <dig> demographics of canine mammary cases
fig.
<dig> representative mammary carcinoma tissues with h&e staining.
a carcinoma complex type with a malignant epithelial component and a benign myoepithelial component.
b carcinoma mixed type with a malignant epithelial component and a benign mesenchymal component .
c carcinoma, tubular type.
the tumor cells are predominantly arranged in a tubular pattern.
d comedocarcinoma.
there are necrotic areas within the center of neoplastic cell aggregates
immunohistochemistry
we were interested in determining whether ezh <dig> is dysregulated in cmts, which are similar to human breast cancers.
in immunohistochemical analyses, there was negative or weak nuclear staining in normal  and non-neoplastic mammary tissues.
clear nuclear staining for ezh <dig> could be observed in cmts .
the intensity of nuclear pattern was especially strong in comedocarcinoma , anaplastic carcinoma , and solid carcinoma.
carcinomas showed higher ezh <dig> expression level than hyperplastic lesions .
high expression of ezh <dig> was found to be associated with mammary carcinoma malignancy .
we sorted each grade of carcinomas according to the expression level of ezh <dig> to clarify the association between grade of malignancy and ezh <dig> expression.
malignancy grade  <dig> was found in  <dig> cases  of ezh <dig> score  <dig>   of ezh <dig> score  <dig>   <dig>  of ezh <dig> score  <dig>  and  <dig>  of ezh <dig> score  <dig>  malignancy grade  <dig> was seen in  <dig> cases  of ezh <dig> score  <dig>   <dig>  of ezh <dig> score  <dig>  and  <dig>  of ezh <dig> score  <dig>  malignancy grade  <dig> was present in no cases of ezh <dig> score  <dig>   <dig>  of ezh <dig> score  <dig>  and  <dig>  of ezh <dig> score  <dig>  according to these criteria, 99 % cases of cmts had elevated ezh <dig> expression.
it did not show any relationship within the most frequent three carcinoma types  of this study .fig.
<dig> immunohistchemistry for ezh <dig>  a normal mammary gland.
immunohistchemistry for ezh <dig> showing negative staining of the mammary gland.
b adenoma.
the intensity score of ezh <dig> is  <dig>  c carcinoma mixed type with malignancy grade  <dig>  the intensity score of ezh <dig> is  <dig>  d carcinoma solid type with malignancy grade  <dig>  diffuse nuclear staining with the intensity score  <dig> of ezh <dig>  e comedocarcinoma.
the intensity score of ezh <dig> is  <dig>  f anaplastic carcinoma.
note the strong nuclear staining of neoplastic cells with intensity score  <dig> of ezh2
fig.
<dig> bar graph of the immunohistochemical intensity scores of ezh <dig>  carcinoma demonstrated higher expression level of ezh <dig> than hyperplasia lesion
fig.
<dig> bar graph of the immunohistochemical intensity scores of ezh <dig>  high malignancy grade tumor showed higher expression level of ezh <dig> than low grade tumor
fig.
<dig> bar graph of the immunohistochemical intensity scores of ezh <dig>  there is no significant difference between three types of carcinoma
western blotting
we used immunoblotting to compare our immunohistochemistry and molecular data and further investigate the correlations between the grade of malignancy and the expression levels of ezh <dig>  western blot analysis showed an increase in the expression of ezh <dig> in the cmt tissues compared with control non-neoplastic mammary tissues.
grade  <dig> carcinomas showed higher expression of ezh <dig> than grades  <dig> and  <dig> .fig.
<dig> representative immunoblots of ezh2
discussion
although increased expression of ezh <dig> has been observed in aggressive solid tumors in humans, the mechanism involved in the mediation of tumor aggressiveness by ezh <dig> remains unclear.
in our current study, we characterized the expression pattern of ezh <dig> in cmts by immunohistochemical staining and immunoblot analysis of cmts and non-neoplastic mammary tissues.
we found that ezh <dig> protein is increased in cmts when compared with non-neoplastic mammary tissues.
in our immunohistochemical analysis, tumor cells exhibited a clear nuclear staining pattern with various expression levels.
when we compared ezh <dig> expression level with the tumor malignancy grade, type of carcinoma, and type of lesion  respectively, only carcinoma malignancy was found to have statistically significant association with ezh <dig> expression.
the proportion of positive ezh <dig> staining increased as the malignancy grade increased.
furthermore, there was strong nuclear staining in anaplastic carcinoma, comedocarcinoma which are regarded as high malignant neoplasms between cmts, and solid carcinoma which reveals poor differentiation.
these results are consistent with a previous report that showed a correlation between the ezh <dig> expression level and aggressiveness or poor differentiation of human breast cancer  <cit> .
we compared the immunohistochemistry results with the immunoblotting results.
ezh <dig> protein showed a higher expression level in grade  <dig> than grade  <dig> mammary cancers.
there were some results that were inconsistent with the tendency for ezh <dig> expression to increase with malignancy.
first, the number of cases was small and not enough to produce a powerful statistic.
we also suspected that one of the reasons for these discrepancies is that there is usually prominent proliferation of the surrounding connective tissue or stroma in cmt tissue.
therefore, homogenization of a small portion of tissue does not always adequately represent the epithelial tumorous component.
nonetheless, there was a tendency for an increased expression of ezh <dig> with cmt malignancy evident in the immunoblotting results, which was consistent with the immunohistochemistry results.
we found no correlation between the carcinoma type and the level of ezh <dig> expression, possibly due to the relatively small number of samples of each type of carcinoma.
murine models such as xenograft and transgenic mouse models have been extremely useful tools in the study of human cancer and have provided valuable insights into cancer biology and biochemistry that could not be readily obtained with other models  <cit> .
despite the importance of these murine models, they have shown a few limitations with respect to some essential features of human cancer, including growth periods, immune function, genomic characterization, and the significant heterogeneity of tumor cells and microenvironments.
even in recently developed patient-derived xenograft models, a higher mutation rate than would arise in the parent tumor, a variable transplantation failure rate, as well as increased costs are major challenges of this approach  <cit> .
accordingly, dogs with naturally occurring tumors are expected to provide an additional value to researchers.
as the cmts in our current study in a canine model showed similarities to human breast cancers in terms of ezh <dig> expression, we suggest that dogs with naturally occurring cmts could be used as animal models in future clinical trials.
canine tumor models may help to identify novel cancer-associated genes, further elucidate molecular pathways in tumors, and be used in the development of novel diagnostic, prognostic, and therapeutic tools  <cit> .
no ezh <dig> inhibitors have been approved for the treatment of human cancers to date.
the methyltransferase activity of ezh <dig> is not required for it to activate certain genes.
therefore, approaches based on disrupting the interaction between ezh <dig> and other factors might be potential therapeutic targets  <cit> .
because ezh <dig> plays a diverse role in cancers, insight into the regulation of its signaling would likely aid the development of ezh2-targeted therapeutics.
ezh <dig> inhibitors are currently being developed and clinical trials for b cell lymphoma are ongoing  <cit> .
to our knowledge, our current study is the first to report on the expression of ezh <dig> in cmts.
our results lend credence to the view that cmts are a valuable model for ezh <dig> studies.
however, we have only just begun to understand the biology and functional role of ezh <dig> in cmts, and we look forward to studies aimed at further elucidating the mechanisms involved.
CONCLUSIONS
ezh <dig> is expressed in cmts, and its levels correlate with carcinoma malignancy.
this leads to a possibility of cmt usage as a model for future ezh <dig> research and clinical trials on breast cancer.

 breast cancer is expected to be the most common malignancy among women in the united states in. although several prognostic markers in human breast cancer have been investigated, only a small number of markers are in clinical use, possibly because of a poor correlation between the findings of animal studies and clinical trials. therefore, there is a need for more appropriate therapeutic targets and additional models to improve the understanding and biological characterization of human breast cancer and therapeutic development. ezh is a catalytic subunit of the epigenetic regulator polycomb repressive complex. prc which includes ezh suppressor of zeste, and embryonic ectoderm development, trimethylates histone lysine residue and leads to silencing of genes involved in processes such as stem cell maintenance and tumor progression without dna sequence modification. recently, overexpression or mutation of ezh have been found in a wide range of human tumors, including breast, prostate, urinary bladder, ovarian, lung, gastric cancer, renal cell carcinoma, and glioblastoma. the evidence indicates that ezh plays a role in the initiation, development, progression, and metastasis of cancer and drug resistance. in particular, ezh has been connected to the aggressiveness of breast cancer. in addition, ezh has been reported to be an adverse prognostic marker for breast cancer and an index of an unfavorable tamoxifen outcome. a correlation between loss of differentiation and deregulated expression of ezh has also been proposed in human breast cancer. recent evidence implicates ezh in transcriptional activation, but the mechanisms are not clearly defined. in order to investigate the feasibility of an additional animal model to improve our understanding of ezh we collected and investigated naturally occurring canine mammary tumors. mammary tumors are the most commonly diagnosed neoplasms in female dogs and nearly 50 % are malignant. one study has found an annual incidence of mammary tumors of –  % and  %. this incidence is greater than that of human breast cancers. dog and human lineages are similar in terms of both nucleotide divergence and rearrangements and dogs have been suggested as additional tumor models. although histomorphological features may differ between human breast cancer and cmts, they share many similarities in terms of age of onset, risk factors, molecular marker expression, behavior, and prognosis. in addition, the incidence of cmts is sufficiently high to secure proper number of subjects in clinical trials and the size of dogs makes multimodality protocols feasible. therefore, it is expected that the study of cancer using mammary tumors of domesticated dog might provide new insights into cancer understanding and therapy development. the purpose of our present study was to investigate ezh in cmts. we found that ezh is overexpressed in clinical samples of canine mammary carcinomas.  histological evaluation the clinical and morphologic features of the mammary gland cases were identified. there were five non-neoplastic lesions including lobular hyperplasia and duct ectasia. sixty-nine cmt cases showed benign morphological features and malignant features such as carcinoma with simple tubular or tubulopapillary type, complex type, mixed type, solid type, anaplastic and inflammatory carcinoma, mucinous carcinoma, lipid-rich carcinoma, and comedocarcinoma. the most common type of carcinoma identified was the complex type, followed by mixed type and simple tubular type. canine mammary carcinomas exhibited a malignancy ranging from 1– as follows:,, and.table demographics of canine mammary cases fig. representative mammary carcinoma tissues with h&e staining. a carcinoma complex type with a malignant epithelial component and a benign myoepithelial component. b carcinoma mixed type with a malignant epithelial component and a benign mesenchymal component. c carcinoma, tubular type. the tumor cells are predominantly arranged in a tubular pattern. d comedocarcinoma. there are necrotic areas within the center of neoplastic cell aggregates immunohistochemistry we were interested in determining whether ezh is dysregulated in cmts, which are similar to human breast cancers. in immunohistochemical analyses, there was negative or weak nuclear staining in normal and non-neoplastic mammary tissues. clear nuclear staining for ezh could be observed in cmts. the intensity of nuclear pattern was especially strong in comedocarcinoma, anaplastic carcinoma, and solid carcinoma. carcinomas showed higher ezh expression level than hyperplastic lesions. high expression of ezh was found to be associated with mammary carcinoma malignancy. we sorted each grade of carcinomas according to the expression level of ezh to clarify the association between grade of malignancy and ezh expression. malignancy grade was found in cases of ezh score of ezh score of ezh score and of ezh score malignancy grade was seen in cases of ezh score of ezh score and of ezh score malignancy grade was present in no cases of ezh score of ezh score and of ezh score according to these criteria, 99 % cases of cmts had elevated ezh expression. it did not show any relationship within the most frequent three carcinoma types of this study.fig. immunohistchemistry for ezh a normal mammary gland. immunohistchemistry for ezh showing negative staining of the mammary gland. b adenoma. the intensity score of ezh is c carcinoma mixed type with malignancy grade the intensity score of ezh is d carcinoma solid type with malignancy grade diffuse nuclear staining with the intensity score of ezh e comedocarcinoma. the intensity score of ezh is f anaplastic carcinoma. note the strong nuclear staining of neoplastic cells with intensity score of ezh2 fig. bar graph of the immunohistochemical intensity scores of ezh carcinoma demonstrated higher expression level of ezh than hyperplasia lesion fig. bar graph of the immunohistochemical intensity scores of ezh high malignancy grade tumor showed higher expression level of ezh than low grade tumor fig. bar graph of the immunohistochemical intensity scores of ezh there is no significant difference between three types of carcinoma western blotting we used immunoblotting to compare our immunohistochemistry and molecular data and further investigate the correlations between the grade of malignancy and the expression levels of ezh western blot analysis showed an increase in the expression of ezh in the cmt tissues compared with control non-neoplastic mammary tissues. grade carcinomas showed higher expression of ezh than grades and.fig. representative immunoblots of ezh2 discussion although increased expression of ezh has been observed in aggressive solid tumors in humans, the mechanism involved in the mediation of tumor aggressiveness by ezh remains unclear. in our current study, we characterized the expression pattern of ezh in cmts by immunohistochemical staining and immunoblot analysis of cmts and non-neoplastic mammary tissues. we found that ezh protein is increased in cmts when compared with non-neoplastic mammary tissues. in our immunohistochemical analysis, tumor cells exhibited a clear nuclear staining pattern with various expression levels. when we compared ezh expression level with the tumor malignancy grade, type of carcinoma, and type of lesion respectively, only carcinoma malignancy was found to have statistically significant association with ezh expression. the proportion of positive ezh staining increased as the malignancy grade increased. furthermore, there was strong nuclear staining in anaplastic carcinoma, comedocarcinoma which are regarded as high malignant neoplasms between cmts, and solid carcinoma which reveals poor differentiation. these results are consistent with a previous report that showed a correlation between the ezh expression level and aggressiveness or poor differentiation of human breast cancer. we compared the immunohistochemistry results with the immunoblotting results. ezh protein showed a higher expression level in grade than grade mammary cancers. there were some results that were inconsistent with the tendency for ezh expression to increase with malignancy. first, the number of cases was small and not enough to produce a powerful statistic. we also suspected that one of the reasons for these discrepancies is that there is usually prominent proliferation of the surrounding connective tissue or stroma in cmt tissue. therefore, homogenization of a small portion of tissue does not always adequately represent the epithelial tumorous component. nonetheless, there was a tendency for an increased expression of ezh with cmt malignancy evident in the immunoblotting results, which was consistent with the immunohistochemistry results. we found no correlation between the carcinoma type and the level of ezh expression, possibly due to the relatively small number of samples of each type of carcinoma. murine models such as xenograft and transgenic mouse models have been extremely useful tools in the study of human cancer and have provided valuable insights into cancer biology and biochemistry that could not be readily obtained with other models. despite the importance of these murine models, they have shown a few limitations with respect to some essential features of human cancer, including growth periods, immune function, genomic characterization, and the significant heterogeneity of tumor cells and microenvironments. even in recently developed patient-derived xenograft models, a higher mutation rate than would arise in the parent tumor, a variable transplantation failure rate, as well as increased costs are major challenges of this approach. accordingly, dogs with naturally occurring tumors are expected to provide an additional value to researchers. as the cmts in our current study in a canine model showed similarities to human breast cancers in terms of ezh expression, we suggest that dogs with naturally occurring cmts could be used as animal models in future clinical trials. canine tumor models may help to identify novel cancer-associated genes, further elucidate molecular pathways in tumors, and be used in the development of novel diagnostic, prognostic, and therapeutic tools. no ezh inhibitors have been approved for the treatment of human cancers to date. the methyltransferase activity of ezh is not required for it to activate certain genes. therefore, approaches based on disrupting the interaction between ezh and other factors might be potential therapeutic targets. because ezh plays a diverse role in cancers, insight into the regulation of its signaling would likely aid the development of ezh2-targeted therapeutics. ezh inhibitors are currently being developed and clinical trials for b cell lymphoma are ongoing. to our knowledge, our current study is the first to report on the expression of ezh in cmts. our results lend credence to the view that cmts are a valuable model for ezh studies. however, we have only just begun to understand the biology and functional role of ezh in cmts, and we look forward to studies aimed at further elucidating the mechanisms involved.  ezh is expressed in cmts, and its levels correlate with carcinoma malignancy. this leads to a possibility of cmt usage as a model for future ezh research and clinical trials on breast cancer. 
investigation of the expression of ezh in canine mammary tumors revealed that ezh protein was overexpressed in canine mammary carcinomas, as in human breast cancer. in our present study, we were interested in determining whether ezh is increased in canine mammary tumors, which show similarities to human breast cancer. this is the first report to describe ezh expression in canine mammary tumors. because the expression of ezh was similar in canine mammary carcinoma and human breast cancer, spontaneous canine mammary tumors may be a suitable model for studying ezh and treatment development. in humans, ezh is linked to oncogenic function in several carcinomas, including breast cancer, and dysregulation of ezh has been particularly associated with loss of differentiation and the development of poorly differentiated breast cancer.
 current studies report that aberrations in epigenetic regulators or chromatin modifications are related to tumor development and maintenance. ezh is one of the catalytic subunits of polycomb repressive complex a crucial epigenetic regulator. ezh has a master regulatory function in such processes as cell proliferation, stem cell differentiation, and early embryogenesis. in humans, ezh is linked to oncogenic function in several carcinomas, including breast cancer, and dysregulation of ezh has been particularly associated with loss of differentiation and the development of poorly differentiated breast cancer. in our present study, we were interested in determining whether ezh is increased in canine mammary tumors, which show similarities to human breast cancer.  investigation of the expression of ezh in canine mammary tumors revealed that ezh protein was overexpressed in canine mammary carcinomas, as in human breast cancer. in addition, the immunohistochemical expression level of ezh was associated with the degree of malignancy in canine mammary carcinoma. this is the first report to describe ezh expression in canine mammary tumors.  because the expression of ezh was similar in canine mammary carcinoma and human breast cancer, spontaneous canine mammary tumors may be a suitable model for studying ezh and treatment development. keywords dogcanine mammary tumorezh2comparative oncologyhttp://dx.doi.org/ /501100004726ministry of health & welfarehi06c0868hi10c2014son woo-chan issue-copyright-statement© the author 2016 