BACKGROUND
cell adhesion to neighbouring cells and the extracellular matrix is a dynamic process and essential for the maintenance of the cellular differentiation status.
in contrast, metastatic progression of epithelial tumours is regularly associated with loss of cell-cell contact and a decrease in cell differentiation.
the same is obviously true for canine mammary tumours that regularly show metastatic spread to distant organs including the lung.
it is therefore not surprising that invasive character and metastatic spread of canine mammary tumours  is associated with decreased expression of cell adhesion associated genes like e-cadherin, connexin  <dig>   <dig> and paxillin  <cit> .
however, these genes not only act as a cellular anchor but also strongly influence cellular proliferation and migratory status through complex downstream signalling cascades  <cit> .
recently we found a significant decrease in expression levels of the cell cycle inhibitor p <dig> in metastatic canine mammary carcinomas and their lymph node metastases  <cit> .
so far, decreased expression of transforming growth factor β  family members has been identified as a possible cause of decreased p <dig> expression levels and increased cellular proliferation of canine mammary tumours  <cit> .
however, due to the ambivalent anti-proliferative and growth stimulatory roles of tgfβ at different stages of mammary tumour carcinogenesis other upstream regulators may be involved in the release of cell cycle brakes like p <dig>  <cit> .
the hepatocyte cell adhesion molecule  <dig>  has been identified as a link between cell cycle arrest at the g2/m junction via p <dig>  p <dig> and p <dig> mediated mechanisms in a breast cancer cell line  <cit> .
however, hepacam <dig> also modulates cell-extracellular matrix interaction and supports breast cancer cell spread  <cit> .
on the transcriptional level hepacam <dig> expression is decreased in several human cancer types including breast cancer but protein expression was not analyzed so far  <cit> .
the role of the hepacam family member  <dig>  in normal and neoplastic mammary epithelial cells is unknown.
due to the findings in human tissues we hypothesized that hepacam <dig> expression might be involved in the decreased p <dig> expression levels in canine mammary tumours.
in the present study we therefore characterized the differential mrna expression of hepacam <dig> and  <dig> in laser microdissected frozen tissue samples and hepacam <dig> protein expression in normal mammary gland epithelium, adenomas and metastatic canine mammary carcinomas, intravascular tumour cells and lymph node metastases.
RESULTS
mrna and protein expression levels of hepacam <dig> in normal gland, primary tumours, intravascular tumour cells and lymph node metastases
hepacam <dig> mrna expression was significantly increased in adenomas when compared to normal gland, carcinomas and lymph node metastases .
specifically, nine out of ten adenomas had mrna expression levels higher than  <dig>  fold of the normal gland of the same dog.
in contrast, hepacam <dig> mrna expression was not significantly changed in carcinomas and their lymph node metastases when compared to normal gland .
ivtc: intravascular tumour cells
* significant differences  in mrna expression levels were observed between microdissected adenomas/carcinomas and adenomas/lymph node metastases
hepacam <dig> protein was expressed at the cell membranes of all normal gland specimens .
furthermore, 94% of carcinomas, 95% of intravascular tumour cells and 89% lymph node metastases had hepacam <dig> protein expression in more than 10% of cells .
surprisingly, only  <dig> percent of adenomas had membrane-bound hepacam <dig> protein expression , despite their increased hepacam <dig> transcription levels.
mrna expression levels of hepacam <dig> in normal gland, primary tumours and metastases
hepacam <dig> mrna expression was significantly increased in microdissected adenomas when compared to normal gland, carcinomas and lymph node metastases .
in detail, seven out of ten adenomas had mrna expression levels higher than  <dig>  fold of the normal gland hepacam <dig> expression of the same dog.
in contrast,  <dig> out of  <dig> carcinomas and  <dig> out of  <dig> lymph node metastases had decreased hepacam <dig> mrna expression levels lower than  <dig>  fold of the normal gland epithelium of the same dog .
discussion
the exact mechanisms of canine mammary tumour metastasis are unknown and this is one reason that there are currently no treatment options available for this tumour type except excision of the primary tumour.
in the present study we therefore asked whether hepacam <dig> is a potential tumour suppressor in canine mammary gland and whether its expression is decreased similar to human breast cancer.
furthermore, we aimed at identifying the hepacam <dig> expression levels at different stages of malignant progression of canine mammary gland epithelium.
normal mammary gland, carcinomas and metastatic cells constantly expressed membrane-bound hepacam <dig>  in contrast, almost none of the adenomas expressed hepacam <dig> protein despite increased hepacam <dig> mrna expression levels.
these surprising findings raise several questions regarding potential roles of hepacam <dig> during malignant transformation of canine mammary tumours.
assuming that the hepacam <dig> protein expressed in carcinomas is functionally active, the role of hepacam <dig> as a tumour suppressor via p <dig>  p <dig> and p <dig> in canine mammary tumours is questionable.
on the contrary, it can be hypothesized that hepacam <dig> expression is associated with increased metastatic spread and cell motility as has been shown for human breast cancer cells before  <cit> .
it will be interesting to analyze in an additional study whether non-metastatic carcinomas differ from metastatic carcinomas by decreased expression levels of hepacam <dig> protein.
however, hepacam  <dig> obviously has also a physiologic role in normal mammary gland cell adhesion and maybe in cell cycle control since virtually all normal gland epithelial cells express the protein.
the differences in hepacam <dig> mrna and protein expression levels in adenomas when compared to carcinomas indicate that decreased hepacam <dig> protein levels in adenomas is caused by failure of mrna translation or increased proteolysis.
however, decreased protein expression levels may induce increased hepacam <dig> transcription in adenomas in a feedback mechanism, although other so far unknown transcription inducers may also cause the increased mrna expression levels.
the function and expression pattern of the expected immunoglobulin-like, membrane protein hepacam <dig> has not been analyzed in any species so far.
in the present study, we found an increased hepacam <dig> expression in adenomas when compared to normal gland.
in contrast, carcinomas and lymph node metastases had a constant and marked decrease in hepacam <dig> expression levels.
hepacam <dig> and  <dig> mrna expression levels are therefore similar in adenomas, while carcinomas and metastases have decreased hepacam <dig> but not hepacam <dig> mrna expression levels.
it can therefore be speculated that hepacam <dig> expression decreases with increasing malignancy of canine mammary tumours as is usually observed for cell-adhesion genes in a wide variety of tumours types.
however, evaluation of the protein expression pattern is needed to support the findings for hepacam <dig> on the transcriptional level.
unfortunately, hepacam <dig> specific antibodies are not available so far for any species.
CONCLUSIONS
a significant decrease in hepacam <dig> protein expression was found in canine mammary adenomas.
in contrast, hepacam <dig> protein was constantly expressed in normal mammary gland, carcinomas, intravascular tumour cells and metastatic cells.
the data question the role of hepacam <dig> as an effective tumour suppressor at late stages of canine mammary carcinogenesis.
in contrast, the hepacam <dig> expression pattern makes it more likely that the protein is involved in physiologic cell adhesion of the canine mammary epithelium and also in metastatic spread of canine mammary tumours.
hepacam <dig> transcription levels were decreased in mammary carcinomas but further analysis of the function and the expression pattern of the hepacam <dig> protein are needed to evaluate its role in normal mammary gland physiology and malignant transformation.

 cell adhesion to neighbouring cells and the extracellular matrix is a dynamic process and essential for the maintenance of the cellular differentiation status. in contrast, metastatic progression of epithelial tumours is regularly associated with loss of cell-cell contact and a decrease in cell differentiation. the same is obviously true for canine mammary tumours that regularly show metastatic spread to distant organs including the lung. it is therefore not surprising that invasive character and metastatic spread of canine mammary tumours is associated with decreased expression of cell adhesion associated genes like e-cadherin, connexin and paxillin. however, these genes not only act as a cellular anchor but also strongly influence cellular proliferation and migratory status through complex downstream signalling cascades. recently we found a significant decrease in expression levels of the cell cycle inhibitor p in metastatic canine mammary carcinomas and their lymph node metastases. so far, decreased expression of transforming growth factor β family members has been identified as a possible cause of decreased p expression levels and increased cellular proliferation of canine mammary tumours. however, due to the ambivalent anti-proliferative and growth stimulatory roles of tgfβ at different stages of mammary tumour carcinogenesis other upstream regulators may be involved in the release of cell cycle brakes like p. the hepatocyte cell adhesion molecule has been identified as a link between cell cycle arrest at the g2/m junction via p p and p mediated mechanisms in a breast cancer cell line. however, hepacam also modulates cell-extracellular matrix interaction and supports breast cancer cell spread. on the transcriptional level hepacam expression is decreased in several human cancer types including breast cancer but protein expression was not analyzed so far. the role of the hepacam family member in normal and neoplastic mammary epithelial cells is unknown. due to the findings in human tissues we hypothesized that hepacam expression might be involved in the decreased p expression levels in canine mammary tumours. in the present study we therefore characterized the differential mrna expression of hepacam and in laser microdissected frozen tissue samples and hepacam protein expression in normal mammary gland epithelium, adenomas and metastatic canine mammary carcinomas, intravascular tumour cells and lymph node metastases.  mrna and protein expression levels of hepacam in normal gland, primary tumours, intravascular tumour cells and lymph node metastases hepacam mrna expression was significantly increased in adenomas when compared to normal gland, carcinomas and lymph node metastases. specifically, nine out of ten adenomas had mrna expression levels higher than fold of the normal gland of the same dog. in contrast, hepacam mrna expression was not significantly changed in carcinomas and their lymph node metastases when compared to normal gland. ivtc: intravascular tumour cells * significant differences in mrna expression levels were observed between microdissected adenomas/carcinomas and adenomas/lymph node metastases hepacam protein was expressed at the cell membranes of all normal gland specimens. furthermore, 94% of carcinomas, 95% of intravascular tumour cells and 89% lymph node metastases had hepacam protein expression in more than 10% of cells. surprisingly, only percent of adenomas had membrane-bound hepacam protein expression, despite their increased hepacam transcription levels. mrna expression levels of hepacam in normal gland, primary tumours and metastases hepacam mrna expression was significantly increased in microdissected adenomas when compared to normal gland, carcinomas and lymph node metastases. in detail, seven out of ten adenomas had mrna expression levels higher than fold of the normal gland hepacam expression of the same dog. in contrast, out of carcinomas and out of lymph node metastases had decreased hepacam mrna expression levels lower than fold of the normal gland epithelium of the same dog. discussion the exact mechanisms of canine mammary tumour metastasis are unknown and this is one reason that there are currently no treatment options available for this tumour type except excision of the primary tumour. in the present study we therefore asked whether hepacam is a potential tumour suppressor in canine mammary gland and whether its expression is decreased similar to human breast cancer. furthermore, we aimed at identifying the hepacam expression levels at different stages of malignant progression of canine mammary gland epithelium. normal mammary gland, carcinomas and metastatic cells constantly expressed membrane-bound hepacam in contrast, almost none of the adenomas expressed hepacam protein despite increased hepacam mrna expression levels. these surprising findings raise several questions regarding potential roles of hepacam during malignant transformation of canine mammary tumours. assuming that the hepacam protein expressed in carcinomas is functionally active, the role of hepacam as a tumour suppressor via p p and p in canine mammary tumours is questionable. on the contrary, it can be hypothesized that hepacam expression is associated with increased metastatic spread and cell motility as has been shown for human breast cancer cells before. it will be interesting to analyze in an additional study whether non-metastatic carcinomas differ from metastatic carcinomas by decreased expression levels of hepacam protein. however, hepacam obviously has also a physiologic role in normal mammary gland cell adhesion and maybe in cell cycle control since virtually all normal gland epithelial cells express the protein. the differences in hepacam mrna and protein expression levels in adenomas when compared to carcinomas indicate that decreased hepacam protein levels in adenomas is caused by failure of mrna translation or increased proteolysis. however, decreased protein expression levels may induce increased hepacam transcription in adenomas in a feedback mechanism, although other so far unknown transcription inducers may also cause the increased mrna expression levels. the function and expression pattern of the expected immunoglobulin-like, membrane protein hepacam has not been analyzed in any species so far. in the present study, we found an increased hepacam expression in adenomas when compared to normal gland. in contrast, carcinomas and lymph node metastases had a constant and marked decrease in hepacam expression levels. hepacam and mrna expression levels are therefore similar in adenomas, while carcinomas and metastases have decreased hepacam but not hepacam mrna expression levels. it can therefore be speculated that hepacam expression decreases with increasing malignancy of canine mammary tumours as is usually observed for cell-adhesion genes in a wide variety of tumours types. however, evaluation of the protein expression pattern is needed to support the findings for hepacam on the transcriptional level. unfortunately, hepacam specific antibodies are not available so far for any species.  a significant decrease in hepacam protein expression was found in canine mammary adenomas. in contrast, hepacam protein was constantly expressed in normal mammary gland, carcinomas, intravascular tumour cells and metastatic cells. the data question the role of hepacam as an effective tumour suppressor at late stages of canine mammary carcinogenesis. in contrast, the hepacam expression pattern makes it more likely that the protein is involved in physiologic cell adhesion of the canine mammary epithelium and also in metastatic spread of canine mammary tumours. hepacam transcription levels were decreased in mammary carcinomas but further analysis of the function and the expression pattern of the hepacam protein are needed to evaluate its role in normal mammary gland physiology and malignant transformation. 
in contrast, metastatic carcinomas, intravascular tumour cells and lymph node metastases had hepacam protein and mrna expression levels similar to normal gland but decreased hepacam mrna expression when compared to normal gland of the same dog. in the present study we quantified gene expression levels of hepacam and to evaluate their possible role during the carcinogenesis of canine mammary tumours. furthermore, it can be speculated, whether hepacam plays a different role in malignancy and metastasis of canine mammary tumours since its transcriptional levels are different in carcinomas and their lymph node metastases when compared to hepacam cell adhesion is an important regulator of cell growth and motility. recently the hepatocyte cell adhesion molecules and, members of the immunoglobulin family of adhesion genes, have been identified. hepacam and seem to be important for cell-cell adhesion of normal and neoplastic canine mammary cells. the loss of hepacam protein expression in adenomas but not in carcinomas questions its role as a tumour suppressor at late stages of malignant transformation and indicates that it might rather be involved in physiologic mammary cell adhesion and canine mammary tumour metastasis. adenomas displayed increased hepacam and mrna expression levels and decreased hepacam protein expression levels when compared to normal gland, carcinomas and lymph node metastases. the role and expression pattern of hepacam has not been analyzed so far.
 cell adhesion is an important regulator of cell growth and motility. recently the hepatocyte cell adhesion molecules and, members of the immunoglobulin family of adhesion genes, have been identified. hepacam is involved in negative cell cycle regulation via p p and p signalling but also mediates increased human breast cancer cell spread. the role and expression pattern of hepacam has not been analyzed so far. in the present study we quantified gene expression levels of hepacam and to evaluate their possible role during the carcinogenesis of canine mammary tumours.  adenomas displayed increased hepacam and mrna expression levels and decreased hepacam protein expression levels when compared to normal gland, carcinomas and lymph node metastases. in contrast, metastatic carcinomas, intravascular tumour cells and lymph node metastases had hepacam protein and mrna expression levels similar to normal gland but decreased hepacam mrna expression when compared to normal gland of the same dog.  hepacam and seem to be important for cell-cell adhesion of normal and neoplastic canine mammary cells. the loss of hepacam protein expression in adenomas but not in carcinomas questions its role as a tumour suppressor at late stages of malignant transformation and indicates that it might rather be involved in physiologic mammary cell adhesion and canine mammary tumour metastasis. furthermore, it can be speculated, whether hepacam plays a different role in malignancy and metastasis of canine mammary tumours since its transcriptional levels are different in carcinomas and their lymph node metastases when compared to hepacam 