Mammary tumors are a major health threat to women and female dogs. In both, metastasis of the primary tumor to distant organs is the most common cause of tumor-related death. Nevertheless, the molecular mechanisms of tumor metastasis are far from being understood, and it is still unknown why some human and canine carcinomas metastasize and others do not. Using 2D-DIGE and MALDI-TOF-MS we identified 21 proteins with significant changes (fold change >1.5; p < 0.05) in protein expression between metastasizing (n = 6) and nonmetastasizing (n = 6) canine mammary carcinomas. Quantitative RT-PCR was used to identify transcriptional or post-transcriptional regulation of protein expression. Up-regulated proteins in metastatic carcinomas included proliferating cell nuclear antigen, ferritin light chain, bomapin, tropomyosin 3, thioredoxin-containing domain C5, adenosin, ornithine aminotransferase, coronin 1A, RAN-binding protein 1,3-phosphoglycerate dehydrogenase, and eukaryotic translation elongation factor 1. Down-regulated proteins in metastatic carcinomas included calretinin, myosin, light chain 2, peroxiredoxin 6, maspin, ibrinogen beta chain, vinculin, isocitrate dehydrogenase 1, tropomyosin 1, annexin A5, and Rho GTPase activating protein 1. Interestingly, 19 of these 21 proteins have been described with a malignancy-associated expression in human breast cancer and other human cancer types before. Further investigations are now necessary to test whether these markers are of prognostic value for canine mammary carcinomas and whether their expression is directly involved in canine mammary carcinogenesis or represent solely a secondary reactive phenotype.
Several markers of malignancy have been proposed for canine mammary tumors on the mRNA and protein levels. However, their association with tumor malignancy applies only for mean values of large groups of tumors, but no single marker identified to date can be used to reliably predict malignancy for individual tumors. A quantitative real-time reverse transcription polymerase chain reaction array was established to quantify the expression levels of 49 genes relevant to carcinogenesis in laser-microdissected tumor cells of 10 benign and 13 metastatic canine mammary tumors. Analysis of variance and discriminant analysis were used to identify relevant gene expression patterns that differentiate adenomas from metastatic carcinomas and their lymph node metastases. Seventeen genes with significant (P < .05) differences in gene expression levels between benign and malignant tumors were identified--including ERBB1, SLIT2, progesterone receptor, MIG6, SATB1, and SMAD6--but correct classification of each tumor as benign or malignant was impossible on the basis of any of these genes alone. However, the combined expression patterns of BMP2, LTBP4, and DERL1 (Derlin-1) correctly classified each individual tumor as benign or malignant. This pilot study identified a complex mRNA expression pattern of 3 genes that was able to identify malignancy in laser-microdissected tumor cells for each individual tumor, instead of group means as used in previous studies.
BackgroundCell adhesion is an important regulator of cell growth and motility. Recently the hepatocyte cell adhesion molecules 1 and 2 (HEPACAM1 and 2), members of the immunoglobulin family of adhesion genes, have been identified. HEPACAM1 is involved in negative cell cycle regulation via p53, p21 and p27 signalling but also mediates increased human breast cancer cell spread. The role and expression pattern of HEPACAM2 has not been analyzed so far. In the present study we quantified gene expression levels of HEPACAM1 and 2 to evaluate their possible role during the carcinogenesis of canine mammary tumours.ResultsAdenomas displayed increased HEPACAM1 and 2 mRNA expression levels and decreased HEPACAM1 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 1 protein and mRNA expression levels similar to normal gland but decreased HEPACAM2 mRNA expression when compared to normal gland of the same dog.ConclusionsHEPACAM1 and 2 seem to be important for cell-cell adhesion of normal and neoplastic canine mammary cells. The loss of HEPACAM1 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 HEPACAM2 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 HEPACAM1.
The molecular mechanisms of the development of canine mammary tumors are still incompletely understood. In the present study we hypothesized that there is a malignant progression from normal gland to malignant carcinomas that is associated with a linear change in protein expression. To this end, the proteome of canine normal mammary gland, adenomas, nonmetastatic carcinomas, and metastatic carcinomas was compared. Application of 2D-DIGE and MALDI-TOF-MS identified 48 proteins with significant changes (fold change >|1.5|; p < 0.05) in expression levels at the different stages of malignant progression. Forty-two of these followed three major stepwise but not linear expression patterns. Thirteen proteins showed the adenoma pattern characterized by a change in protein expression levels during progression from normal gland to adenomas which persisted on the same level at the subsequent stages of malignancy. Nine proteins followed the carcinoma pattern with an up- or down-regulation between adenomas and carcinomas. The majority of 20 proteins followed the metastasis pattern with a significant change of protein expression levels between nonmetastatic and metastatic carcinomas. The present study therefore shows that differences in malignancy are associated with a stepwise but not linear change in protein expression levels, which does not finally confirm or disapprove the existence of a malignant progression in canine mammary tumors. In addition, the acquisition of metastatic potential seems to be associated with the strongest changes in protein expression levels.
Insulin receptor (INSR) or insulin-like growth factor (IGF) signalling is speculated to be involved in mammary tumour development. Expression levels of members of the insulin receptor family (INSR, IGF1R, IGF2R, GHR) and their ligands IGF1and IGF2 were quantified in macro-and microdissected tissue samples of normal canine mammary gland, adenomas, carcinomas and their lymph node metastases to evaluate their potential impact on the carcinogenesis of canine mammary tumours. Normal mammary gland and adenomas had strong INSR expression, while carcinomas and metastases had significantly decreased expression. No differences were observed for IGF1R expression. IGF1, IGF2 and GHR mRNA expressions were strongly decreased in adenomas, carcinomas and metastases. INSR and IGF1R are therefore expressed in normal gland and adenomas and an increased stimulus by their ligands may be a proliferative stimulus in those tissues. However, decreased INSR expression carcinomas and their metastases render questionable its impact at late stages of carcinogenesis.
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