The constitutively active Stat5 and its C-terminally truncated variant have been implicated in cancer in laboratory animals and humans. Here we established a model system, based on mice expressing the individual transgenic STAT5 variants, to study the distinct variants' oncogenic role in the mammary gland.
Stat5 mediates proliferation in the mammary gland of pregnant females [46
], and different effects of its two variants have been demonstrated in transgenic mice during the reproductive cycle: STAT5ca-induced proliferation during pregnancy and delayed apoptosis and tissue remodeling during involution [11
]. The opposite has been shown for the STAT5Δ750 variant [11
]. This diversity results from the structural differences between the STAT5 variants which involves either forced activation or lack of TAD-mediated interactions with the proteins, such as CBP/p300, that recruit acetylases [47
]. Both STAT5 variants seem to have a persistent effect, probably on chromatin structure and accessibility [16
], which results in different profiles of gene expression in the developing tumors.
Differential expression of 364 genes was found between mammary tumors developed in BLG/STAT5ca- and BLG/STAT5Δ750-transgenic mice. These genes were involved in a set of cellular activities, many of which could still be associated with the two main processes mediated by Stat5 in the intact tissue: cell proliferation and cell death. The individual genes that established the specific mark of the STAT5 variants in the tumors were identified by gene-array analysis and their differential expression in the population was confirmed in a distinct "test set" of tumors using a different detection method, real-time PCR. Of special interest was the gene encoding the proto-oncogene Met, which was expressed in the STAT5Δ750-induced tumors at a 22-fold higher level than in their STAT5ca-induced counterparts. The product of Met is the hepatocyte growth factor receptor which encodes tyrosine-kinase activity. The ligand-activated cytoplasmic domain of the c-Met receptor induces growth motility, morphogenesis and angiogenesis [50
]. In breast cancer, c-Met overexpression is associated with tumor progression (reviewed in [51
]) and has an independent predictive value for poor survival, even in early-stage patients with negative lymph nodes [52
]. Expression levels of Met were upregulated relative to intact tissue in the STAT5Δ750-induced tumors and downregulated in tumors developed in mice carrying the BLG/STAT5ca transgene. This differential expression suggests a more aggressive downstream cascade in the former. Other genes with exceptionally high expression levels in the STAT5Δ750-induced tumors, which were not affected by the STAT5ca variant, were proliferin and Igf2. Proliferin is a member of the prolactin family which is involved in progenitor cell expansion along the luminal and myoepithelial lineage [53
] and Igf2 plays a pivotal role in fetal and cancer development by signaling via the IGF-I and insulin receptors, and activating the estrogen-signaling cascade [54
]. Unfortunately, studies on these genes [55
] do not include or base additional lists of genes with altered expression profiles that might be compared to our data and aid in delineating the pathway(s) involved in the tumorigenic effect of STAT5Δ750.
A group of genes regulating carbohydrate metabolism and transport could also be discerned. These genes were not associated with Stat5 effects in the intact mammary gland but were differentially expressed in the two sets of tumors. Their role in the tumors could be related to altered levels of metabolism (i.e. higher metabolic rate in the STAT5Δ750-induced tumors) rather than cancer growth per se.
Expression of an additional set of 14 genes linked the effects of the BLG/STAT5Δ750 and the BLG/STAT5ca transgenes to the resulting phenotypes of poorly differentiated carcinoma or highly differentiated papillary adenocarcinoma, respectively (r = 0.97) [see Additional file 1
, data sheet G and ]. These genes cover a wide variety of cellular functions: calcium sensitivity of the myofibrils (troponin I and tropomyosin 1), interaction between the cell and the extracellular matrix (endomucin), normal adipose tissue development (lipin 3), lipid metabolism, cellular growth and apoptosis (caveolin 2), mRNA metabolism (5'-3' exoribonuclease 1), mitochondrial fatty acid oxidation (acyl coenzyme A dehydrogenase), as well as kinase and protease activities (TAU tubulin kinase 1 and corin, respectively). Taken together, their diverse expression is most likely involved in determining tumor phenotype, i.e. associating higher proportions of the poorly differentiated carcinomas or the highly differentiated papillary adenocarcinomas with the expression of STAT5Δ750 or STAT5ca, respectively [15
Overall, 94% of the genes specifically affected by STAT5ca were downregulated relative to their expression in the host tissue. This contrasts with the more equal specific effect of the STAT5Δ750 variant and provides additional evidence for the distinct routes via which the two STAT5 variants impose their mark on tumor growth and maintenance. The high number of tumor and growth suppressors defined among the STAT5ca-downregulated genes vs. the potent oncogenes (Met, Igf2) that were induced by the STAT5Δ750 variant may serve to further distinguish the routes via which they initiate and maintain tumorigenesis.
Substantial downregulation of gene expression has been demonstrated in breast cancers with bone marrow (BM) micrometastasis [58
], and during the molecular transition from organ-confined to metastatic prostate cancer [59
]. Apparently, transcription repression is important for the metastatic process in these tissues. When compared with the list of genes downregulated by STAT5ca-, no overlap was observed for those associated with BM micrometastasis, and only four genes (Ptn, Cflar, Cnn1 and Mpdz) shared the list of downregulated genes mediating prostate metastasis. Thus, the resultant tumor characteristics are probably determined by a combination of the phenomenon of gene downregulation per se
and the properties of the specifically affected genes (in this case by STAT5ca).
Our attempt to characterize the different roles of the STAT5 variants in mammary cancer development did not produce any evidence for the effect of transgenic STAT5 expression in the tumors. Neither distinct metabolic pathways nor a central mediator(s) were located. This suggests domination of an earlier STAT5 effect on the resulting gene-expression profiles. Several groups of genes, each composed of three to eight members, with significant internal correlations were identified among the sets specifically affected by STAT5ca and STAT5Δ750. This correlation was not associated with physical linkage. Within a single group, genes with correlated expression might control a distinct range of cellular functions such as angiogenesis (Ptn and Tyrp1 [60
]), apoptosis (Cflar, [62
]) and morphogenesis (Ebf2, [63
]). Their comparable levels of expression, defined by their location in a confined region within the cluster, suggest that although these genes are mapped to several loci, they may colocalize to a shared transcription site. The concept of several loci being targeted to a shared transcription site where they generate "transcription factories" with similar levels of expression was proposed a few years ago and is reviewed in [64
]. The relevance of this biological system to the specific STAT5 effects presented in this study remains to be determined. Regardless of the detailed mechanism involved in the effect of STAT5Δ750 and STAT5ca on tumor growth and maintenance, this study establishes the feasibility of identifying and distinguishing mammary tumors according the variants' signature on specific gene-expression profiles. To the best of our knowledge, this signature is specific to the effect of the STAT5 variants. However, its uniqueness will only be confirmed by the contribution of further gene-expression profiles that are specific for the effects of other oncogenes.