Sca-1 is widely accepted as a stem/progenitor cell marker in normal mouse tissues 
. However, Sca-1eGFP/eGFP
mice did not exhibit a reproducible phenotype on mammary gland development in our laboratory (unpublished data). Previous studies have shown that Sca-1 positive cells are expanded in Wnt/β-catenin induced mammary tumors 
. Additionally, a Ly6 family member, Ly-6D is upregulated in a variety of murine tumors and triple-negative breast cancers 
. Despite these associations there is limited knowledge of functional role of Sca-1. Our findings indicate that Sca-1 plays an important role in mammary tumorigenesis as revealed using a novel cell line derived from MMTV-Wnt-1 mouse mammary tumors. First, Sca-1 promotes cell migration and affects cell adhesion to several ECM substrates in vitro. Second, Sca-1 regulates the frequency of tumor propagating cells and tumor cell proliferation in early lesions. These studies point to epithelial-ECM interactions as mediators of Sca-1 function; however, direct effects on downstream signaling and their relationship to tumor latency have not yet been determined.
There are several plausible explanations for our observations. First, Sca-1 may directly (or indirectly) interact with integrins modulating their ability to heterodimerize and bind ECM proteins, and/or modulating the strength of integrin-ECM interactions. The increased expression of α5-integrin in shSca-1 cells likely accounts for the increased adhesion to fibronectin via the α5β1 heterodimer. α5-integrin has been implicated as a suppressor of metastasis and α6-integrin promotes metastasis in breast cancer cell lines 
. Since both of these integrins are expressed at similar levels in the Wnt1-YL cells, further investigation is required to fully define the relationship between Sca-1 and the role of integrins in this system. Next, Sca-1 may interact with non-integrin receptors such as growth factor receptors that cooperate with integrin signaling 
. Alternatively, Sca-1 may alter interactions with cell surface receptors that act independently of integrin signaling. Additionally, Sca-1 may regulate the activation of MMPs leading to the release/activation of growth factors stimulating proliferation of tumor cells as observed in skeletal muscle cell regeneration 
The Wnt1-YL cells exhibited collective cell migration as a sheet in a scratch monolayer assay. In transwell migration assays, evaluating single cell migration across a porous membrane we did not show statistically significant differences in migration (data not shown). Furthermore, when the cells were seeded in matrigel (laminin-rich matrix) for a 3D morphogenesis/invasion assay the cells did not exhibit differences in terms of acini/colony formation frequency, size, morphology or invasive properties (data not shown). These observations suggest that Sca-1 is responsible for subtle changes in cell-cell and cell-ECM interactions in this cell line. Deciphering these subtleties in the context of cell migration and invasion may require further investigation of this cell line on matrices of single ECM substrates.
Interestingly, repression of Sca-1 alters chemokine expression, influencing the recruitment of inflammatory infiltrates. Since immune cells influence many processes including angiogenesis, cell invasion, matrix remodeling, interactions between tumor cells and the immune system have become of increasing interest in the past decade. Immune cells in both the innate and adaptive immune systems have proved to be important in tumor development and metastasis 
. Chemokine secretion from shSca-1 cells may recruit immune cells with pro-tumor activities accounting for the accelerated tumor growth. Furthermore, Sca-1 not only regulates chemokine expression, but Wnt1-YL cells grown in culture show differential secretion of both cytokines and chemokines (data not shown). Also, insulin degrading enzyme (Ide-1), a protein that physically interacts with Sca-1 to regulate differentiation skeletal muscle cells 
, was down regulated in shSca-1 cells. Ide-1 catalyzes the degradation of mitogenic peptides attenuating proliferative signals. Reduction in this activity may also account for proliferative response seen the shSca-1 tumor development. Additionally, Fgf20, a Wnt/β-catenin target gene, was up regulated in response to Sca-1 repression. Cooperation between the Wnt/β-catenin and FGF signaling pathways has been reported in human cancers and our laboratory has previously shown a strong association leading to rapid proliferation upon simultaneous activation of these pathways 
. Thus, Sca-1 potentially regulates multiple aspects of tumor development. The impact of these changes in mRNA expression needs to be determined with regard to protein expression and activity to better understand the role of Sca-1 in tumorigenesis.
Recently, Upadhyay and colleagues showed that Sca-1 inhibited TGF-β signaling by disrupting the heterodimerization of the TGF-β receptors and repressing expression of Gfd10, a TGF-β ligand, in a mammary adenocarcinoma cell line induced by medroxyprogestrone (MP) and 7,12-dimethylbenz(a)anthracene (DMBA) 
. Their tumor outgrowth data indicate that repression of Sca-1 reduces tumorigenicity or outgrowth potential as observed in normal mammary epithelial cells. Similarly, another report shows delayed tumor development in MP/DMBA induced tumors in Sca-1 knock-out mice 
. In this case, the delay in tumor development was attributed to the upregulation and activation of PPARγ. In contrast, our data indicate that Sca-1 may restrict cell growth. There are several explanations for these discrepancies. First, the tumors were developed under different conditions likely driven by different signaling pathways, which have been shown to yield very different tumor histopathologies 
. Second, the relative level of Sca-1 on the cell surface is likely to govern how Sca-1 regulates signaling activities 
. This may also account for the lack of overlapped genes in the microarrays when comparing the data of Upadhyay et. al. and our data set. Yuan et. al. only shared 15 genes in common with our data set with a p<0.01 and a fold change >1.5 (Table S2
). These genes were all upregulated, but did not reveal an enrichment of a common functional pathway. Since the tumor cell models employed in the two studies were developed using different methods, it is likely that they express Sca-1 at different levels. Furthermore, it is unlikely that the efficiency of Sca-1 repression is the same as different shRNA constructs were used. Cell context no doubt plays an important role in influencing the effects of Sca-1 in tumors that may have been derived from very different cell lineages. For instance, CD24high
luminal mammary epithelial cells (MECs) do not express ER and PR and have increased in vitro
progenitor activity in contrast to CD24high
luminal MECs that are ER and PR positive with reduced in vitro progenitor activity 
. Nevertheless, these studies highlight that Sca-1 likely regulates multiple cellular processes.
In conclusion, we provide evidence that Sca-1 regulates multiple cellular functions in mammary tumor cells. Our data highlight the importance of studying Sca-1 in the context of tumor development. To definitively differentiate the roles that Sca-1 plays in tumor initiation and tumor progression it will be necessary to use a conditional system in which Sca-1 can be knocked at various stages of tumor development. Additionally, it may be necessary to evaluate the role of Sca-1 in tumor subpopulations in models in which tumor-initiating cells are present. Further investigations along these lines will lead to a better understand of GPI anchored protein functions in tumors.