Only the combination of deregulated ERα and loss of Stat5a resulted in a significant delay in TEB differentiation. Stat5a is linked to terminal differentiation of mammary epithelial cells during pregnancy (15
). Mammary epithelial transplant studies reveal an ‘ample’ TEB number in the Stat5a−/−-transplanted epithelium of 11-week-old recipient mice and showed the effects of Stat5a deficiency on differentiation are restricted to the epithelium (17
). Here, we demonstrate for the first time that deregulated ERα can collaborate with Stat5a deficiency to impair differentiation of TEB mammary epithelial cells.
HANs have been likened to human hyperplastic epithelial lobular units (4
). Deregulated ERα expression significantly increased HAN prevalence, nuclear-localized Stat5a was expressed in the lesions, and Stat5a loss abrogated HAN development in nulliparous mice, suggesting that Stat5a-mediated signaling pathways contribute to ERα-initiated preneoplasia. Previous studies in which Stat5a loss compromised cancer progression initiated by non-hormonal mechanisms implicated impaired mammary epithelial cell survival as a mechanism (30
). We found apoptotic rates low in the mammary epithelial cells of mice with deregulated ERα that were not further reduced by Stat5a loss. This does not exclude the possibility that Stat5a loss may selectively impair survival of HAN progenitor cells. Stat5a also is known to mediate cell proliferation in hematopoietic cells (37
). Deregulated ERα significantly increased mammary epithelial cell proliferation (6
) but this was not reduced by loss of Stat5a in the normal appearing mammary epithelium. Therefore, Stat5a was not required for the abnormal increase in proliferation initiated by deregulated ERα, at least in the normal appearing mammary epithelial cells. It does not exclude that Stat5a loss could lead to significant changes in cell proliferation only in hyperplasia or may act to extend the cell cycle, which could explain the delay in TEB differentiation. Even though Stat5a is a transcription factor, the gene signature was not changed except for downregulation of β-casein. This does not rule out the involvement of altered gene transcription but does indicate that differences were not profound and may involve only a subpopulation of mammary epithelial cells. Loss of Stat5a has a defined role on lobuloalveolar but not ductal development (39
), possibly explaining the paucity of gene expression changes in the non-pregnant mammary glands composed primarily of ductal epithelium. Moreover, the mice were not exposed to increased levels of prolactin or other growth factors through pregnancy or exogenous stimulation. It is possible that fewer gene expression changes are found when Stat5a is only basally activated in ductal cells than when Stat5a is highly activated by prolactin or other ligands in lobuloalveolar cells.
The experiments excluded changes in apoptosis, proliferation or measurable gene transcription as reasons for the absence of HANs in the CERM/Stat5a−/− mice leaving open the hypothesis that HAN precursor cells could selectively require Stat5a for their survival or proliferation. Despite being highly conserved, Stat5a and Stat5b exert non-redundant functions (40
) and exhibit different potencies on similar effects (41
). However, in the mammary gland, Stat5b activation through serial pregnancy has been shown to rescue the lactational defect of Stat5a−/− mice indicating that Stat5b can compensate for at least some functions of Stat5a in mammary epithelium (17
). If the same were true for HAN precursor cells, then HANs would be predicted to develop in serially pregnant CERM/Stat5a−/− mice, which occurred (data not shown), supporting the concept that either of the two Stat5 homologs could support HAN development if sufficiently activated. Stat5 may contribute to the development of specific mammary cancer precursor cell lineages in a manner paralleling the established role of Stat5 in determining normal hematopoietic and mammary epithelial cell lineages (39
The experimental results led to a conundrum. Loss of Stat5a in the context of deregulated ERα delayed TEB differentiation, a condition hypothesized to increase cancer risk (11
), but Stat5a loss decreased the risk of preneoplasia development in nulliparous mice. To more rigorously test if Stat5a loss was cancer protective, 4-month-old mice were exposed to DMBA, the time point when impaired TEB differentiation was documented. HAN prevalence was reduced with loss of Stat5a in the mice with deregulated ERα but not absent. This is consistent with the notion that Stat5a might play a supportive role in survival or proliferation of HAN precursor cells but also indicated that Stat5a is not mandatory. This suggests that other factors could compensate for its absence. One compensating factor could be Stat5b (17
) acting to increase cyclin D1, a Stat5a and Stat5b target gene (45
). Nuclear-localized Stat5b was documented in all cancers and the percentage of cells with nuclear-localized cyclin D1 was significantly increased in cancers as compared with normal epithelium. The increased percentage of cells with cyclin D1 expression in the cancers is consistent with the fact that the percentage of cyclin D1-positive cells is higher in hyperplasia than normal epithelium in CERM mice (6
). Cyclin D1 loss in CERM mice interrupts mammary epithelial cell survival (7
). Cyclin D1 may play a role with Stat5 in development of the hyperplasias and cancers studied here.
DMBA exposure experiments revealed that Stat5a loss did not absolutely protect from cancer promotion and that the impact of Stat5a loss was context dependent. When deregulated ERα was present, Stat5a loss reduced HAN development but in the framework of normal mammary epithelial cells, Stat5a loss increased HAN development and cancer appeared. This could relate to a differentiation defect in the Stat5a−/− mice, independent from TEB differentiation, that increased susceptibility to DMBA-induced carcinogenesis (49
). It also illustrated that cancer formed even though enzyme activity required for DMBA metabolism into the active form might be decreased by Stat5a loss (50
). The reason Stat5a loss resulted in a different HAN prevalence following DMBA treatment dependent on the presence or absence of deregulated ERα is not clear. However, mammary cells expressing deregulated ERα demonstrate a differential dependence upon cyclin D1 for survival that is not found in normal mammary cells (7
) and this may represent another example of how mammary epithelial cells with deregulated ERα react differently to genetic changes than normal mammary epithelial cells.
Both ERα/PR-positive and ERα/PR-negative mammary cancers developed in the mice with deregulated ERα, similar to when deregulated ERα is combined with Brca1 loss and p53 haploinsufficiency (9
). ERα/PR-positive adenocarcinoma developed with Stat5a−/− loss and no deregulated ERα. The origins of ERα/PR-negative mammary cancers and their relationship to estrogen signaling are an active area of investigation with evidence for generation from either ERα-positive precursor (51
) or ERα-negative precursor (52
) cells. These mouse models are tools to further investigate the origins of ERα-positive and ERα-negative cancers.
In summary, the impact of Stat5a loss on mammary carcinogenesis was context dependent (). Although absence of Stat5a in the background of deregulated ERα reduced the prevalence of preneoplasia, this did not extend to protection from DMBA-induced cancer. In the absence of deregulated ERα, Stat5a loss appeared to increase susceptibility to carcinogen-induced preneoplasia and was associated with cancer development. It is possible that the same interplay may be found in the human breast. This will need to be explored if and when Stat5a targeted therapies for humans become available.
Fig. 5. Stage-dependent effects of Stat5a with respect to differentiation, hyperplasia and cancer formation in mouse models with and without ERα deregulation. Deregulated ERα (A) and loss of Stat5a demonstrated persistent TEBs by 4 months of age, (more ...)