The TBX3 T-box transcription factor plays an important role in early mammary development [3, 7, 12, 40]. Mutations that cause haploinsufficiency of Tbx3 result in mammary gland hypoplasia in both mice and human [3, 12, 13]. On the other hand, Tbx3 is over-expressed in a variety of cancers, including breast cancer [16, 18, 26, 28]. Although Tbx3 over-expression has been associated with oncogenesis by its known ability to inhibit P14ARF expression and bypass senescence or by contributing to breast cancer cell migration [11, 17, 19], no direct evidence has been shown to suggest that over-expression of TBX3, alone, can induce tumor formation within the mammary gland. In this study, we over-expressed TBX3 within the mammary glands of mice, using a tissue-specific, doxycycline inducible transgenic system. Transgenic mouse models using constitutive promoters have provided information about specific genes and breast cancer development, particularly oncogene function [41, 42]. However, there are significant limitations to these systems due to the lack of control of transgene expression. The ability to control TBX3 expression is critical since homozygous Tbx3 knockout is embryonic lethal and constitutive over-expression is potentially toxic [12, 43]. We implemented a Tet-On system in our transgenic mouse model so that TBX3 transgene expression is inducible in a time and tissue-specific manner , enabling us to test possible TBX3 function in tumorigenesis in the mammary glands. An advantage of our mouse model is the ability to use luciferase expression as an indication of TBX3 transgene expression (Figure 1A). In this way, we are able to monitor TBX3 expression without sacrificing the animal. Using in vivo imaging as well as a luciferase assay, we were able to show that transgene expression is tightly controlled by doxycycline administration (Figure 1B). Our results show that this system is reliable and transgene expression could be induced in all five pairs of mammary glands.
Previous studies have shown that the five pairs of mouse mammary glands are differentially regulated by Tbx3 during early development. For example, in Tbx3 knockout studies, homozygous mutations resulted in the absence of mammary placodes, except for an occasional induction of the second pair of mammary placodes . Heterozygous mutations of Tbx3 caused decreased branching morphogenesis in the first three pairs of mammary glands, but had no significant impact on the fourth and fifth pairs of mammary glands . In 18.5 day old Tbx3 heterozygous embryos, 75% of the first pair of mammary glands was missing with no nipple or ductal tree formation while the second pair of mammary glands was affected to a lesser extent . Although these studies suggest that Tbx3 regulates murine mammary glands differently, we found that over-expression of TBX3 promotes accelerated mammary gland development in both the first and fourth mammary glands (Figure 2A and 2B) as well as the second, third and fifth mammary glands (data not shown).
Research has solidified a role for Tbx3 in the early development of the mammary gland. Tbx3 homozygous mutant mice results in mammary gland hypoplasia while heterozygous mutations of Tbx3 caused decreased branching morphogenesis in mammary glands [12, 21]. Our research complements these previous studies showing that TBX3 over-expression within the mammary glands causes hyperplasia, promoting increased secondary and tertiary branching as well as accelerated ductal elongation. It is also important to discuss that we have over-expressed human TBX3 within the mammary glands of mice. It has been shown that human TBX3 and mouse Tbx3 are 97% homologous at the protein level. Our group and others have demonstrated that human TBX3 is functional in mouse cells [9, 12, 16, 20]. Furthermore, aTbx3 knockout mouse model was able to recapitulate the phenotype seen in humans with Ulnar Mammary Syndrome (UMS). In a study performed by Papaioannou et al., a mutation in the mouse Tbx3 gene that closely corresponds to truncation mutations seen in some individuals with UMS resulted in a deficiency in mammary placode induction and the absence or reduction of mammary buds in mutant embryos, corresponding to the mammary gland hypoplasia seen in patients with UMS. Moreover, the deficiency in the development of limb elements in individuals with UMS was also reflected in limb abnormalities in the Tbx3 mutant mice. Mutant mice had deformities in the forelimb digits, foot and fibula resulting from a failure in the development of posterior limb elements. This study exemplifies that the Tbx3 protein plays a similar role in the development of the mammary glands in both human and mice. The mechanism by which TBX3 over-expression promotes hyperplasia in mammary glands needs to be elucidated. Using an Edu cell proliferation assay, we showed that over-expression of TBX3 resulted in a dramatic increase in cell proliferation within the mammary glands of pregnant doxycycline induced double transgenic mice at 10.5 dpc (Figure 3). Although cell proliferation was not directly quantified for the other developmental time points (i.e. 7-weeks, 10-weeks, and 12-weeks), the similarity in the observed accelerated mammary gland development suggests that the increase in cell proliferation at 10.5 dpc may also play a role in causing the accelerated branching and elongation of ducts during the other phases of mammary gland growth. A study has shown that mammary epithelia lacking the gene encoding NFκBIA contained increased NFkB activity as well as increased ductal branching and widespread intraductal hyperplasia , similar to results seen in our study. Furthermore, aberrant activation of NF-κB increased cell proliferation and breast cancer progression . In this study, we found that TBX3 inhibits the promoter activity of NFκBIB in vitro (Figure 4A). Upon further analysis, in vivo, we observed that Nfκbib expression was dramatically reduced in doxycycline induced double transgenic mice as compared to its un-induced double transgenic littermate controls (Figure 4B). Taken together, our results suggest a mechanism by which TBX3 over-expression represses NFKBIB/Nfkbib expression to enhance cell proliferation and promote mammary gland hyperplasia. However, TBX3 is a multifunctional transcription factor and the NFkB pathway could be one of many pathways regulated by TBX3. Wnt signaling has also been shown to play a major role in regulating mammary gland development . A TBX3-/- mouse model lacked expression of LEF1 and Wnt10b , suggesting that Wnt signaling is a downstream target of TBX3 and that TBX3 may regulate mammary gland development via the Wnt signaling pathway. Additional experiments can be done to further elucidate other mechanisms by which TBX3 over-expression promotes mammary hyperplasia.
Studies have suggested a role for Tbx3/TBX3 in regulating the self-renewal of mouse embryonic stem (ES) cells as well as breast cancer stem-like cells [36, 45–47]. Mouse ES cells require leukemia inhibitory factor (LIF) to maintain their undifferentiated state . Mouse ES cells genetically modified to over-express Tbx3 and grown in culture without LIF were able to maintain their undifferentiated state . Knockdown of Tbx3 expression in mouse ES cells resulted in a loss of self-renewal, causing these cells to differentiate . These findings suggest that Tbx3 expression is necessary to maintain mouse ES cells in their undifferentiated state and plays a functional role to promote self-renewal. A recent study has proposed a model in which the expression of TBX3 in cancer cells promotes the expansion of cancer stem-like cells through paracrine fibroblast growth factor (FGF) signaling . Over-expression of TBX3 increased the proportion of cancer stem-like cells in MCF7 cells by nine-fold as well as lead to an increase in tumorsphere formation and tumor initiation , suggesting that TBX3 is sufficient to promote normal and cancer stem like cell phenotypes. Due to its role in promoting proliferation of mouse ES cells and breast cancer stem-like cells as well as its requirement for early mammary gland development, TBX3 may also play a role in regulating mammary stem cell proliferation. Mammary glands consist of two cell lineages: myoepithelial and luminal epithelial cells. Both of them arise from a common progenitor, the mammary stem cell. Research has shown that a single Lin-CD24+CD29high cell is able to generate a functional mammary gland, suggesting that these cells are mammary stem cells . To determine whether over-expression of TBX3 affects mammary stem cell proliferation, we performed FACS analysis of the stem-like cell population, Lin-CD24+CD29high, in doxycycline induced double transgenic mice and their un-induced littermate controls. We found that over-expression of TBX3 significantly increased the frequency of Lin-CD24+CD29high stem-like cell population (Figure 5A and 5B), indicating that TBX3 expression is associated with an increased number of mammary stem-like cells. This could explain another mechanism by which TBX3 over-expression can cause hyperplasia and accelerated mammary gland development. Further studies of the mechanisms by which TBX3 regulates mammary stem-like cells are required to improve our understanding of mammary gland development and TBX3 function.