STUDY OF THE FUNCTION OF TBX3 TRANSCRIPTION FACTOR IN MAMMARY NORMAL AND CANCER STEM CELLS

The TBX3 gene, a member of the T-box family of transcription factors encodes for a protein involved in early embryonic cell fate decisions, including embryonic patterning, extra-embryonic structure regulation and organogenesis. Mutations in human TBX3 cause the Ulnar-Mammary Syndrome (UMS, OMIM 181450), an autosomal dominant disorder that affects limb, apocrine-gland, hair, genital and dental development. In addition, TBX3 has a role in the posterior limb mesoderm and the dorsal/ventral axis of the forelimb specification. TBX3 acts as a repressor or an activator of gene expression. The first TBX3 target to be identified was p19ARF, a gene that regulates senescence and apoptosis in mouse fibroblast and neural cells. In mammary epithelial cells, TBX3 also promotes cell proliferation by repressing p19ARF through a pathway alternative to Mdm2-p53 [12]. Recently, TBX3 over expression in melanoma cell lines was found to enhance cell invasiveness by repressing the expression of E-Cadherin [13]. As TBX3 was also found over expressed in a subset of breast cancer cell lines, this suggested that aberrant TBX3 expression may have a role in oncogenesis. Our data suggest that TBX3 is a key gene in the regulation of mammary gland development due to its expression being necessary for the maintenance of the mammary stem cell pool. In agreement with our work in mouse embryonic stem cells (mESCs), TBX3 was described as a stem cell factor that is sufficient to maintain pluripotency in the absence of the leukaemia inhibitory factor (LIF) [87]. Other recent research suggests that embryonic stem cell associated genes and pathways are aberrantly regulated in mammary stem cell malignancy. To elucidate the role of TBX3 in mammary gland development and cancer initiation and progression, we initiated studies using rat (LA7) and human mammary cancer stem cells (CSCs) isolated from patients. LA7 CSCs were isolated by Dr. Renato Dulbecco from a breast adenocarcinoma, and at the single cell level have multi-lineage differentiation, tubulo-alveolar-like structure and self-renewal capacity and the potential generate heterogenous tumors indefinitely [67, 68]. Quantitative Real PCR showed that TBX3 is expressed in LA7 and human CSCs and is down regulated during the onset of CSC differentiation, suggesting that TBX3 has a role in the CSC maintenance, while its down regulation induces CSC differentiation. It is likely that the role of TBX3 that we have uncovered is also valid in normal mammary SCs and other types of SCs. Through optimization of the culture conditions that allowed for expansion and isolation of populations of cancer cells from human mammary gland of patients we identified in breast tumors a rare subpopulation of cells with stem-cell properties and designated these cells as human CSCs. Tumor samples were kindly provided by hospitals of Pavia and Crema, Italy. This rare subpopulation of cells had the potential to form mammospheres in non-adherent culture conditions and, when embedded in Matrigel, form tubular-like structures that recapitulate the branching structure of the mammary gland tree. TBX3 down-regulation by TBX3-siRNA treatment resulted in the permanent loss of the capacity of LA7 and human CSCs to generate both mammospheres and tubular structures and the up regulation of mammary differentiation markers, demonstrating that TBX3 down regulation induced loss of CSC self-renewal and loss of multilineage differentiation. Moreover, by screening candidate miRNAs from a microRNA gene chip array performed using LA7 cells cultured in non-differentiating and differentiating conditions, we identified a micro-RNA (miRNATBX3) that repressed TBX3 expression in mammary CSCs. We determined that TBX3 is the primary target of miRNATBX3 since down-regulation of TBX3 by itself was sufficient to induce CSC differentiation, and that inhibition of TBX3 down-regulation resulted in the repression of CSC differentiation and increased CSC proliferation. These results suggest that the normal role of TBX3 is to regulate the number of SCs in the normal mammary gland SC niche by modulating the rate of SC proliferation and repressing SC differentiation. In collaboration with Hans Schöler’s laboratory, we investigated the effects of TBX3 modulation in mESCs that were engineered to express the green fluorescent protein (GFP) under the control of OCT4 promoter, a gene essential for the maintenance of pluripotency. The down-regulation of TBX3 in undifferentiated mESCs lead to the differentiation of the mESCs that was readily observable since the colonies displayed morphologies typical of differentiated mESCs and had lost the GFP expression. In order to obtain a deeper insight into the mechanisms by which TBX3 regulates mammary CSCs, we initiated studies to identify the target genes of TBX3. A set of candidate target genes such as GLI2 and GATA3, were confirmed to be TBX3 direct targets by chromatin immunoprecipitation (CHIP) analysis [118]. By global expression analysis with microarrays we determined that TBX3 is involved in a common pathway that regulates the inhibition of mESCs and mammary CSCs differentiation. A parallel circuit of LIF signalling in mES cells which maintains pluripotency involving TBX3 has recently been identified that leads to the activation of NANOG and SOX2 and the regulation of OCT3/4. By the silencing of TBX3 we observed down-regulation of both NANOG and SOX2 in LA7 and human CSCs. This preliminary result suggested that SOX2 could be a direct target of TBX3 and is supported by the overlap in the genes up or down -regulated both after TBX3 and SOX2 silencing. The gene overlap was identified by comparing microarray data generated from siRNA-TBX3 LA7CSC treated cells and mESCs in which SOX2 was down regulated. Collectively, my thesis research suggests that TBX3 is an inhibitor of pluripotent ESC, mammary stem cell (SC) and cancer stem cell (CSC) differentiation and may represent a common master gene by which regulation of stem cell self-renewal is maintained in other types of stem cells. Our results reveal an unknown link between a regulatory network controlling self-renewal in mammary CSCs and pluripotency in mouse embryonic stem cell (mESC). We hypothesize that the “normal” function of Tbx3 is maintain SCs in an equilibrium between a state of quiescence and a state that allows SCs to replenish themselves for the purpose of tissue expansion, regeneration and differentiation.