Our finding that Bmi-1 loss induces an exhaustion of PrSCs and a depletion of the LSC stem cell fraction as well as the p63+
primitive cells is similar to what is seen in postnatal hematopoiesis, where loss of Bmi-1 significantly reduces the number of HSCs (Park et al., 2003
). Bmi-1 is also required for the self-renewal of stem cells in the peripheral and central nervous system, and loss of Bmi-1 results in postnatal growth retardation and neurological defects (Molofsky et al., 2005
). These results highlight the conserved role of Bmi-1 across cells derived from all three germ layers.
Bmi-1 has been shown to regulate leukemic stem cell self-renewal as well (Lessard and Sauvageau, 2003
). Loss of Bmi-1 in HSCs did not prevent primary leukemia formation. Only when leukemic cells were transplanted into secondary recipients was there a difference in leukemiagenesis. In contrast, loss of Bmi-1 completely prevented the formation of prostate hyperplasia in the FGF10 model, and significantly attenuated the primary outgrowth of prostate cancer induced by Pten deletion.
Bmi-1 expression patterns also vary between tissues. Bmi-1 lineage tracing experiments in pancreas show that Bmi-1+
cells are differentiated and co-express lineage markers and Ki67, indicating that Bmi-1 is present in more differentiated proliferating cells. The detection of Bmi-1+
cells one year after pulse-chase marking experiments indicates that Bmi-1 is also present in a subpopulation of self-renewing pancreatic acinar cells (Sangiorgi and Capecchi, 2009
). In contrast, Bmi-1 is only expressed in the stem cells of the intestine, and the Bmi-1+
cells do not co-stain for differentiation markers (Sangiorgi and Capecchi, 2008
). Our cell fractionation studies reveal that Bmi-1 expression is highest in the prostate basal/stem cell enriched fraction. This fraction can be further divided into Trop2hi
stem- and Trop2lo
basal-subpopulations (Goldstein et al., 2008
). Bmi-1 was highest in the Trop2hi
stem cell fraction, but was still present in the Trop2lo
basal cell population as well (data not shown), suggesting that Bmi-1 is not solely expressed by the most primitive cells of the prostate. Weissman and colleagues used a Bmi-1-GFP mouse to show similar results in the hematopoietic system, where Bmi-1 is highest in the HSCs, but also present in certain progenitors (Hosen et al., 2007
). Occasionally, Bmi-1 was found in a few adluminal cells in the proximal region of the prostate. These cells may be related to the recently characterized CARNs cells, which represent rare luminal stem cells that have tumor initiating capacities (Wang et al., 2009
P63 is a transcription factor belonging to the P53 super-family whose deletion causes a failure to develop stratified epithelia and epithelial appendages in mice, in addition to prostate agenesis (Yang et al., 1999
). Signoretti et al. complemented p63−/− blastocysts with p63+/+ galactosidase+ embryonic stem cells and found that luminal cells of the prostate originated only from p63+ donors, which suggests the defects of the p63
-null stem cells in proliferation and self-maintenance (Signoretti et al., 2000
). p63 also marks the more stem-like cells in the prostate (Xin et al., 2007
In our study, Bmi-1 over-expression led to an expansion of these p63+
cells in p53−/−
prostate spheres. Furthermore, we observed a breakdown in cellular organization of the spheres, where the p63+
cells normally confined to the outer rim of spheres expanded in towards the middle (). Such adluminal expansion of p63+
cells has also been observed in several models of murine prostate cancer (Memarzadeh et al., 2007
; Wang et al., 2006
). The FGF10 model of prostate hyperplasia is an over-growth and excessive branching of the basal/stem cell population of prostate cells, characterized by a concomitant expansion of p63+
cells. The most primitive prostate cells, which are also p63+
are the most susceptible to such transformation (Lawson et al., 2010
). Our data suggests that inhibition of Bmi-1 was able to prevent this form of hyperplasia because loss of Bmi-1 caused a depletion of this p63+
LSC population. This finding may have important implications in other epithelial cancers, as many are thought to initiate from stem cells.
The evolutionarily conserved pathways Wnt, Sonic Hh (Shh), Notch and the Polycomb Group gene Bmi-1 are consistently found involved in stem cell self-renewal from Drosophila to humans. Interactions between these pathways have been well documented. Both Wnt and Shh signaling are important for neural tube development, neural stem cell proliferation and self-renewal (Michaelidis and Lie, 2008
; Patten and Placzek, 2000
; Wechsler-Reya and Scott, 1999
). Wnt pathway inhibitors can regulate threshold responses to Shh target genes, demonstrating the existence of mechanisms that integrate graded Shh and Wnt signaling to determine cell fate decisions in the neural tube (Lei et al., 2006
). Wnt-1, Hh and Bmi-1 have all been shown in mammary stem cells to influence self-renewal and regenerative capacities (Asselin-Labat et al., 2008
). Increase in Hh signaling directly up-regulates Bmi-1 expression, and it is the level of Bmi-1 that controls mammary stem cell self-renewal (Liu et al., 2006
). Attempts were made to test the importance of Bmi-1 for Hh driven self-renewal in the prostate. In contrast to mammary stem cells, Hh over-activation through Lentiviral overexpression of Gli-1, and the addition of rShh to PrSCs did not result in a sustained increase in sphere forming and self-renewal activity in our assay (data not shown). Therefore the effects of Bmi-1 inhibition were not tested in these conditions. Interestingly, it also took an extra generation to see significant differences in sphere forming activity in response to Bmi-1 over-expression, while mammary sphere formation increased in generation 1. Both of these findings could be due to the fact that prostate cells are more sensitive to oncogene induced senescence.
β-catenin stabilization did increase self-renewal in the prostate, and Bmi-1 loss reversed this gain of sphere forming activity. In contrast to the interactions between Hh and Bmi-1 in the mammary, β-catenin signaling does not appear to activate Bmi-1 signaling directly and Bmi-1 is likely not downstream of β-catenin. These findings suggest that Bmi-1 and β-catenin are acting in parallel to control self-renewal, and Bmi-1 is necessary for β-catenin to affect this process. Bmi-1 expression and resulting chromosomal modifications may set the cell to a specific state that allows for self-renewal and proliferation. Even if several developmental pathways are active in certain epithelial stem cells, controlling Bmi-1 may be enough to modulate self-renewal. With the close relationship between stem cell self-renewal and cancer progression, this finding has important implications for controlling cancer growth as well.