Lrp5 in Mammary Stem Cells Figure 7. Wnt Signaling of Whole Populations is not Significantly Affected by the Absence of Lrp5. A) Quantification of Lrp5 and Lrp6 mRNA levels in Lrp5 +/+ and 2/2 MECs. B) Quantification of expression of the Wnt target gene, Axin2, mRNA in Lrp5 +/+ and 2/2 MECs. C) Quantification of Axin2 and Gpr49 mRNA expression levels in Lrp5 +/+ and 2/2 MECs treated with Wnt3a or control conditioned media. All samples were cultured for three days and mRNA levels were normalized to the expression of the housekeeping genes, TBP and HPRT (as described in the Methods section). Normalized expression = 22DCt (see Methods S1). Data shown in A were compared by unpaired Student’s t-test. Data shown in B and C were compared by Wilcoxon Rank Sum Test. Significance was established at p,0.05. doi:10.1371/journal.pone.0006594.g007 Moreover, we found the maintenance of Wnt signaling in Lrp52/2 MECs was not due to the up-regulation of Lrp6 in response to culture conditions, since Lrp6 mRNA levels remained relatively unchanged throughout the culture period (Figure S7). Taken together, these results indicate that although there was a significant change in the physiology of Lrp52/2 glands, the canonical Wnt response was largely normal. Potential explanations for that are described in the Discussion section. Discussion Previously, we have shown that Wnt1-induced, hyperplastic mammary glands accumulate undifferentiated cells (of both basal and luminal lineages) and ductal regenerative/stem cells (as a proportion of total) [9]. Here, we show an increased proportion of basal cells (measured by their CD24/CD49f profile and higher relative expression of basal marker proteins/total protein) and increased expression of a basal cell-associated p63 variant, DNp63, associated with proliferative function in basal cell lineages. These glands subsequently develop solitary differentiated tumors with multiple lineage characteristics. We show here (and Lindvall et al, 2006) that loss of Lrp5 generates the reciprocal phenotype – slower ductal outgrowth, the accumulation of peri-senescent cells (of both lineages), almost total depletion of adult regenerative cells from the ductal tree, a reduced proportion of basal cells compared to luminal, and increased expression of the TAp63 isoform, associated with senescence. We predict that these glands will be highly tumor resistant (for tumors arising in a basal cell precursor). We have shown that Lrp5 is expressed together with Lrp6 on most/all basal cells of the mammary gland. However, most basal cells have a low cell surface expression of Lrp5, and only cells with a high level have enriched stem cell function. This fraction includes 80% of the mammary stem cells. Lrp5 is unique amongst the markers described so far for mammary epithelial populations for providing significant levels of stem cell enrichment without combining it with other markers. Wnt1-induced mouse mammary tumors share a transcriptional signature with Brca1+/2 and carcinogen-induced tumors [39], and these in turn share components of their basaloid signature with human basaloid tumors [40]. Characteristically, all of these tumors have residual basal cells, and are likely to derive from the basal lineage. It is perhaps not surprising then that the key components of the oncogenic Wnt signaling pathway are specifically expressed by basal cells. There are data to suggest that gain of function of Lrp5 or -6 is important to breast cancer. Some human breast cancer cells have PLoS ONE | www.plosone.org 9 August 2009 | Volume 4 | Issue 8 | e6594