Adiponectin and Breast Cancer Figure 8. Tumor cells derived from PyVT(+/2)/ADN(+/2) mice showed increased sensitivity to adiponectin-mediated inhibition of Akt/GSK3beta/beta-catenin signaling and cell proliferation. Tumor cells isolated from both PyVT(+/2)/ADN(+/+) and PyVT(+/2)/ADN(+/2) mice were pre-treated with 15 mg/ml of adiponectin for 24 hr in 0.5% FBS DMEM. The serum-stimulated phosphorylation changes of Akt, GSK3beta and beta-catenin were analyzed by Western Blotting (A) as described previously [28]. The nuclear beta-catenin activities were assayed using the TOPflash/FOPflash reporter assay (B). Cell proliferation was evaluated for both types of tumor cells under the indicated treatment conditions using 3H- thymidine incorporation assay (C). *, P,0.05 and **, P,0.01 vs corresponding vehicle control (n = 3, from three independent experiments). doi:10.1371/journal.pone.0004968.g008 hyper-activation of beta-catenin signalling associated with adipo- nectin haplodeficient tumors (Figure 7). Notably, the cross-talks between PI3K/Akt and the canonical Wnt/beta-catenin signaling pathways have been demonstrated by a number of studies from independent groups (23). Overexpression of PTEN inhibits Wnt-1 induced beta-catenin stabilization and mammary tumorigenesis in mice [51]. PI3K/Akt pathway is involved in Wnt3a-induced proliferation and beta-catenin nuclear accumulation in NIH3T3 cells [52]. In HT29 colorectal adenocarcinoma cells, inhibition of PI3K was accompanied by a considerably reduced expression level of beta-catenin [53]. The linkage between Wnts and PI3K/Akt signalling have also been found in the regulation of bone mass, osteoblast progenitor proliferation, differentiation and osteoblast apoptosis, as well as cardiomyogenesis [54]. The protein levels of p110alpha subunits are elevated in tumor cells isolated from adiponectin haplodeficient PyVT mice. However, adiponectin treatment has no effects on p110alpha, despite that it can inhibit Akt phosphorylation and nuclear beta-catenin activities (Figure 7), suggesting that the inhibitory effects of this hormone is downstream of PI3K and upstream of Akt, possibly through modulating PTEN’s activities. Insufficiency in adiponectin production might promote mam- mary tumor formation from distinct type of cells, as suggested by the consistent morphological and gene expression differences between tumors derived from PyVT(+/2)/ADN(+/+) and PyVT(+/2)/ADN(+/2) mice (Figure 3). Adiponectin haplodefi- cient tumor is more related to a basal-like subtype, which is characterized by high proliferative activity and unfavorable PLoS ONE | www.plosone.org 10 March 2009 | Volume 4 | Issue 3 | e4968