Adiponectin and Breast Cancer Figure 4. A basal-like subtype of mammary tumors derived from adiponectin haplodeficient MMTV-PyVT mice. A, Quantitative RT-PCR analysis of the expression levels of gene markers associated with different subtypes of breast tumors. The RNA was extracted from cultured primary tumor cells isolated from 14-week old PyVT(+/2)/ADN(+/+) and PyVT(+/2)/ADN(+/2) female FVB/N mice and quantitative PCR analysis performed as described in the Methods. *, P,0.01 vs PyVT(+/2)/ADN(+/+) group, n = 10. B, Morphological features of tumors derived from 14-week old female PyVT(+/2)/ADN(+/+) and PyVT(+/2)/ADN(+/2) mice of the FVB/N background. Distinct morphologies were observed between tumors collected from mice with normal and reduced adiponectin levels. Note that central necrosis and geographic tumor necrosis, as well as stromal lymphocytic responses represented the typical basal-like subtype of breast tumors in PyVT(+/2)/ADN(+/2) mice. C, The protein levels of p53 were much higher in tumors derived from PyVT(+/2)/ADN(+/+) mice compared to those of the PyVT(+/2)/ADN(+/2) mice as measured by Western Blotting using specific antibodies purchased from Cell Signaling Biotechnology. doi:10.1371/journal.pone.0004968.g004 proliferation of tumor cells derived from PyVT (+/2)/ADN(+/2) mice, their inhibitors were used for cell proliferation measurement using [3H]-thymidine incorporation assay. Importantly, the general inhibitor LY294002 and Akti-1/2 showed greater extent of attenuation on the cell growth at all time points, whereas the p110alpha-selective inhibitor PIK75 was more potent than the other two inhibitors (Figure 7D), suggesting that blockade of PI3K or Akt reversed the proliferative advantage of adiponectin haplodeficient tumors. Adiponectin treatment significantly atten- uated phosphorylations of Akt and GSK3beta and beta-catenin protein levels and nuclear activities, as well as inhibited cell proliferation to a greater extent in PyVT (+/2)/ADN(+/2) tumor cells (Figure 8). On the other hand, it had little effects on p110alpha levels. These results implicated that the activation of PI3K/Akt pathway might contribute to the elevated beta-catenin signalling cascades in adiponectin haplodeficient mammary tumors. Decreased PTEN activities caused by altered redox environment in adiponectin haplodeficient PyVT tumors PTEN is one of the most frequently mutated tumor suppressors that can prevent the activation of the cell survival PI3K/Akt signaling pathway [44]. In the absence of PTEN function, cells exhibit elevated Akt activities. It has been reported that PTEN could bind to Trx1 in the cytosol, resulting in a functional loss of its lipid phosphatase and membrane binding activity [45]. Interestingly, PTEN activities were decreased by more than 50% in PyVT (+/2)/ADN(+/2) tumor cells (Figure 9A), whereas its total protein amount was not significantly different (Figure 9B). The activities of both Trx1 and its upstream binding enzyme, TrxR1, were augmented by nearly 40% in PyVT(+/2)/ADN(+/ 2) tumor cells (Figure 9A). While the protein levels of Trx1 were similar between PyVT(+/2)/ADN(+/+) and PyVT(+/2)/ADN(+/ 2) tumors, the total amount of TrxR1 was increased in PyVT(+/ 2)/ADN(+/2) tumor cells (Figure 8B). Surprisingly, co-immuno- precipitation experiment revealed that the amounts of Trx1-bound PTEN were dramatically increased in tumor cells derived from the adiponectin haplodeficient PyVT(+/2) mice (Figure 9C). Treat- ment with curcumin, an irreversible inhibitor of TrxR1 (40), elevated PTEN activity by nearly 3 folds in PyVT(+/2)/ADN(+/ 2) tumor cells, which was accompanied by the decreased activities of both TrxR1 and Trx1 (Figure 9A). A stimulatory effect on PTEN activity was also observed in cells treated with adiponectin (Figure 9A). In PyVT(+/2)/ADN(+/2) tumor cells, the TrxR1 promoter-driven reporter activity was ,1.8 fold higher than that of PyVT(+/2)/ADN(+/+) tumor cells (Figure 9D). Treatment with adiponectin for 24 hrs significantly reduced the reporter activities by ,60% in PyVT(+/2)/ADN(+/2) tumor cells but had no significant effects on PyVT(+/2)/ADN(+/+) tumor cells. Similar effects were also observed for TrxR1 mRNA levels in tumor cells treated with or without adiponectin (Figure 9D). Taken together, PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4968