with breast cancer risk has been observed in both pre- and post- menopausal women, independent of body mass index and other known risk factors [17,18,19,20,21,22,23,24]. Moreover, mam- mary tumors arising in women with low serum adiponectin levels are more likely to show a biologically aggressive and poor prognosis phenotype. These epidemiological evidences suggest that reduced adiponectin expression might be causally involved in obesity-related carcinogenesis. In line with these clinical findings, numerous experimental evidences support the role of adiponectin as an inhibitory factor for breast cancer development [25,26,27,28,29,30,31,32,33]. Adiponectin at physiological concentrations suppresses the prolif- eration and causes cell cycle arrest in both estrogen receptor (ER)- negative and ER-positive human breast carcinoma cells. It inhibits insulin- and growth factors-stimulated growth of ER-positive breast cancer cells [28]. Adiponectin replenishment suppresses mammary tumorigenesis of MDA-MB-231 cells in nude mice [28]. Cell-type dependent signalling mechanisms have been suggested to mediate the growth inhibitory effects of adiponectin. In MCF-7 cells, adiponectin induces AMP-activated protein kinase (AMPK) phosphorylation and inactivates p42/p44 MAPki- nase (ERK1/2) [29]. By contrast, the inhibitory effects of adiponectin on T47D cell growth are associated with inactivation of ERK1/2 but not AMPK or p38 MAPK [18,28]. In MDA-MB- 231 cells with ectopic ER over-expression, globular adiponectin inhibits cell proliferation by blocking JNK2 signalling [26]. In ER- negative MDA-MB-231 cells, adiponectin could modulate the glycogen synthase kinase-3beta (GSK3beta)/beta-catenin signal- ing pathway [28]. Prolonged treatment with adiponectin markedly reduces serum-induced phosphorylation of GSK3beta, decreases intracellular accumulation and nuclear translocation of beta- catenin, and suppresses cyclin D1 expression. Despite of these progresses, whether adiponectin deficiency is a direct contributor to the pathogenesis of breast cancer remain elusive. In this study, we investigated the effects of reduced adiponectin expression on mammary tumor development in MMTV-PyVT transgenic mice. Mice with reduced adiponectin expressions were established in both FVB/N and C57BL/6J backgrounds. Adiponectin haploinsufficiency significantly reduced tumor latency and promoted mammary tumor development in both female and male animals. The results demonstrated that inadequate adipo- nectin production might alter the stromal microenvironment towards more pro-proliferative and pro-tumorigenic in mammary tissue, by triggering the abnormal redox activities that led to the inhibition of tumor suppressor PTEN and hyperactivation of PI3K/Akt signaling pathways in mammary tumor cells. Results Adiponectin haploinsufficiency promotes mammary tumor development in MMTV-PyVT mice We generated MMTV-PyVT transgenic mice with reduced adiponectin expressions in both FVB/N and C57BL/6J back- grounds. PyVT transgenic mice with complete loss of the adiponectin alleles could not be born alive across all generations due to embryonic lethality. On the other hand, the knockout genotypes were found in male and female PyVT(2/2) litters. Therefore, mice with normal PyVT(+/2)/ADN(+/+) and reduced PyVT(+/2)/ADN(+/2) adiponectin expressions were used in the present study. The heterozygotes showed a 4–5 folds reduction of adiponectin levels (Figure 1), which were more relevant to those breast cancer patients with decreased adiponectin levels. Tumor development of these mice was closely monitored every 2–3 days. All mice carrying the PyVT transgene developed mammary Adiponectin and Breast Cancer tumors. Tumor onset was recorded as the age of the animal at which palpable abnormal masses were detected (Figure 2). The overall median age of tumor latency in PyVT(+/2)/ADN(+/2) mice of FVB/N background were 58 days for female (n = 20) and 115 days for male (n = 23) mice respectively, which were significantly earlier than those of PyVT(+/2)/ADN(+/+) mice (66 days for female and 133.5 days for male mice, n = 23 and 24 respectively, p,0.0001). Similar phenomena were also observed in mice of C57BL/6J background. The overall median tumor latency of female and male adiponectin haplodeficient PyVT mice (66 and 114 days respectively, n = 19) was significantly reduced comparing with those of mice having normal adiponectin expression levels (73 and 137 days respectively, n = 19, p,0.0001). Tumor develop- ment was monitored twice per week up to 14 and 28 weeks for female and male mice respectively (Figure 3). No tumors were found in PyVT(2/2) mice up to 60 weeks, irrespective of their adiponectin levels. Tumor growth was significantly accelerated in both female and male adiponectin haplodeficient PyVT mice compared to PyVT(+/2)/ADN(+/+) mice. At the time of sacrifice, the total wet weights of tumors in PyVT(+/2)/ADN(+/2) mice was over 2-fold heavier than those with normal adiponectin expression levels (Table 1). The mean tumor weight of female PyVT mice of FVB/N background when sacrificed at 14 wks of age was 9.88963.189 g in ADN(+/2) animals compared to 4.48361.645 g in ADN(+/+) animals. Similarly, in male FVB/N PyVT mice sacrificed at 22 wks of age, the mean tumor weight was 6.85761.262 g in ADN(+/2) animals compared to that of 3.68761.483 g in ADN(+/+) animals. On the other hand, although the wet weights of lung tissues in female and male PyVT(+/2) mice were heavier than those in non-transgenic PyVT(2/2) mice (data not shown), they were not significantly different between mice with reduced and normal adiponectin expressions. Distinct basal-like subtype of tumors in adiponectin haplodeficient PyVT mice Five subtypes of breast carcinoma with different outcomes, including luminal A, luminal B, HER2+/ER2, basal-like and normal breast-like, were revealed by microarray studies [42]. Luminal A and B are ER positive tumors, whereas the other three subtypes are ER negative. Our preliminary microarray analysis suggested that the molecular profiles of tumor cells derived from PyVT(+/2)/ADN(+/2) mice were very different from those of PyVT(+/2)/ADN(+/+) mice and could be clustered separately (data not shown). To further validate such an observation, gene markers associated with different tumor subtypes were quantified by real-time PCR analysis. In PyVT(+/2)/ADN(+/2) tumors, basal-like subtype genes, including KRT17, KRT5, MFGE8 and FZD7, were significantly up-regulated, whereas HER2+/ER2 subtype-related genes, ERBB2 and MED1, were dramatically down-regulated (Figure 4A). Histological analysis demonstrated typical morphologic features associated with the basal-like subtype, including markedly elevated geographic tumor necrosis, ribbon- like architecture associated with central necrosis, pushing margin of invasion, and stromal lymphocytic response in tumors from PyVT(+/2)/ADN(+/2) mice [43] (Figure 4B). We could not detect these morphological features in any of PyVT(+/2)/ADN(+/ +) mice or the original PyVT mice, which in contrast showed a well-structured and organized morphology, suggesting that the phenotype differences may not be tumor developmental stage dependent. Moreover, the protein levels of p53, a characteristic associated with tumors overexpressing ERBB2, was significantly higher in the PyVT(+/2)/ADN(+/+) tumors comparing with PyVT(+/2)/ADN(+/2) tumors (Figure 4C). These and the above PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4968