the patients , methodology of tumor fine - needle aspiration biopsy , rna purification , microarray hybridization , and generation of gene - expression profiles ( public gene expression omnibus dataset gse-20194 ) were described previously ( 1012 ) . additional data were retrieved from medical records under a protocol approved by the institutional review board in compliance with health insurance portability and accountability act regulations . one hundred thirty - seven estrogen receptor  positive ( er+ ) bc patients with available body mass index ( bmi ) information were classified as nonobese ( bmi < 30kg / m ; n = 94 patients ) and obese ( bmi  30kg / m ; n = 43 patients ) . no patient was excluded based on research - based prediction analysis of microarray 50 ( pam50 ) subtype predictor . patients provided written informed consent to obtain tumor biopsy for genomic studies ( institutional review board protocol lab99 - 402 , uso-02 - 103 , 2003 - 0321 , i - spy-1 ) ( 1012 ) . mcf7 , t47d , and 3t3-l1 cells were obtained from american type culture collection ( manassas , va ) . the mouse mammary tumor eo771 cells ( c57bl6 genetic background ) ( 13 ) were kindly provided by dr xiaohong leng ( md anderson cancer center , houston , tx ) . cell culture conditions , differentiation of 3t3-l1 into adipocytes , and in vitro cell culture experiments are described in the supplementary methods ( available online ) . cell viability was determined by mtt ( 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium ) assay , as described previously ( 14 ) . each treatment set was made in at least three separate independent cell population ( n = 3 ) to allow for statistical evaluation and repeated at least twice to ensure reproducibility . generation of mmtv - tgf;a 
 /a mouse model of er+ bc and obesity is described in the supplementary methods ( available online ) . body fat percentage was determined as described in the supplementary methods ( available online ) . oral glucose tolerance test and insulin tolerance test were performed as previously described ( 15 ) . mice were killed , and tumors ( one - third ) were collected for total rna extraction by trizol ( invitrogen , grand island , ny ) as previously described ( 16 ) . total rna samples were sent to the genomics core facility at the university of texas md anderson cancer center for gene expression analysis using the platform array illumina mousewg-6_v2_0_r3 ( illumina , san diego , ca ) . orthotopic / syngeneic allografting experiments were performed by injecting eo771-fg12 cells in matrigel ( bd bioscience , san jose ca ) into the left fourth mammary fat pad . generation of eo771-fg12 cells and in vivo imaging are described in the supplementary methods ( available online ) . all animal experiments were conducted in accordance with american association for laboratory animal science regulations and the approval of the university of texas md anderson cancer center institutional animal care & use committee . microarray data were analyzed using nexus expression 3 ( biodiscovery , hawthorne , ca ) . downstream effects functional analysis was completed using ingenuity pathway analysis ( ingenuity systems , www.igenuity.com ) software , which uses the ingenuity knowledge base to analyze transcriptomic dataset and identify the expected functions ( eg , biological processes and disease or toxicological functions ) that are increased or decreased ( p < .01 ) because of the observed changes in gene expression ( http://ingenuity.force.com/ipa/articles/ feature_description / accessing - and - using - functional - analysis ) . the width of the links and the relationship of the biological processes with cancer hallmarks were determined using the z scores and illustrated in a circos plot ( 9,17 ) . sections were stained with hematoxylin and eosin or ki67 for pathological evaluation as previously described ( 18 ) . all protein analyses were performed using lysates from whole - cell pellets or mouse tumor samples in radioimmunoprecipitation assay buffer as previously described ( 19 ) . protein samples were used for multiplex immunosorbent assay and western blot analysis as described in the supplementary methods ( available online ) . serum insulin , insulin - like growth factor 1 ( igf-1 ) , and estradiol levels were measured and compared using enzyme - linked immunosorbent assay kits ( r&d systems , minneapolis , mn ) . adipokines expression was measured using the proteome profiler mouse adipokine array ( r&d systems ) . breslow test was also used for survival comparisons because of little early censoring among the patients . for survival comparisons of mice , the assumption of proportionality was verified by the likelihood ratio test and the global  test . for the mouse studies , breast tumor specific deaths were the primary endpoints and deaths due to other causes ( eg , fighting , excessive cystic skin lesions , and killed for inability to roll over or obtain food and water because of excessive obesity ) were the competing risks events . gray competing risk analysis was performed using r software ( version 2.13.00 ; r foundation for statistical computing , vienna , austria ) . statistical differences were assessed by two - tailed t test or 1-way analysis of variance with post hoc multiple comparisons ( holm  sidak method ) when appropriate . all other statistical analyses were performed using sigmaplot ( version 12.3 ; systat , san jose , ca ) or spss ( pc version 18 ; ibm spss , armonk , new york ) software . all error bars represent 95% confidence intervals ( cis ) . all results were considered statistically significant when p was less than .05 when calculated with the appropriate statistical test . the patients , methodology of tumor fine - needle aspiration biopsy , rna purification , microarray hybridization , and generation of gene - expression profiles ( public gene expression omnibus dataset gse-20194 ) were described previously ( 1012 ) . additional data were retrieved from medical records under a protocol approved by the institutional review board in compliance with health insurance portability and accountability act regulations . one hundred thirty - seven estrogen receptor  positive ( er+ ) bc patients with available body mass index ( bmi ) information were classified as nonobese ( bmi < 30kg / m ; n = 94 patients ) and obese ( bmi  30kg / m ; n = 43 patients ) . no patient was excluded based on research - based prediction analysis of microarray 50 ( pam50 ) subtype predictor . patients provided written informed consent to obtain tumor biopsy for genomic studies ( institutional review board protocol lab99 - 402 , uso-02 - 103 , 2003 - 0321 , i - spy-1 ) ( 1012 ) . mcf7 , t47d , and 3t3-l1 cells were obtained from american type culture collection ( manassas , va ) . the mouse mammary tumor eo771 cells ( c57bl6 genetic background ) ( 13 ) were kindly provided by dr xiaohong leng ( md anderson cancer center , houston , tx ) . cell culture conditions , differentiation of 3t3-l1 into adipocytes , and in vitro cell culture experiments are described in the supplementary methods ( available online ) . cell viability was determined by mtt ( 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium ) assay , as described previously ( 14 ) . each treatment set was made in at least three separate independent cell population ( n = 3 ) to allow for statistical evaluation and repeated at least twice to ensure reproducibility . generation of mmtv - tgf;a 
 /a mouse model of er+ bc and obesity is described in the supplementary methods ( available online ) . body fat percentage was determined as described in the supplementary methods ( available online ) . oral glucose tolerance test and insulin tolerance test were performed as previously described ( 15 ) . mice were killed , and tumors ( one - third ) were collected for total rna extraction by trizol ( invitrogen , grand island , ny ) as previously described ( 16 ) . total rna samples were sent to the genomics core facility at the university of texas md anderson cancer center for gene expression analysis using the platform array illumina mousewg-6_v2_0_r3 ( illumina , san diego , ca ) . orthotopic / syngeneic allografting experiments were performed by injecting eo771-fg12 cells in matrigel ( bd bioscience , san jose ca ) into the left fourth mammary fat pad . generation of eo771-fg12 cells and in vivo imaging are described in the supplementary methods ( available online ) . all animal experiments were conducted in accordance with american association for laboratory animal science regulations and the approval of the university of texas md anderson cancer center institutional animal care & use committee . microarray data were analyzed using nexus expression 3 ( biodiscovery , hawthorne , ca ) . downstream effects functional analysis was completed using ingenuity pathway analysis ( ingenuity systems , www.igenuity.com ) software , which uses the ingenuity knowledge base to analyze transcriptomic dataset and identify the expected functions ( eg , biological processes and disease or toxicological functions ) that are increased or decreased ( p < .01 ) because of the observed changes in gene expression ( http://ingenuity.force.com/ipa/articles/ feature_description / accessing - and - using - functional - analysis ) . the width of the links and the relationship of the biological processes with cancer hallmarks were determined using the z scores and illustrated in a circos plot ( 9,17 ) . sections were stained with hematoxylin and eosin or ki67 for pathological evaluation as previously described ( 18 ) . all protein analyses were performed using lysates from whole - cell pellets or mouse tumor samples in radioimmunoprecipitation assay buffer as previously described ( 19 ) . protein samples were used for multiplex immunosorbent assay and western blot analysis as described in the supplementary methods ( available online ) . serum insulin , insulin - like growth factor 1 ( igf-1 ) , and estradiol levels were measured and compared using enzyme - linked immunosorbent assay kits ( r&d systems , minneapolis , mn ) . adipokines expression was measured using the proteome profiler mouse adipokine array ( r&d systems ) . breslow test was also used for survival comparisons because of little early censoring among the patients . for survival comparisons of mice , the assumption of proportionality was verified by the likelihood ratio test and the global  test . for the mouse studies , breast tumor specific deaths were the primary endpoints and deaths due to other causes ( eg , fighting , excessive cystic skin lesions , and killed for inability to roll over or obtain food and water because of excessive obesity ) were the competing risks events . fine gray competing risk analysis was performed using r software ( version 2.13.00 ; r foundation for statistical computing , vienna , austria ) . statistical differences were assessed by two - tailed t test or 1-way analysis of variance with post hoc multiple comparisons ( holm  sidak method ) when appropriate . all other statistical analyses were performed using sigmaplot ( version 12.3 ; systat , san jose , ca ) or spss ( pc version 18 ; ibm spss , armonk , new york ) software . all results were considered statistically significant when p was less than .05 when calculated with the appropriate statistical test . to examine the impact of obesity on gene expression in er+ bc , we identified and reviewed the medical records of 137 patients in a prospective cohort of er+ bc patients ( supplementary table 1 , available online ) . meier analysis showed that obese patients had decreased overall survival ( p = .02 ) and progression - free survival ( p = .03 ) compared with nonobese patients ( figure 1a ) . a multivariable cox regression model showed that obesity was a statistically significant predictor of progression - free survival ( hazard ratio = 1.95 ; 95% ci = 1.02 to 3.75 ; p = .04 ) ( supplementary table 2 , available online ) . therefore , this particular cohort is representative of er+ bc patients that exhibit the association of obesity with decreased survival . transcriptomic changes and adverse clinical outcomes associated with obesity in a prospective cohort of estrogen receptor  positive ( er+ ) breast cancer ( bc ) patients . a ) kaplan  meier analysis of the overall survival ( left panel ) and progression - free survival ( right panel ) of untreated er+ bc patients in gene expression omnibus dataset gse-20194 who were obese ( body mass index [ bmi ]  30.0kg / m ; red ; n = 43 ) or nonobese ( bmi < 30kg / m ; black ; n = 94 ) . b ) heat map of the 130 gene probes with statistically significant changed expression ( p  .01 ; log ratio > 0.1 ) appearing in the same order as in supplementary table 3 ( available online ) . c ) venn diagram of microarray data of pretreatment tumor biopsies , which identified 112 genes with statistically significantly changed expression ( p  .01 ; log ratio > 0.1 ) between obese and nonobese patients . d ) circos plot of the connections of statistically significantly changed biological processes ( bp01 to bp50 ; p<.05 ) ( see also supplementary table 4 , available online ) to cancer hallmarks ( symbols and color - coded labels at left ) . the widths of the connectors represent the absolute values of the biological process z scores . e ) gene set enrichment analyses ( gseas ) for akt activation target genes ( left panel ) and genes involve in the epithelial  mesenchymal transition ( emt ) and metastasis ( right panel ) . gsea results with gene symbol , gene name , and gene enrichment scores of all genes included in each gene set are listed in supplementary table 5 and supplementary table 8 ( available online ) . transcriptomic analysis of breast tumor biopsy from this patient cohort showed that obese patients had statistically significantly increased expression of 62 genes and statistically significantly decreased expression of 50 genes ( p < .01 ) ( figure 1 , b and c ; supplementary table 3 , available online ) . functional transcriptomic analysis identified 59 biological processes that were statistically significantly different ( p < .05 ) between the obese and nonobese groups ( supplementary table 4 , available online ) . the relationships among these biological processes and cancer hallmarks were visualized in a circos plot ( 9,17 ) ( figure 1d ) . these data provide direct evidence that insulin signaling and inflammation are mechanisms that mediate the effect of obesity on human er+ bc . a more specific transcriptomic analysis , gene set enrichment analysis ( 20 ) , showed statistically significant upregulation of protein kinase b ( akt ) target genes ( p = .04 ) , genes involved in the glucose metabolism ( p = .02 ) , genes involved in the generation of precursors of metabolites and energy ( p < mesenchymal transition and metastasis ( p = .03 ) ( figure 1e ; supplementary figure 1 and supplementary tables 58 , available online ) . to gather evidence for a causal relationship between obesity and accelerated carcinogenesis and subsequent cancer progression , we generated the oncogene - induced bc obese mouse model mmtv - tgf;a 
 /a . compared with the lean littermates ( mmtv - tgf;a / a ) , this obese mouse model recapitulates the human obesity - induced phenotype and endocrine profile with higher body fat , glucose intolerance ( p < .001 ) , and insulin resistance ( p < .001 ) ( figure 2a ; supplementary figure 2 , available online ) , as well as statistically significantly higher mean circulating serum levels of fasting insulin ( 1.727 vs 0.472ng / ml ; p < .001 ) , igf-1 ( 1416.1 vs 1108.9ng / ml ; p < .001 ) and estradiol ( 65.23 vs 40.32 pg / ml ; p = .049 ) ( figure 2b ) . mmtv - tgf:a 
 /a obese mice : a genetic mouse model of obesity and breast cancer ( bc ) . a ) plotted body weight of mmtv - tgf;a 
 /a obese mice ( red ) and mmtv - tgf;a / a lean mice ( blue ) ( left panel ) , oral glucose tolerance test results ( middle panel ; n = 11 mice per group ) , and insulin tolerance test results ( right panel ; n = 13 mice per group ) . statistical significance was calculated by two - tailed t test of the area under the curves from the two groups of mice . b ) box plots of hormone levels for obese mice ( red ) and lean mice ( blue ) . these transgenic obese mice had a statistically significantly shorter median overall survival ( 277 vs 451 days ; p < .001 by log - rank test ) and higher breast tumor  specific death cumulative incidence based on fine  gray competing risk analysis ( p = .002 ) ( figure 3a ) ( 21 ) . a cross - sectional comparison at the age of 5 months showed that the mammary fat pads of obese mice had ductal hyperplasia and increased proliferation of mammary ductal epithelium , whereas those of lean mice were morphologically normal ( figure 3b ) . at the age of 7 months , whole mounts of mammary glands revealed visible tumors in obese mice , whereas the lean mice had none ( figure 3c ) . at 9.5 months , obese mice showed adenocarcinoma , as compared with normal mammary tissue from their lean littermates ( figure 3d ) . gross pathology of mice at the age of 9 months revealed bigger breast lesions and increased solid breast tumor growth in obese mice than in lean mice ( p = .002 ) ( figure 3e ) . multiplex protein profiling showed increased phospho - protein levels of the akt / mammalian target of rapamycin ( mtor ) signaling members in tumors from obese mice compared with tumors from lean mice ( figure 3f ) . we also found that the mrna level of the tripartite motif - containing protein 72 ( trim72 , also mg53 ) , which is an e3 ubiquitin ligase that promotes the degradation of the insulin receptor and insulin receptor substrate 1 ( 22 , 23 ) , is decreased in tumor tissues from the obese mice ( supplementary figure 3 , available online ) . this result shows congruence with the observed steady level of insulin receptor and insulin receptor substrate 1 and increased activation of the akt / mtor signaling in these tumors . in contrast , the mrna level of traf6 , which is an e3 ubiquitin ligase that promotes the activation of akt ( 24 ) , is increased in these tumors from obese mice . moreover , akt target genes are induced in tumor tissue from obese mice , confirming downstream activation of the akt / mtor signaling pathway ( supplementary figure 3 , available online ) . a ) kaplan  meier analysis of overall survival ( left panel ) and fine  gray competing risk analysis of breast tumor  specific death ( right panel ) . two - sided log - rank test ( left panel ) and fine  gray competing risk analysis ( right panel ) were used to calculate the p values . b ) representative hematoxylin and eosin ( h&e)stained slides ( top panels ) and ki67 immunohistochemical analysis ( bottom panels ) of mammary ductal epithelium . c ) representative pictures of fourth mammary fat pads illustrating a difference in tumor formation . e ) tumor weights of obese mice ( blue ) and lean mice ( red ) killed at the age of 9 months in the cross - sectional study . f ) phospho - protein levels of members of the akt / mtor signaling pathway from tumor lysates of mmtv - tgf;a 
 /a obese mice ( n = 7 ) plotted relative to those from mmtv - tgf;a / a lean mice ( n = 5 ) . error bars in panel ( f ) and supplementary figure 3 represent 95% confidence intervals . transcriptomic analysis showed that , compared with those from mmtv - tgf;a / a lean mice , breast tumors from mmtv - tgf;a 
 /a obese mice had statistically significantly increased expression of 688 genes and statistically significantly decreased expression of 915 genes ( p  .01 ) ( figure 4 , a and b ; supplementary table 9 , available online ) . functional transcriptomic analysis identified 42 biological processes that were statistically significantly different ( p < .01 ) between the obese and nonobese mice ( supplementary table 10 , available online ) . based on the downstream effects analysis by the ingenuity pathway analysis software , we identified 85 biological functions ( p < .01 ) in both humans and mice ( supplementary table 11 , available online ) . many of these functions concordantly affected by obesity in humans and mice were linked to hallmarks of cancer , as illustrated with a circos plot ( figure 4c ) ( 9,17 ) . these transcriptomic analyses suggest that obesity promotes functions associated primarily with metastasis and invasion , tumor - promoting inflammation , resistance to cell death and , above all , sustained proliferation in er+ bc of both humans and mice . transcriptomic changes associated with obesity by comparing breast cancers ( bcs ) from obese and lean mice , and comparison of overall transcriptomic landscape with that of human estrogen receptor  positive ( er+ ) bcs . a ) heat map of differentially expressed genes in bcs showed clear clustering with sharp differences between mmtv - tgf;a 
 /a obese mice ( n = 5 ) and mmtv - tgf;a / a lean mice ( n = 5 ) . b ) venn diagram of microarray data of transgenic mouse tumors identified 1603 genes with statistically significantly changed expression ( n = 5 mice per group ) . c ) circos plot ( http://mkweb.bcgsc.ca/tableviewer ) of the relationships among biological functions ( bf01 to bf85 ; supplementary table 7 , available online ) affected by obesity in both humans and mice and cancer hallmarks . the width of each link represents the average of the absolute values of the z scores for humans and mice . obesity promoted functions associated with sustained proliferation , resistance to cell death , tumor - promoting inflammation , metastasis and invasion , and so on in er+ bcs of both humans ( obese : n = 43 ; nonobese : n = 94 ) and mice ( n = 5 mice per group ) . bioinformatic analysis of the direct and indirect interactions of estrogen , insulin , and adipokines revealed a complex web of cross - talk graphically presented as a hive plot ( supplementary figure 4 , available online ) ( 25 ) . comparison of this network with the upstream regulators in the human transcriptomic provided strong evidence for the involvement of signaling by estrogen , insulin , igf-1 , and adipokines ( eg , vascular endothelial growth factor a , tumor necrosis factor alpha , interleukin 6 , oncostatin - m , chemokine ligand 5 , leptin , lif , c - reactive protein , adiponectin , and interleukin 10 ) in mediating the influence of obesity on patients with er+ bc . a network was also constructed linking the upstream regulatory network to 10 genes associated with each cancer hallmark in humans ( supplementary figure 5 , available online ) and in mice ( supplementary figure 6 , available online ) . overlay of the human er+ bc transcriptomic data and mouse transcriptomic data showed upregulation of some cancer hallmarks . the relative expression levels suggested that estrogen signaling and leptin signaling play major roles in both humans and mice and that igf-2 , ccl2 ( mcp-1 ) , and tissue inhibitor of metalloproteinase 1 ( timp-1 ) signaling have important roles in mouse tumor growth . to further investigate whether obesity could accelerate cancer progression and the role of akt / mtor on this effect , we used the orthotopic / syngeneic allograft model using in vivo bioluminescent imaging of tumors after injection of syngeneic eo771-fg12 bc cells . our results support that obesity accelerates tumor growth 3.7- to 7.0-fold ( p < .001 ) ( figure 5 , a and b ; supplementary figure 7 , available online ) and increases tumor volume 2.5-fold ( p = .002 ) ( figure 5c ) of er+ bc . inhibition of the akt downstream effector mtor by everolimus or treatment with metformin statistically significantly reduced this obesity - induced tumor growth ( 0.3-fold with p < .001 or 0.5-fold with p = .04 ) ( figure 5b ; supplementary figure 7 , available online ) and decreased obesity - induced increase in tumor volume ( 0.5-fold with p<.001 or 0.6-fold with p = .009 ) ( figure 5c ) . tumors from these obese mice showed increased activation of the akt / mtor signaling members , whereas treatment with metformin or everolimus showed decreased activation ( figure 5 , d and e ) . thus , obesity - induced tumor growth is dependent on the akt / mtor signaling , and metformin and everolimus may be treatment alternatives for bc patients with obesity . effect of obesity on breast cancer progression and tumor growth in orthotopic / syngeneic mice . a ) representative in vivo bioluminescent imaging of tumors performed 4 weeks after orthotopic / syngeneic allografting of eo771-fg12 cells into female transgenic lean and obese mice negative for the mmtv - tgf transgene ( n = 7 mice per group ) . b ) representative in vivo bioluminescent imaging of tumors performed 4 weeks after orthotopic / syngeneic allografting of eo771-fg12 cells into female a / a lean mice ( n = 7 ) , a 
 /a obese mice ( n = 6 ) , a 
 /a obese mice treated with metformin ( 300mg / kg daily ; n = 6 ) and a 
 /a obese mice treated with everolimus ( 4mg / kg daily ; n = 8) . c ) representative images of syngeneic allografted tumors harvested from randomized lean , obese , metformin - treated , and everolimus - treated obese female mice ( scale bars represent 5 mm ; left panel ) . a bar graph illustrates the mean tumor weights from the same experiment ( n = 68 mice per group ; right panel ) . d ) phospho - protein levels of members of the akt / mtor signaling pathway from syngeneic allografted tumor lysates from obese mice plotted relative to those from lean mice ( n = 5 ) . e ) western blot analysis of total and phospho - akt ( ser473 ) , total and phospho - mtor ( ser2448 ) , and total and phospho - p70s6k ( thr389 ) . error bars in panels ( b ) and ( d ) represent 95% confidence intervals . adipokine profiles from serum of both obese mouse models ( figure 6 ) , together with the hormone data ( figure 2b ) , corroborated the importance of estradiol , insulin , hepatic growth factor , igf-1 , leptin , and timp-1 as important upstream regulators in obese er+ bc . obese mice treated with metformin or everolimus showed a decrease in serum adipokines ( eg , fetuin - a , igf-1 , mcp-1 , timp-1 ) , but indirect inhibition of the phosphatidylinositide-3-kinase ( pi3k ) pathway by rosiglitazone only suppressed serum hepatic growth factor , resistin , and timp-1 ( figure 6 ) . in the context of obesity , mature adipocytes humorally accelerated mcf7 ( p < .001 ) ( figure 7a ) and eo771 ( p = .03 ) ( figure 7b ) bc cell proliferation in vitro through secreted mediators ( p = .004 ) ( figure 7c ) , whereas metformin inhibited the adipocyte - accelerated cell proliferation in a dose  dependent manner ( 0.1 mm , p = .02 ; 1 mm and 10 mm , p < .001 ) ( figure 7d ) . to identify adipocyte - secreted factors contributing to bc cell growth , we measured the level of secreted adipokines into the adipocyte - conditioned media and found increased levels of several adipokines but a decreased level of adiponectin relative to the pre - adipocyte - conditioned media ( figure 7e ) . in contrast , metformin treatment statistically significantly increased adipocytes secretion of adiponectin ( p < .01 ) and fibroblast growth factor 21 ( p < .001 ) but suppressed secretion of igf-1 , igf-2 , leptin , and timp-1 ( p < .001 ) ( figure 7e ) . moreover , metformin decreased lipid accumulation during adipocytes differentiation ( supplementary figure 8 , available online ) . relative protein level presented as log ratio of integrated optical density of the microarray dot blot in the mmtv - tgf;a 
 /a obese mouse pooled sera array relative to that in the mmtv - tgf;a / a lean mouse pooled sera array is plotted ( n = 3 mice per group composite ) for each adipokine tested ( left panel ) . protein level presented as log ratio of integrated optical density of the microarray dot blot in the metformin - treated obese mouse pooled sera array relative to that in the untreated obese mouse pooled sera array is plotted ( n = 3 mice per group composite ) for each adipokine tested ( right panel ) . relative protein level presented as log ratio of integrated optical density of the microarray dot blot in the a 
 /a obese mouse pooled sera array relative to that in the a / a lean mouse pooled sera array is plotted ( n = 3 mice per group composite ) for each adipokine tested ( left panel ) . protein level presented as log ratio of integrated optical density of the microarray dot blot in the metformin - treated and everolimus - treated obese mouse pooled sera arrays relative to that in the untreated obese mouse pooled sera array is plotted ( n = 3 mice per group composite ) for each adipokine tested ( right panel ) . a ) mcf7 cell proliferation after having been cultured alone , in coculture with 3t3-l1 fibroblasts , or in coculture with 3t3-l1 mature adipocytes for 4 days . b ) eo771 cell proliferation after having been cultured alone or in coculture with 3t3-l1 mature adipocytes for 3 days with high glucose dulbecco s modified eagle medium and 10% bovine calf serum . c ) cell viability of t47d bc cells after incubation with undifferentiated 3t3-l1 pre - adipocyte - conditioned media or with differentiated 3t3-l1 mature adipocyte - conditioned media . d ) mcf7 cell proliferation after metformin treatment cultured in coculture with 3t3-l1 mature adipocytes for 4 days . e ) adipokine profile array of conditioned media supernatants from 3t3-l1 mature adipocytes cultured for 24 hours relative to 3t3-l1 pre - adipocytes ( n = 3 composite ; left panel ) . adipokines of conditioned media supernatants from 3t3-l1 mature adipocytes cultured with metformin for 24 hours relative to nontreated 3t3-l1 adipocytes ( n = 3 ; right panel ) . asterisks represent statistical significance ( * p<.05 , * * p<.01 , * * * p<.001 ) , one - way analysis of variance . f ) mcf7 cell proliferation after being cocultured with 3t3-l1 mature adipocytes for 4 days alone or with timp-1 neutralizing antibody ( 2 g / ml ; r&d systems ) . g ) mcf7 cell proliferation after being cultured for 4 days alone or with human timp-1 ( 100ng / ml ; millipore , billerica , ma ) . h ) representative photomicrographs of invasion assay of mcf7 cells , which were cultured for 4 days alone or with timp-1 ( 100ng / ml ) . error bars in panels ( a  g ) represent 95% confidence intervals ( n  3 ) . statistical significances were calculated by one - way analysis of variance for panels ( a ) and ( d ) and by two - tailed t test for panels ( c ) , ( f ) , and ( g ) . interestingly , our transcriptomic analysis showed timp-1 as an upstream regulator related to the impact of obesity on bc . investigating the role of timp-1 on the adipocyte - induced bc cell proliferation , we found that adipocyte - induced mcf7 cell proliferation was statistically significantly reduced by a timp-1 neutralizing antibody ( p = .04 ) ( figure 7f ) . in addition , treatment with timp-1 induced mcf-7 cell proliferation ( p = .03 ) ( figure 7 g ) and extracellular matrix invasion ( figure 7h ) . to examine the impact of obesity on gene expression in er+ bc , we identified and reviewed the medical records of 137 patients in a prospective cohort of er+ bc patients ( supplementary table 1 , available online ) . meier analysis showed that obese patients had decreased overall survival ( p = .02 ) and progression - free survival ( p = .03 ) compared with nonobese patients ( figure 1a ) . a multivariable cox regression model showed that obesity was a statistically significant predictor of progression - free survival ( hazard ratio = 1.95 ; 95% ci = 1.02 to 3.75 ; p = .04 ) ( supplementary table 2 , available online ) . therefore , this particular cohort is representative of er+ bc patients that exhibit the association of obesity with decreased survival . transcriptomic changes and adverse clinical outcomes associated with obesity in a prospective cohort of estrogen receptor  positive ( er+ ) breast cancer ( bc ) patients . a ) kaplan  meier analysis of the overall survival ( left panel ) and progression - free survival ( right panel ) of untreated er+ bc patients in gene expression omnibus dataset gse-20194 who were obese ( body mass index [ bmi ]  30.0kg / m ; red ; n = 43 ) or nonobese ( bmi < 30kg / m ; black ; n = 94 ) . b ) heat map of the 130 gene probes with statistically significant changed expression ( p  .01 ; log ratio > 0.1 ) appearing in the same order as in supplementary table 3 ( available online ) . c ) venn diagram of microarray data of pretreatment tumor biopsies , which identified 112 genes with statistically significantly changed expression ( p  .01 ; log ratio > 0.1 ) between obese and nonobese patients . d ) circos plot of the connections of statistically significantly changed biological processes ( bp01 to bp50 ; p<.05 ) ( see also supplementary table 4 , available online ) to cancer hallmarks ( symbols and color - coded labels at left ) . the widths of the connectors represent the absolute values of the biological process z scores . e ) gene set enrichment analyses ( gseas ) for akt activation target genes ( left panel ) and genes involve in the epithelial  mesenchymal transition ( emt ) and metastasis ( right panel ) . gsea results with gene symbol , gene name , and gene enrichment scores of all genes included in each gene set are listed in supplementary table 5 and supplementary table 8 ( available online ) . transcriptomic analysis of breast tumor biopsy from this patient cohort showed that obese patients had statistically significantly increased expression of 62 genes and statistically significantly decreased expression of 50 genes ( p < .01 ) ( figure 1 , b and c ; supplementary table 3 , available online ) . functional transcriptomic analysis identified 59 biological processes that were statistically significantly different ( p < .05 ) between the obese and nonobese groups ( supplementary table 4 , available online ) . the relationships among these biological processes and cancer hallmarks were visualized in a circos plot ( 9,17 ) ( figure 1d ) . these data provide direct evidence that insulin signaling and inflammation are mechanisms that mediate the effect of obesity on human er+ bc . a more specific transcriptomic analysis , gene set enrichment analysis ( 20 ) , showed statistically significant upregulation of protein kinase b ( akt ) target genes ( p = .04 ) , genes involved in the glucose metabolism ( p = .02 ) , genes involved in the generation of precursors of metabolites and energy ( p < mesenchymal transition and metastasis ( p = .03 ) ( figure 1e ; supplementary figure 1 and supplementary tables 58 , available online ) . to gather evidence for a causal relationship between obesity and accelerated carcinogenesis and subsequent cancer progression , we generated the oncogene - induced bc obese mouse model mmtv - tgf;a 
 /a . compared with the lean littermates ( mmtv - tgf;a / a ) , this obese mouse model recapitulates the human obesity - induced phenotype and endocrine profile with higher body fat , glucose intolerance ( p < .001 ) , and insulin resistance ( p < .001 ) ( figure 2a ; supplementary figure 2 , available online ) , as well as statistically significantly higher mean circulating serum levels of fasting insulin ( 1.727 vs 0.472ng / ml ; p < .001 ) , igf-1 ( 1416.1 vs 1108.9ng / ml ; p < .001 ) and estradiol ( 65.23 vs 40.32 pg / ml ; p = .049 ) ( figure 2b ) . mmtv - tgf:a 
 /a obese mice : a genetic mouse model of obesity and breast cancer ( bc ) . a ) plotted body weight of mmtv - tgf;a 
 /a obese mice ( red ) and mmtv - tgf;a / a lean mice ( blue ) ( left panel ) , oral glucose tolerance test results ( middle panel ; n = 11 mice per group ) , and insulin tolerance test results ( right panel ; n = 13 mice per group ) . statistical significance was calculated by two - tailed t test of the area under the curves from the two groups of mice . b ) box plots of hormone levels for obese mice ( red ) and lean mice ( blue ) . these transgenic obese mice had a statistically significantly shorter median overall survival ( 277 vs 451 days ; p < .001 by log - rank test ) and higher breast tumor  specific death cumulative incidence based on fine  gray competing risk analysis ( p = .002 ) ( figure 3a ) ( 21 ) . a cross - sectional comparison at the age of 5 months showed that the mammary fat pads of obese mice had ductal hyperplasia and increased proliferation of mammary ductal epithelium , whereas those of lean mice were morphologically normal ( figure 3b ) . at the age of 7 months , whole mounts of mammary glands revealed visible tumors in obese mice , whereas the lean mice had none ( figure 3c ) . at 9.5 months , obese mice showed adenocarcinoma , as compared with normal mammary tissue from their lean littermates ( figure 3d ) . gross pathology of mice at the age of 9 months revealed bigger breast lesions and increased solid breast tumor growth in obese mice than in lean mice ( p = .002 ) ( figure 3e ) . multiplex protein profiling showed increased phospho - protein levels of the akt / mammalian target of rapamycin ( mtor ) signaling members in tumors from obese mice compared with tumors from lean mice ( figure 3f ) . we also found that the mrna level of the tripartite motif - containing protein 72 ( trim72 , also mg53 ) , which is an e3 ubiquitin ligase that promotes the degradation of the insulin receptor and insulin receptor substrate 1 ( 22 , 23 ) , is decreased in tumor tissues from the obese mice ( supplementary figure 3 , available online ) . this result shows congruence with the observed steady level of insulin receptor and insulin receptor substrate 1 and increased activation of the akt / mtor signaling in these tumors . in contrast , the mrna level of traf6 , which is an e3 ubiquitin ligase that promotes the activation of akt ( 24 ) , is increased in these tumors from obese mice . moreover , akt target genes are induced in tumor tissue from obese mice , confirming downstream activation of the akt / mtor signaling pathway ( supplementary figure 3 , available online ) . a ) kaplan  meier analysis of overall survival ( left panel ) and fine  gray competing risk analysis of breast tumor  specific death ( right panel ) . two - sided log - rank test ( left panel ) and fine  gray competing risk analysis ( right panel ) were used to calculate the p values . b ) representative hematoxylin and eosin ( h&e)stained slides ( top panels ) and ki67 immunohistochemical analysis ( bottom panels ) of mammary ductal epithelium c ) representative pictures of fourth mammary fat pads illustrating a difference in tumor formation . e ) tumor weights of obese mice ( blue ) and lean mice ( red ) killed at the age of 9 months in the cross - sectional study . f ) phospho - protein levels of members of the akt / mtor signaling pathway from tumor lysates of mmtv - tgf;a 
 /a obese mice ( n = 7 ) plotted relative to those from mmtv - tgf;a / a lean mice ( n = 5 ) . error bars in panel ( f ) and supplementary figure 3 represent 95% confidence intervals . transcriptomic analysis showed that , compared with those from mmtv - tgf;a / a lean mice , breast tumors from mmtv - tgf;a 
 /a obese mice had statistically significantly increased expression of 688 genes and statistically significantly decreased expression of 915 genes ( p  .01 ) ( figure 4 , a and b ; supplementary table 9 , available online ) . functional transcriptomic analysis identified 42 biological processes that were statistically significantly different ( p < .01 ) between the obese and nonobese mice ( supplementary table 10 , available online ) . based on the downstream effects analysis by the ingenuity pathway analysis software , we identified 85 biological functions ( p < .01 ) in both humans and mice ( supplementary table 11 , available online ) . many of these functions concordantly affected by obesity in humans and mice were linked to hallmarks of cancer , as illustrated with a circos plot ( figure 4c ) ( 9,17 ) . these transcriptomic analyses suggest that obesity promotes functions associated primarily with metastasis and invasion , tumor - promoting inflammation , resistance to cell death and , above all , sustained proliferation in er+ bc of both humans and mice . transcriptomic changes associated with obesity by comparing breast cancers ( bcs ) from obese and lean mice , and comparison of overall transcriptomic landscape with that of human estrogen receptor  positive ( er+ ) bcs . a ) heat map of differentially expressed genes in bcs showed clear clustering with sharp differences between mmtv - tgf;a 
 /a obese mice ( n = 5 ) and mmtv - tgf;a / a lean mice ( n = 5 ) . b ) venn diagram of microarray data of transgenic mouse tumors identified 1603 genes with statistically significantly changed expression ( n = 5 mice per group ) . c ) circos plot ( http://mkweb.bcgsc.ca/tableviewer ) of the relationships among biological functions ( bf01 to bf85 ; supplementary table 7 , available online ) affected by obesity in both humans and mice and cancer hallmarks . the width of each link represents the average of the absolute values of the z scores for humans and mice . obesity promoted functions associated with sustained proliferation , resistance to cell death , tumor - promoting inflammation , metastasis and invasion , and so on in er+ bcs of both humans ( obese : n = 43 ; nonobese : n = 94 ) and mice ( n = 5 mice per group ) . bioinformatic analysis of the direct and indirect interactions of estrogen , insulin , and adipokines revealed a complex web of cross - talk graphically presented as a hive plot ( supplementary figure 4 , available online ) ( 25 ) . comparison of this network with the upstream regulators in the human transcriptomic provided strong evidence for the involvement of signaling by estrogen , insulin , igf-1 , and adipokines ( eg , vascular endothelial growth factor a , tumor necrosis factor alpha , interleukin 6 , oncostatin - m , chemokine ligand 5 , leptin , lif , c - reactive protein , adiponectin , and interleukin 10 ) in mediating the influence of obesity on patients with er+ bc . a network was also constructed linking the upstream regulatory network to 10 genes associated with each cancer hallmark in humans ( supplementary figure 5 , available online ) and in mice ( supplementary figure 6 , available online ) . overlay of the human er+ bc transcriptomic data and mouse transcriptomic data showed upregulation of some cancer hallmarks . the relative expression levels suggested that estrogen signaling and leptin signaling play major roles in both humans and mice and that igf-2 , ccl2 ( mcp-1 ) , and tissue inhibitor of metalloproteinase 1 ( timp-1 ) signaling have important roles in mouse tumor growth . to further investigate whether obesity could accelerate cancer progression and the role of akt / mtor on this effect , we used the orthotopic / syngeneic allograft model using in vivo bioluminescent imaging of tumors after injection of syngeneic eo771-fg12 bc cells . our results support that obesity accelerates tumor growth 3.7- to 7.0-fold ( p < .001 ) ( figure 5 , a and b ; supplementary figure 7 , available online ) and increases tumor volume 2.5-fold ( p = .002 ) ( figure 5c ) of er+ bc . inhibition of the akt downstream effector mtor by everolimus or treatment with metformin statistically significantly reduced this obesity - induced tumor growth ( 0.3-fold with p < .001 or 0.5-fold with p = .04 ) ( figure 5b ; supplementary figure 7 , available online ) and decreased obesity - induced increase in tumor volume ( 0.5-fold with p<.001 or 0.6-fold with p = .009 ) ( figure 5c ) . tumors from these obese mice showed increased activation of the akt / mtor signaling members , whereas treatment with metformin or everolimus showed decreased activation ( figure 5 , d and e ) . thus , obesity - induced tumor growth is dependent on the akt / mtor signaling , and metformin and everolimus may be treatment alternatives for bc patients with obesity . effect of obesity on breast cancer progression and tumor growth in orthotopic / syngeneic mice . a ) representative in vivo bioluminescent imaging of tumors performed 4 weeks after orthotopic / syngeneic allografting of eo771-fg12 cells into female transgenic lean and obese mice negative for the mmtv - tgf transgene ( n = 7 mice per group ) . b ) representative in vivo bioluminescent imaging of tumors performed 4 weeks after orthotopic / syngeneic allografting of eo771-fg12 cells into female a / a lean mice ( n = 7 ) , a 
 /a obese mice ( n = 6 ) , a 
 /a obese mice treated with metformin ( 300mg / kg daily ; n = 6 ) and a 
 /a obese mice treated with everolimus ( 4mg / kg daily ; n = 8) . c ) representative images of syngeneic allografted tumors harvested from randomized lean , obese , metformin - treated , and everolimus - treated obese female mice ( scale bars represent 5 mm ; left panel ) . a bar graph illustrates the mean tumor weights from the same experiment ( n = 68 mice per group ; right panel ) . d ) phospho - protein levels of members of the akt / mtor signaling pathway from syngeneic allografted tumor lysates from obese mice plotted relative to those from lean mice ( n = 5 ) . e ) western blot analysis of total and phospho - akt ( ser473 ) , total and phospho - mtor ( ser2448 ) , and total and phospho - p70s6k ( thr389 ) . error bars in panels ( b ) and ( d ) represent 95% confidence intervals . adipokine profiles from serum of both obese mouse models ( figure 6 ) , together with the hormone data ( figure 2b ) , corroborated the importance of estradiol , insulin , hepatic growth factor , igf-1 , leptin , and timp-1 as important upstream regulators in obese er+ bc . obese mice treated with metformin or everolimus showed a decrease in serum adipokines ( eg , fetuin - a , igf-1 , mcp-1 , timp-1 ) , but indirect inhibition of the phosphatidylinositide-3-kinase ( pi3k ) pathway by rosiglitazone only suppressed serum hepatic growth factor , resistin , and timp-1 ( figure 6 ) . in the context of obesity , .001 ) ( figure 7a ) and eo771 ( p = .03 ) ( figure 7b ) bc cell proliferation in vitro through secreted mediators ( p = .004 ) ( figure 7c ) , whereas metformin inhibited the adipocyte - accelerated cell proliferation in a dose  dependent manner ( 0.1 mm , p = .02 ; 1 mm and 10 mm , p < .001 ) ( figure 7d ) . to identify adipocyte - secreted factors contributing to bc cell growth , we measured the level of secreted adipokines into the adipocyte - conditioned media and found increased levels of several adipokines but a decreased level of adiponectin relative to the pre - adipocyte - conditioned media ( figure 7e ) . in contrast , metformin treatment statistically significantly increased adipocytes secretion of adiponectin ( p < .01 ) and fibroblast growth factor 21 ( p < .001 ) but suppressed secretion of igf-1 , igf-2 , leptin , and timp-1 ( p < .001 ) ( figure 7e ) . moreover , metformin decreased lipid accumulation during adipocytes differentiation ( supplementary figure 8 , available online ) . relative protein level presented as log ratio of integrated optical density of the microarray dot blot in the mmtv - tgf;a 
 /a obese mouse pooled sera array relative to that in the mmtv - tgf;a / a lean mouse pooled sera array is plotted ( n = 3 mice per group composite ) for each adipokine tested ( left panel ) . protein level presented as log ratio of integrated optical density of the microarray dot blot in the metformin - treated obese mouse pooled sera array relative to that in the untreated obese mouse pooled sera array is plotted ( n = 3 mice per group composite ) for each adipokine tested ( right panel ) . relative protein level presented as log ratio of integrated optical density of the microarray dot blot in the a 
 /a obese mouse pooled sera array relative to that in the a / a lean mouse pooled sera array is plotted ( n = 3 mice per group composite ) for each adipokine tested ( left panel ) . protein level presented as log ratio of integrated optical density of the microarray dot blot in the metformin - treated and everolimus - treated obese mouse pooled sera arrays relative to that in the untreated obese mouse pooled sera array is plotted ( n = 3 mice per group composite ) for each adipokine tested ( right panel ) . a ) mcf7 cell proliferation after having been cultured alone , in coculture with 3t3-l1 fibroblasts , or in coculture with 3t3-l1 mature adipocytes for 4 days . b ) eo771 cell proliferation after having been cultured alone or in coculture with 3t3-l1 mature adipocytes for 3 days with high glucose dulbecco s modified eagle medium and 10% bovine calf serum . c ) cell viability of t47d bc cells after incubation with undifferentiated 3t3-l1 pre - adipocyte - conditioned media or with differentiated 3t3-l1 mature adipocyte - conditioned media . d ) mcf7 cell proliferation after metformin treatment cultured in coculture with 3t3-l1 mature adipocytes for 4 days . e ) adipokine profile array of conditioned media supernatants from 3t3-l1 mature adipocytes cultured for 24 hours relative to 3t3-l1 pre - adipocytes ( n = 3 composite ; left panel ) . adipokines of conditioned media supernatants from 3t3-l1 mature adipocytes cultured with metformin for 24 hours relative to nontreated 3t3-l1 adipocytes ( n = 3 ; right panel ) . asterisks represent statistical significance ( * p<.05 , * * p<.01 , * * * p<.001 ) , one - way analysis of variance . f ) mcf7 cell proliferation after being cocultured with 3t3-l1 mature adipocytes for 4 days alone or with timp-1 neutralizing antibody ( 2 g / ml ; r&d systems ) . g ) mcf7 cell proliferation after being cultured for 4 days alone or with human timp-1 ( 100ng / ml ; millipore , billerica , ma ) . h ) representative photomicrographs of invasion assay of mcf7 cells , which were cultured for 4 days alone or with timp-1 ( 100ng / ml ) . error bars in panels ( a  g ) represent 95% confidence intervals ( n  3 ) . statistical significances were calculated by one - way analysis of variance for panels ( a ) and ( d ) and by two - tailed t test for panels ( c ) , ( f ) , and ( g ) . interestingly , our transcriptomic analysis showed timp-1 as an upstream regulator related to the impact of obesity on bc . investigating the role of timp-1 on the adipocyte - induced bc cell proliferation , we found that adipocyte - induced mcf7 cell proliferation was statistically significantly reduced by a timp-1 neutralizing antibody ( p = .04 ) ( figure 7f ) . in addition , treatment with timp-1 induced mcf-7 cell proliferation ( p = .03 ) ( figure 7 g ) and extracellular matrix invasion ( figure 7h ) . obesity induces overall transcriptional changes , enhancing the cancer hallmarks of tumors from er+ bc patients before any treatment . our findings give a comprehensive overview as well as direct evidence for the mechanisms associated with the obesity - induced poor clinical outcomes ( eg , overall survival , progression - free survival , aggressiveness , metastasis ) . the bioinformatics approach provided the complete landscape of functional changes affected by the upstream regulators . thus , we have provided strong evidence for the involvement of adipokines in mediating the influence of obesity on patients with er+ bc . previous in vivo studies of obesity and cancer have used lepr 
  or lepr 
  mice that lack functional leptin signaling ( 26,27 ) and diet - induced obesity ( 2830 ) . high - fat diet induces obesity , but it is not the only driver of the metabolic changes underlying obesity ( 3133 ) . diet - induced obesity models can not exclude the contribution of excessive dietary fat percentage on carcinogenesis and cancer progression . our model did not demonstrate that obesity by itself could initiate the carcinogenic process , but both our longitudinal and cross - sectional data did demonstrate in vivo that obesity accelerates oncogene - driven breast carcinogenesis and tumor growth . functional transcriptomic analysis of bc from obese and lean mice also recapitulated many of the biological functions affected by obesity from the human dataset . in agreement with the human transcriptomic analysis , tumors from obese mice showed increased phospho - protein levels of the pi3k / akt / mtor signaling pathway members ( figure 3 g ) , which is an indicator of increased anabolic metabolism , cancer cell proliferation , and survival ( 34 ) . our results supported that obesity accelerates carcinogenesis and tumor growth in mice and that this obesity - induced effect is dependent on the akt / mtor signaling . moreover , our data suggest that metformin and everolimus may serve as treatments of choice or in combination with other standard cancer treatment for bc patients with obesity . in the context of obesity , our studies show that adipocytes and adipokines promote cancer development and accelerate cancer cell growth after transformation . in contrast , metformin or everolimus suppresses mouse serum adipokines ( eg , timp-1 ) , which correlates with the decrease in tumor growth . timp-1 has been related with poor prognosis in bc ( 35,36 ) , and it is recently known to activate the pi3k / akt pathway ( 37 ) . our findings showed timp-1 as an upstream regulator with an important role in obesity - induced bc . our study has provided a better understanding of the mechanism involved in the effect of obesity on cancer and has set the ground for targeting appropriate specific patient population for treatment with metformin and everolimus . it has provided the obesity - induced functional transcriptomic changes and signaling mechanisms involved in obesity - accelerated breast cancer in both humans and mice in the context of their contributions to specific cancer hallmarks . however , a major limitation in our study is that the transcriptomic changes reflect the mixed response of cancer cells and stromal cells to obesity . the relative contribution of stroma to the transcriptome of the tissue samples may vary . future research with new methodology may be able to overcome this limitation . in conclusion , our data from humans provide direct evidence for the mechanistic involvement of adipokines in addition to estrogen , insulin , or igf-1 signaling . the results from our mouse model corroborate human data and provide prospective data to support the conclusion that obesity accelerates er+ breast carcinogenesis and cancer progression through adipokines and pi3k / akt / mtor signaling . our mouse model will be a useful tool for future research on therapeutic strategies that would block or reverse the effect of obesity on cancer ( 38 ) . this work was supported by a susan g. komen for the cure promise grant ( kg081048 to scy , mhl , fje , and wlm ) ; and the national cancer institute at the national institutes of health ( ro1-ca089266 to mhl ; cancer center support grant 
 p30-ca16672 to the university of texas md anderson cancer center ) . efm was supported by the national cancer institute at the national institutes of health training grant program in molecular genetics ( t32-ca009299 ) and national institutes of health loan repayment program . gvt was supported by a national institutes of health cancer prevention fellowship ( r25 t ca57730 ) and then by a national institutes of health minority supplement ( 3-r01ca089266 - 08s1 , 3-r01ca089266 - 09s1 , and 3-r01ca089266 - 10s1 ; pi : mhl ) . lph was supported by the vietnam education foundation , the rosalie b. hite foundation , and the department of defense breast cancer research program ( w81xwh-100171 ) .
backgroundobesity increases the risk of cancer death among postmenopausal women with estrogen receptor  positive ( er+ ) breast cancer , but the direct evidence for the mechanisms is lacking . 
 the purpose of this study is to demonstrate direct evidence for the mechanisms mediating this epidemiologic phenomenon.methodswe analyzed transcriptomic profiles of pretreatment biopsies from a prospective cohort of 137 er+ breast cancer patients . 
 we generated transgenic ( mmtv - tgf;a 
 y 
 /a ) and orthotopic / syngeneic ( a 
 y 
 /a ) obese mouse models to investigate the effect of obesity on tumorigenesis and tumor progression and to determine biological mechanisms using whole - genome transcriptome microarrays and protein analyses . 
 we used a coculture system to examine the impact of adipocytes / adipokines on breast cancer cell proliferation . 
 all statistical tests were two-sided.resultsfunctional transcriptomic analysis of patients revealed the association of obesity with 59 biological functional changes ( p < .05 ) linked to cancer hallmarks . gene enrichment analysis revealed enrichment of akt - target genes ( p = .04 ) and epithelial  mesenchymal transition genes ( p = .03 ) in patients . 
 our obese mouse models demonstrated activation of the akt / mtor pathway in obesity - accelerated mammary tumor growth ( 3.7- to 7.0-fold ; p < .001 ; n = 67 mice per group ) . 
 metformin or everolimus can suppress obesity - induced secretion of adipokines and breast tumor formation and growth ( 0.5-fold , p = .04 ; 0.3-fold , p < .001 , respectively ; n = 68 mice per group ) . 
 the coculture model revealed that adipocyte - secreted adipokines ( eg , timp-1 ) regulate adipocyte - induced breast cancer cell proliferation and invasion . 
 metformin suppress adipocyte - induced cell proliferation and adipocyte - secreted adipokines in vitro.conclusionsadipokine secretion and akt / mtor activation play important roles in obesity - accelerated breast cancer aggressiveness in addition to hyperinsulinemia , estrogen signaling , and inflammation . 
 metformin and everolimus have potential for therapeutic interventions of er+ breast cancer patients with obesity .