David A. Bader

High-fat maternal diet during pregnancy persistently alters the offspring microbiome in a primate model

The intestinal microbiome is a unique ecosystem and an essential mediator of metabolism and obesity in mammals. However, studies investigating the impact of the diet on the establishment of the gut microbiome early in life are generally lacking, and most notably so in primate models. Here we report that a high-fat maternal or postnatal diet, but not obesity per se, structures the offspring’s intestinal microbiome in Macaca fuscata (Japanese macaque). The resultant microbial dysbiosis is only partially corrected by a low-fat, control diet after weaning. Unexpectedly, early exposure to a high-fat diet diminished the abundance of non-pathogenic Campylobacter in the juvenile gut, suggesting a potential role for dietary fat in shaping commensal microbial communities in primates. Our data challenge the concept of an obesity-causing gut microbiome, and rather provide evidence for a contribution of the maternal diet in establishing the microbiota, which in turn affects intestinal maintenance of metabolic health.

Inflammation-Mediated Genetic and Epigenetic Alterations Drive Cancer Development in the Neighboring Epithelium upon Stromal Abrogation of TGF-β Signaling

Deletion of tumor suppressor genes in stromal fibroblasts induces epithelial cancer development, suggesting an important role of stroma in epithelial homoeostasis. However, the underlying mechanisms remain to be elucidated. Here we report that deletion of the gene encoding TGFβ receptor 2 (Tgfbr2) in the stromal fibroblasts (Tgfbr2fspKO) induces inflammation and significant DNA damage in the neighboring epithelia of the forestomach. This results in loss or down-regulation of cyclin-dependent kinase inhibitors p15, p16, and p21, which contribute to the development of invasive squamous cell carcinoma (SCC). Anti-inflammation treatment restored p21 expression, delayed tumorigenesis, and increased survival of Tgfbr2fspKO mice. Our data demonstrate for the first time that inflammation is a critical player in the epigenetic silencing of p21 in tumor progression. Examination of human esophageal SCC showed a down-regulation of TGFβ receptor 2 (TβRII) in the stromal fibroblasts, as well as increased inflammation, DNA damage, and loss or decreased p15/p16 expression. Our study suggests anti-inflammation may be a new therapeutic option in treating human SCCs with down-regulation of TβRII in the stroma.

Inhibition of the hexosamine biosynthetic pathway promotes castration-resistant prostate cancer

The precise molecular alterations driving castration-resistant prostate cancer (CRPC) are not clearly understood. Using a novel network-based integrative approach, here, we show distinct alterations in the hexosamine biosynthetic pathway (HBP) to be critical for CRPC. Expression of HBP enzyme glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is found to be significantly decreased in CRPC compared with localized prostate cancer (PCa). Genetic loss-of-function of GNPNAT1 in CRPC-like cells increases proliferation and aggressiveness, in vitro and in vivo. This is mediated by either activation of the PI3K-AKT pathway in cells expressing full-length androgen receptor (AR) or by specific protein 1 (SP1)-regulated expression of carbohydrate response element-binding protein (ChREBP) in cells containing AR-V7 variant. Strikingly, addition of the HBP metabolite UDP-N-acetylglucosamine (UDP-GlcNAc) to CRPC-like cells significantly decreases cell proliferation, both in-vitro and in animal studies, while also demonstrates additive efficacy when combined with enzalutamide in-vitro. These observations demonstrate the therapeutic value of targeting HBP in CRPC.

The miRNA Interactome in Metabolic Homeostasis.

Global expression analyses demonstrate that alterations in miRNA levels correlate with various metabolic diseases. miRNAs regulate central metabolic pathways and thus play vital roles in maintaining organismal energy balance and metabolic homeostasis. Here we highlight novel sequencing technologies used to comprehensively define the target spectrum of miRNAs in metabolic disease that complement recent literature reporting physiologic roles for miRNAs in the regulation of glucose and lipid metabolism in peripheral tissues of animal models of metabolic dysfunction. These emerging technologies help decipher the complexity of the miRNA interactome and enrich our understanding of how miRNAs mediate physiologic effects by targeting a spectrum of gene transcripts simultaneously. miRNA-based therapeutics emerge as a viable strategy for treating metabolic diseases.

The Landscape of microRNA Targeting in Prostate Cancer Defined by AGO-PAR-CLIP.

MicroRNA (miRNA) deregulation in prostate cancer (PCa) contributes to PCa initiation and metastatic progression. To comprehensively define the cancer-associated changes in miRNA targeting and function in commonly studied models of PCa, we performed photoactivatable ribonucleoside-enhanced cross-linking immunoprecipitation of the Argonaute protein in a panel of PCa cell lines modeling different stages of PCa progression. Using this comprehensive catalogue of miRNA targets, we analyzed miRNA targeting on known drivers of PCa and examined tissue-specific and stage-specific pathway targeting by miRNAs. We found that androgen receptor is the most frequently targeted PCa oncogene and that miR-148a targets the largest number of known PCa drivers. Globally, tissue-specific and stage-specific changes in miRNA targeting are driven by homeostatic response to active oncogenic pathways. Our findings indicate that, even in advanced PCa, the miRNA pool adapts to regulate continuing alterations in the cancer genome to balance oncogenic molecular changes. These findings are important because they are the first to globally characterize miRNA changes in PCa and demonstrate how the miRNA target spectrum responds to staged tumorigenesis.

Mitochondrial activity in human white adipocytes is regulated by the ubiquitin carrier protein 9/microRNA-30a axis

The acquisition of beige adipocyte features by white fat cells corresponds to protection against obesity-induced metabolic diseases in humans and animal models of type 2 diabetes. In adipose tissue, expression of the E2 small ubiquitin-like modifier ligase ubiquitin carrier protein 9 (Ubc9) is positively correlated with markers of insulin resistance and corresponds with impaired browning of human white adipocytes. However, the molecular regulation of Ubc9 expression in adipocytes and other cells remains unclear. In this study, we demonstrate that the mRNA and protein expression of Ubc9 are regulated by the microRNA miRNA-30a (miR-30a) in human subcutaneous adipocytes. Ubc9 and miR-30a exhibit inverse expression in adipose tissue, with miR-30a robustly elevated in brown fat. Depletion of Ubc9 by siRNA or enforced expression of a miR-30a mimic augments mitochondrial volume and respiration in human white adipocytes, reflecting features of brown fat cells. Furthermore, Ubc9 depletion induces a brown fat gene program in human subcutaneous adipocytes. Induction of the beige-selective gene program corresponds to stabilization of the PR domain-containing 16 (PRDM16) protein, an obligate transcriptional regulator of the brown/beige fat metabolic program in white adipocytes that interacts with Ubc9. Taken together, our data demonstrate a previously unappreciated molecular axis that controls browning of human white adipocytes.

Ubc9 Impairs Activation of the Brown Fat Energy Metabolism Program in Human White Adipocytes

Insulin resistance and type 2 diabetes mellitus (T2DM) result from an inability to efficiently store and catabolize surplus energy in adipose tissue. Subcutaneous adipocytes protect against insulin resistance and T2DM by coupling differentiation with the induction of brown fat gene programs for efficient energy metabolism. Mechanisms that disrupt these programs in adipocytes are currently poorly defined, but represent therapeutic targets for the treatment of T2DM. To gain insight into these mechanisms, we performed a high-throughput microscopy screen that identified ubiquitin carrier protein 9 (Ubc9) as a negative regulator of energy storage in human sc adipocytes. Ubc9 depletion enhanced energy storage and induced the brown fat gene program in human sc adipocytes. Induction of adipocyte differentiation resulted in decreased Ubc9 expression commensurate with increased brown fat gene expression. Thiazolidinedione treatment reduced the interaction between Ubc9 and peroxisome proliferator-activated receptor (PPAR)γ, suggesting a mechanism by which Ubc9 represses PPARγ activity. In support of this hypothesis, Ubc9 overexpression remodeled energy metabolism in human sc adipocytes by selectively inhibiting brown adipocyte-specific function. Further, Ubc9 overexpression decreased uncoupling protein 1 expression by disrupting PPARγ binding at a critical uncoupling protein 1 enhancer region. Last, Ubc9 is significantly elevated in sc adipose tissue isolated from mouse models of insulin resistance as well as diabetic and insulin-resistant humans. Taken together, our findings demonstrate a critical role for Ubc9 in the regulation of sc adipocyte energy homeostasis.

miR-30a Remodels Subcutaneous Adipose Tissue Inflammation to Improve Insulin Sensitivity in Obesity

Chronic inflammation accompanies obesity and limits subcutaneous white adipose tissue (WAT) expandability, accelerating the development of insulin resistance and type 2 diabetes mellitus. MicroRNAs (miRNAs) influence expression of many metabolic genes in fat cells, but physiological roles in WAT remain poorly characterized. Here, we report that expression of the miRNA miR-30a in subcutaneous WAT corresponds with insulin sensitivity in obese mice and humans. To examine the hypothesis that restoration of miR-30a expression in WAT improves insulin sensitivity, we injected adenovirus (Adv) expressing miR-30a into the subcutaneous fat pad of diabetic mice. Exogenous miR-30a expression in the subcutaneous WAT depot of obese mice coupled improved insulin sensitivity and increased energy expenditure with decreased ectopic fat deposition in the liver and reduced WAT inflammation. High-throughput proteomic profiling and RNA-Seq suggested that miR-30a targets the transcription factor STAT1 to limit the actions of the proinflammatory cytokine interferon-γ (IFN-γ) that would otherwise restrict WAT expansion and decrease insulin sensitivity. We further demonstrated that miR-30a opposes the actions of IFN-γ, suggesting an important role for miR-30a in defending adipocytes against proinflammatory cytokines that reduce peripheral insulin sensitivity. Together, our data identify a critical molecular signaling axis, elements of which are involved in uncoupling obesity from metabolic dysfunction.

Abstract 5431: Androgen receptor regulates the mitochondrial pyruvate carrier to fuel oncometabolism in prostate cancer

The metabolic underpinnings of androgen receptor (AR)-driven growth in prostate cancer (PCa) are underexplored, hindering the development of strategies to leverage the metabolic properties differentiating PCa from normal tissue. To this end, we discovered a subunit of the mitochondrial pyruvate carrier (MPC), MPC2, is a direct AR target gene that is increased in PCa specimens and associated with clinical outcomes. Our observation suggests a targetable link between AR signaling and carbon trafficking in PCa. Indeed, MPC inhibition delays proliferation and alters metabolic properties in hormone sensitive and castrate resistant AR-driven PCa models. Next, using 13C metabolic flux analysis coupled with a reverse phase protein array, we determined MPC inhibition results in activation of the eIF2α/ATF4 integrated stress response pathway to partially compensate for MPC inhibition by coordinating increased glutamine incorporation into the citric acid cycle. Therefore, to amplify the effect of MPC inhibition, we restricted glutamine availability during MPC inhibition and found this combination resulted in proliferative arrest. Last, we extended our efforts in-vivo and demonstrated combinatorial MPC/glutaminase inhibition resulted in a significant reduction in castrate-resistant prostate tumor xenograft growth in mice with no overt host toxicity. We anticipate our findings will accelerate the development of clinical strategies to therapeutically manipulate carbon flux to starve clinically lethal castrate-resistant PCa. Citation Format: David A. Bader, Nagireddy Putluri, Sean M. Hartig, Sean E. McGuire. Androgen receptor regulates the mitochondrial pyruvate carrier to fuel oncometabolism in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5431. doi:10.1158/1538-7445.AM2017-5431

Abstract 50: Abrogation of stromal TGF-β signaling induces DNA damage and genetic/epigenetic alterations in neighboring epithelia

Inactivation of tumor suppressor genes in stromal fibroblast induces epithelial cancer development, suggesting an important role of stroma in epithelial homoeostasis. We investigated the underlying mechanisms in squamous cell carcinoma (SCC) development due to loss of TGF- signaling in FSP1+ fibroblasts (Tgfbr2 fspKO ). We found that stroma specific deletion of Tgfbr2 resulted a loss of p15 and p16, the cyclin dependent kinase (CDK) inhibitors in adjacent epithelia. In addition, there was an increased p21 methylation in the promoter region of the p53 binding site. These data suggest that multiple cell cycle checkpoints were compromised in the epithelia of Tgfbr2 fspKO mice. Consistent with these observations, there was an increased cell proliferation in the epithelial compartment. The mechanisms mediating the cross talk between the epithelia and the stroma apparently involved inflammation that occurred only in the Tgfbr2 fspKO mice but not in Tgfbr2 flox/flox mice. This is characterized by an increased CD45+ leukocyte infiltration, increased cytokines expression (IL-10, IFN-γ TNF-α and GM-CSF) and elevated levels of iNOS, COX2 and NFκB. Interestingly, a significant DNA damage, featured by 8-Oxo-dG DNA adduct formation, and elevated expression of γ-H2AX, a histone protein in response to DNA double strand break, was observed in Tgfbr2 fspKO mice. Inhibition of inflammation with COX2 inhibitor (Celecoxib) or germ free housing condition, significantly delayed epithelial hyperplasia/dysplasia and reduced DNA damage in Tgfbr2 fspKO mice. Examination of human esophageal cancers showed a downregulation of TβR2 in the stromal fibroblast, and an increased inflammation and DNA damage similar to Tgfbr2 fspKO mice. Together, our data demonstrate that attenuation of stromal TGF- signaling induces inflammation that in turn causes DNA damage, epigenetic and genetic alterations in epithelia. Therefore, therapeutic targeting of inflammation and tumor microenvironment may be useful in treating cancers with downregulation of TGF- signaling in stroma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 50. doi:1538-7445.AM2012-50