Subramaniam, Malayannan

Metabolic dysfunction underlies several chronic diseases, many of which are exacerbated by obesity. Dietary interventions can reverse metabolic declines and slow aging, although compliance issues remain paramount. 17α-estradiol treatment improves metabolic parameters and slows aging in male mice. The mechanisms by which 17α-estradiol elicits these benefits remain unresolved. Herein, we show that 17α-estradiol elicits similar genomic binding and transcriptional activation through estrogen receptor α (ERα) to that of 17β-estradiol. In addition, we show that the ablation of ERα completely attenuates the beneficial metabolic effects of 17α-E2 in male mice. Our findings suggest that 17α-E2 may act through the liver and hypothalamus to improve metabolic parameters in male mice. Lastly, we also determined that 17α-E2 improves metabolic parameters in male rats, thereby proving that the beneficial effects of 17α-E2 are not limited to mice. Collectively, these studies suggest ERα may be a drug target for mitigating chronic diseases in male mammals.

Abstract Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of BC patients, yet accounts for a disproportionately higher rate of BC morbidity and mortality, in part due to lack of targeted therapies. We have shown that estrogen receptor beta (ERβ) is expressed in approximately 20% of TN breast tumors and that ligand-mediated activation of ERβ with estradiol (E2) or ERβ-selective agonists decreases tumor cell proliferation, invasion and migration in vitro and in vivo. Therefore, we aimed to elucidate the mechanisms by which ERβ elicits its anti-cancer effects in TNBC. RNAseq analysis of ERβ-expressing MDA-MB-231 cells demonstrated that ERβ significantly downregulates NFκB signaling in the presence of E2. ChIPseq for ERβ in these cells revealed that ERβ primarily associated with estrogen response elements, but 12% of all ERβ binding sites were located at NFκB consensus motifs. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. RNAseq analysis of MDA-MB-231-ERβ cells treated with TNFα or E2+TNFα revealed substantial global inhibition of TNFα regulated genes in the presence of E2. ChIPseq for NFκB demonstrated that ERβ significantly alters NFκB’s cistrome whereby it can both diminish NFκB binding and redistribute NFκB throughout the genome. ChIPseq also demonstrated that ligand-mediated activation of ERβ significantly diminished an activating histone mark (H3K27Ac) at many of these NFκB target genes while enhancing a repressive mark (H3K27Me3). The addition of H3K27Me3 at these loci was shown to occur through the recruitment of the histone methyltransferase, EZH2. Drug-mediated blockade of EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Knockdown of NFκB or Mutation of ERβ’s DNA binding domain rendered ERβ incapable of associating with DNA, recruiting EZH2, methylating NFκB target gene loci, repressing NFκB target gene expression and inhibiting proliferation. Interestingly, ERβ was shown to elicit more potent anti-cancer effects in TNBC cells expression a constitutively active form of NFκB. These finding suggest that a primary mechanism by which ERβ functions as a tumor suppressor is through inhibition of NFκB pathway activity. Our studies have also revealed that ERβ functions as a molecular switch for EZH2 and repurposes it for tumor suppressive activities, as EZH2 has previously been reported to enrich NFκB signaling in TNBC. These findings could address the paradox that high EZH2 expression is associated with worse TNBC patient outcomes, while high H3K27Me3 expression is associated with improved patient outcomes. Currently, a Mayo Clinic Breast Cancer SPORE prospective phase II clinical trial is underway to investigate the efficacy of estradiol for the treatment of metastatic ERβ+TNBC and to further evaluate the cross-talk between ERβ, EZH2 and NFκB signaling.

AbstractDeletion of TGFβ inducible early gene‐1 (TIEG) in mice results in an osteopenic phenotype that exists only in female animals. Molecular analyses on female TIEG knockout (KO) mouse bones identified increased expression of sclerostin, an effect that was confirmed at the protein level in serum. Sclerostin antibody (Scl‐Ab) therapy has been shown to elicit bone beneficial effects in multiple animal model systems and human clinical trials. For these reasons, we hypothesized that Scl‐Ab therapy would reverse the low bone mass phenotype of female TIEG KO mice. In this study, wildtype (WT) and TIEG KO female mice were randomized to either vehicle control (Veh, n = 12/group) or Scl‐Ab therapy (10 mg/kg, 1×/wk, s.c.; n = 12/group) and treated for 6 weeks. Following treatment, bone imaging analyses revealed that Scl‐Ab therapy significantly increased cancellous and cortical bone in the femur of both WT and TIEG KO mice. Similar effects also occurred in the vertebra of both WT and TIEG KO animals. Additionally, histomorphometric analyses revealed that Scl‐Ab therapy resulted in increased osteoblast perimeter/bone perimeter in both WT and TIEG KO animals, with a concomitant increase in P1NP, a serum marker of bone formation. In contrast, osteoclast perimeter/bone perimeter and CTX‐1 serum levels were unaffected by Scl‐Ab therapy, irrespective of mouse genotype. Overall, our findings demonstrate that Scl‐Ab therapy elicits potent bone‐forming effects in both WT and TIEG KO mice and effectively increases bone mass in female TIEG KO mice.

AbstractAimTieg1 is involved in multiple signalling pathways, human diseases, and is highly expressed in muscle where its functions are poorly understood.MethodsWe have utilized Tieg1 knockout (KO) mice to identify novel and important roles for this transcription factor in regulating muscle ultrastructure, metabolism and mitochondrial functions in the soleus and extensor digitorum longus (EDL) muscles. RNA sequencing, immunoblotting, transmission electron microscopy, MRI, NMR, histochemical and mitochondrial function assays were performed.ResultsLoss of Tieg1 expression resulted in altered sarcomere organization and a significant decrease in mitochondrial number. Histochemical analyses demonstrated an absence of succinate dehydrogenase staining and a decrease in cytochrome c oxidase (COX) enzyme activity in KO soleus with similar, but diminished, effects in the EDL. Decreased complex I, COX and citrate synthase (CS) activities were detected in the soleus muscle of KO mice indicating altered mitochondrial function. Complex I activity was also diminished in KO EDL. Significant decreases in CS and respiratory chain complex activities were identified in KO soleus. 1H‐NMR spectra revealed no significant metabolic difference between wild‐type and KO muscles. However, 31P spectra revealed a significant decrease in phosphocreatine and ATPγ. Altered expression of 279 genes, many of which play roles in mitochondrial and muscle function, were identified in KO soleus muscle. Ultimately, all of these changes resulted in an exercise intolerance phenotype in Tieg1 KO mice.ConclusionOur findings have implicated novel roles for Tieg1 in muscle including regulation of gene expression, metabolic activity and organization of tissue ultrastructure. This muscle phenotype resembles diseases associated with exercise intolerance and myopathies of unknown consequence.

Significance Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and patients exhibit high rates of recurrence and mortality in part due to lack of treatment options beyond standard-of-care chemotherapy regimens. In the subset of TNBCs that express estrogen receptor beta (ERβ), ligand-mediated activation of ERβ elicits potent anticancer effects. We report here the elucidation of the ERβ cistrome and transcriptome in TNBC and identify a mechanism whereby ERβ induces cystatin gene expression resulting in inhibition of canonical TGFβ signaling and a blockade of metastatic phenotypes. These findings suggest that ERβ-targeted therapies represent a treatment option for the subset of women with ERβ-expressing TNBC.

TIEG knockout (KO) mice exhibit a female‐specific osteopenic phenotype and altered expression of TIEG in humans is associated with osteoporosis. Gene expression profiling studies identified sclerostin as one of the most highly up‐regulated transcripts in the long bones of TIEG KO mice relative to WT littermates suggesting that TIEG may regulate SOST expression. TIEG was shown to substantially suppress SOST promoter activity and the regulatory elements through which TIEG functions were identified using promoter deletion and chromatin immunoprecipitation assays. Knockdown of TIEG in IDG‐SW3 osteocyte cells using shRNA and CRISPR‐Cas9 technology resulted in increased SOST expression and delayed mineralization, mimicking the results obtained from TIEG KO mouse bones. Given that TIEG is an estrogen regulated gene, and as changes in the hormonal milieu affect SOST expression, we performed ovariectomy (OVX) and estrogen replacement therapy (ERT) studies in WT and TIEG KO mice followed by miRNA and mRNA sequencing of cortical and trabecular compartments of femurs. SOST expression levels were considerably higher in cortical bone compared to trabecular bone. In cortical bone, SOST expression was increased following OVX only in WT mice and was suppressed following ERT in both genotypes. In contrast, SOST expression in trabecular bone was decreased following OVX and significantly increased following ERT. Interestingly, a number of miRNAs that are predicted to target sclerostin exhibited inverse expression levels in response to OVX and ERT. These data implicate important roles for TIEG and estrogen‐regulated miRNAs in modulating SOST expression in bone.

Abstract TGFβ–SMAD signaling exerts a contextual effect that suppresses malignant growth early in epithelial tumorigenesis but promotes metastasis at later stages. Longstanding challenges in resolving this functional dichotomy may uncover new strategies to treat advanced carcinomas. The Krüppel-like transcription factor, KLF10, is a pivotal effector of TGFβ/SMAD signaling that mediates antiproliferative effects of TGFβ. In this study, we show how KLF10 opposes the prometastatic effects of TGFβ by limiting its ability to induce epithelial-to-mesenchymal transition (EMT). KLF10 depletion accentuated induction of EMT as assessed by multiple metrics. KLF10 occupied GC-rich sequences in the promoter region of the EMT-promoting transcription factor SLUG/SNAI2, repressing its transcription by recruiting HDAC1 and licensing the removal of activating histone acetylation marks. In clinical specimens of lung adenocarcinoma, low KLF10 expression associated with decreased patient survival, consistent with a pivotal role for KLF10 in distinguishing the antiproliferative versus prometastatic functions of TGFβ. Our results establish that KLF10 functions to suppress TGFβ-induced EMT, establishing a molecular basis for the dichotomy of TGFβ function during tumor progression. Cancer Res; 77(9); 2387–400. ©2017 AACR.

Abstract Background: The primary mechanism by which tamoxifen (Tam) and its metabolites exert their biologic effects is through estrogen receptor (ER) binding and inhibition of ER signaling. We and others demonstrated that endoxifen (Endx) has greater antitumor activity in vitro and in vivo compared to Tam and the first-in-human Endx phase I study demonstrated its antitumor activity in patients with prior progression on Tam (Goetz SABC 2015). PKCs are a family of serine/threonine-specific protein kinases that regulate signaling pathways involved in cell proliferation and tumorigenic transformation. Our prior protein docking studies suggested endoxifen may be a substrate for PKCs. Here we report the effects of Tam and Endx on PKCβ1 binding, kinase activity, as well as interactions between PKCβ1 and ERα. Methods: Surface Plasmon Resonance (SPR, Biacore T200, GE Healthcare) was used to evaluate binding of Tam, N-desmethyl Tam (NDMT), 4-HT, and Endx to PKCβ1 and PKCβ2. The effects of Tam and Endx on PKCβ1 kinase activity were determined. Proliferation and colony formation in MCF7 parental and PKCβ1 overexpressing cells were evaluated. siRNA silencing was used to knockdown PKCβ1 expression in the following cells: MCF7 aromatase expressing cells that were either sensitive (MCF7/AC1) or resistant to letrozole (MCF7/AC1 L-resistant); T47D; and MDA-MB-361. Coimmunoprecipitation assay and DUOlink in situ proximity ligation were used to investigate the interaction between PKCβ1 and ERα. Results: Endx more potently inhibited PKCβ1 kinase activity compared to Tam ( IC50 350 nM vs 47.8 μM ) with KDs for PKCβ1 binding as follows: Endx (100 nM ), Tam ( 2 μM ), 4-HT ( 2 μM ) and NDMT (> 7 μM ). None of the SERMs exhibited PKCβ2 binding. In the MCF7/AC1 and MCF7/AC1 L-resistant cells, PKCβ1 knockdown resulted in ERα degradation and potently inhibited cell proliferation. These results were confirmed in T47D and MDA-MB-361 cells. Notably, PKCβ1 knockdown in MCF7/AC1 cells resulted in significantly greater E2 induced proliferation comparing siRNA knockdown vs. control. To further explore these effects, we evaluated the effects of PKCβ1 overexpression in MCF7 cells and demonstrated that PKCβ1 overexpression reduced cell proliferation and colony formation compared to parental MCF-7 cells without affecting ERα protein stability. Coimmunoprecipitation assays in transient transfected MCF-7 cells with exogenous PKCβ1 as well as PKCβ1 expressing MDA-MB-231 cells transiently or stably transfected with ERα demonstrated PKCβ1 and ERα interaction, with confirmation by Duolink assay that this interaction occurs in the cytoplasm. Conclusions: Our findings demonstrated that endoxifen binds and inhibits PKCβ1 at relevant concentrations achieved in the endoxifen clinical trial studies. PKCβ1 interacts with cytoplasmic ERα and PKCβ1 knockdown inhibits cell proliferation and enhances ERα turnover. However, in PKCβ1 overexpressing cells, PKCβ1 may exhibit tumor suppressive effects. These data suggest a complex interaction between PKCβ1 and ERα and that endoxifen's effects on PKCβ1 may alter drug response of endocrine therapy. Further studies are ongoing to characterize the role of PKCβ1 and its role in ER biology and response to endoxifen. Citation Format: Guo C, Kuffel MJ, Kudgus RA, Huang Z, Bode AM, Cheng J, Suman VJ, Reid JM, Bruinsma ES, Subramaniam M, Ames MM, Hawse JR, Goetz MP. Identification and characterization of a novel endoxifen substrate, PKCβ1, and its interaction with the estrogen receptor [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-03.