Keshav Gopal

Lipotoxicity in obesity and diabetes-related cardiac dysfunction.

Patients with type 2 diabetes (T2D) are at increased risk for cardiovascular diseases including diabetic cardiomyopathy, which is ventricular dysfunction independent of underlying coronary artery disease and/or hypertension. With numerous advancements in our ability to detect ventricular dysfunction, as well as the molecular mechanisms contributing to ventricular dysfunction in diabetic patients, it is now appreciated that diabetic cardiomyopathy is becoming more prevalent in our population. In spite of these advancements, we do not have any specific therapies currently approved for treating this condition. As obesity increases the risk for both T2D and cardiovascular disease, it has been postulated that obesity-mediated alterations in myocardial lipid metabolism are critical to the pathophysiology of diabetic cardiomyopathy. Indeed, animal studies have provided strong evidence that alterations in either myocardial fatty acid uptake or fatty acid β-oxidation lead to the accumulation of various lipid intermediates including triacylglycerol, diacylglycerol, ceramide, long-chain acyl CoA, acylcarnitine, and many others that are tightly linked to the progression of ventricular dysfunction. We review herein why lipid intermediates accumulate in the heart during obesity and/or T2D, with a focus on which of these various lipid intermediates may be responsible for cardiac lipotoxicity, and whether findings in animal models are relevant to humans. An improved understanding of how these lipid intermediates accumulate in the heart and how they produce cardiac toxicity may lead to the discovery of novel targets to pursue for the treatment of human diabetic cardiomyopathy. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis.

β-Catenin, a Sox2 binding partner, regulates the DNA binding and transcriptional activity of Sox2 in breast cancer cells

Sox2, an embryonic stem cell marker, has been recently implicated in the pathogenesis of breast cancer (BC). Using liquid chromatography-mass spectrometry and co-immunoprecipitation, we identified β-catenin as a Sox2 binding partner in MCF7 cells. The interaction between Sox2 and β-catenin was substantially different between the two cell subsets separated based on their differential responsiveness to a Sox2 reporter. Specifically, while β-catenin binds to Sox2 in the nuclear fraction of cells showing reporter-responsiveness (i.e. RR cells), this interaction was not detectable in those that were reporter-unresponsive (i.e. RU cells). In RR but not in RU cells, siRNA knockdown of β-catenin significantly upregulated the Sox2 transcriptional activity, enhanced its DNA binding and increased the expression of its target genes. Correlating with these findings, while inhibition of β-catenin significantly downregulated the mammosphere formation efficiency in RU cells, this treatment paradoxically increased that of RR cells. To conclude, we identified that β-catenin is an important binding partner of Sox2 and a regulator of its transcriptional activity in a small subset of BC cells. The interaction between Sox2 and β-catenin provides a novel mechanism underlying the functional dichotomy of BC cells, which carries potential therapeutic implications.

The opposing function of STAT3 as an oncoprotein and tumor suppressor is dictated by the expression status of STAT3β in esophageal squamous cell carcinoma

Purpose: STAT3 is known to have both oncogenic and tumor suppressive effects, but the regulation of these opposing effects is elusive. We hypothesized that STAT3β, one of the two STAT3 isoforms, is the key determinant in this context. Experimental Design: The prognostic significance of STAT3β and phospho-STAT3αY705 (pSTAT3αY705) was evaluated in 286 cases of patients with esophageal squamous cell carcinoma (ESCC). STAT3β-induced changes in the chemosensitivity to cisplatin and 5-fluorouracil were assessed both in vitro and in vivo. STAT3β-induced changes in the frequency of cancer stem cells were evaluated using Hoechst and CD44 staining. How STAT3β regulates STAT3α was determined using immunoprecipitation, confocal microscopy, DNA-binding, and chromatin immunoprecipitation-PCR. Results: STAT3β expression is an independent protective prognostic marker in patients with ESCC, which strongly correlated with longer overall survival (P = 0.0009) and recurrence-free survival (P = 0.0001). STAT3β significantly decreased the cancer stem cell population, and sensitized ESCC cells to cisplatin and 5-fluorouracil in tumor xenografts. Mechanistically, STAT3β markedly attenuated the transcription activity of STAT3α via inducing STAT3α:STAT3β heterodimers. However, the heterodimer formation decreased the binding between STAT3α and PTPN9 (better known as PTP-MEG2), a protein tyrosine phosphatase, thereby promoting the phosphorylation of STAT3αY705 and enhancing its nuclear translocation and DNA binding. Correlating with this, high STAT3β expression converts the prognostic value of pSTAT3αY705 from unfavorable to favorable in patients with ESCC. Conclusions: STAT3β suppresses chemoresistance and cancer stemness by blocking the transcriptional activity of STAT3α. The paradoxical increase in pSTAT3αY705 induced by STAT3β carries important implications as to how the biologic and prognostic significance of STAT3 in cancers should be interpreted. Clin Cancer Res; 22(3); 691–703. ©2015 AACR.

FoxO1 regulates myocardial glucose oxidation rates via transcriptional control of pyruvate dehydrogenase kinase 4 expression

Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation and a critical regulator of metabolic flexibility during the fasting to feeding transition. PDH is regulated via both PDH kinases (PDHK) and PDH phosphatases, which phosphorylate/inactivate and dephosphorylate/activate PDH, respectively. Our goal was to determine whether the transcription factor forkhead box O1 (FoxO1) regulates PDH activity and glucose oxidation in the heart via increasing the expression of Pdk4, the gene encoding PDHK4. To address this question, we differentiated H9c2 myoblasts into cardiac myocytes and modulated FoxO1 activity, after which Pdk4/PDHK4 expression and PDH phosphorylation/activity were assessed. We assessed binding of FoxO1 to the Pdk4 promoter in cardiac myocytes in conjunction with measuring the role of FoxO1 on glucose oxidation in the isolated working heart. Both pharmacological (1 µM AS1842856) and genetic (siRNA mediated) inhibition of FoxO1 decreased Pdk4/PDHK4 expression and subsequent PDH phosphorylation in H9c2 cardiac myocytes, whereas 10 µM dexamethasone-induced Pdk4/PDHK4 expression was abolished via pretreatment with 1 µM AS1842856. Furthermore, transfection of H9c2 cardiac myocytes with a vector expressing FoxO1 increased luciferase activity driven by a Pdk4 promoter construct containing the FoxO1 DNA-binding element region, but not in a Pdk4 promoter construct lacking this region. Finally, AS1842856 treatment in fasted mice enhanced glucose oxidation rates during aerobic isolated working heart perfusions. Taken together, FoxO1 directly regulates Pdk4 transcription in the heart, thereby controlling PDH activity and subsequent glucose oxidation rates.NEW & NOTEWORTHY Although studies have shown an association between FoxO1 activity and pyruvate dehydrogenase kinase 4 expression, our study demonstrated that pyruvate dehydrogenase kinase 4 is a direct transcriptional target of FoxO1 (but not FoxO3/FoxO4) in the heart. Furthermore, we report here, for the first time, that FoxO1 inhibition increases glucose oxidation in the isolated working mouse heart.

Oxidative stress induces the acquisition of cancer stem-like phenotype in breast cancer detectable by using a Sox2 regulatory region-2 (SRR2) reporter

We have previously identified a novel intra-tumoral dichotomy in breast cancer based on the differential responsiveness to a Sox2 reporter (SRR2), with cells responsive to SRR2 (RR) being more stem-like than unresponsive cells (RU). Here, we report that RR cells derived from MCF7 and ZR751 displayed a higher tolerance to oxidative stress than their RU counterparts, supporting the concept that the RR phenotype correlates with cancer stemness. Sox2 is directly implicated in this differential H2O2 tolerance, since siRNA knockdown of Sox2 in RR cells leveled this difference. Interestingly, H2O2 converted a proportion of RU cells into RR cells, as evidenced by their expression of luciferase and GFP, markers of SRR2 activity. Compared to RU cells, converted RR cells showed a significant increase in mammosphere formation and tolerance to H2O2. Converted RR cells also adopted the biochemical features of RR cells, as evidenced by their substantial increase in Sox2-SRR2 binding and the expression of 3 signature genes of RR cells (CD133, GPR49 and MUC15). Lastly, the H2O2-induced RU/RR conversion was detectable in a SCID mouse xenograft model and primary tumor cells. To conclude, the H2O2-induced RU/RR conversion has provided a novel model to study the acquisition of cancer stemness and plasticity.

Profiling gene promoter occupancy of Sox2 in two phenotypically distinct breast cancer cell subsets using chromatin immunoprecipitation and genome-wide promoter microarrays

IntroductionAberrant expression of the embryonic stem cell marker Sox2 has been reported in breast cancer (BC). We previously identified two phenotypically distinct BC cell subsets separated based on their differential response to a Sox2 transcription activity reporter, namely the reporter-unresponsive (RU) and the more tumorigenic reporter-responsive (RR) cells. We hypothesized that Sox2, as a transcription factor, contributes to their phenotypic differences by mediating differential gene expression in these two cell subsets.MethodsWe used chromatin immunoprecipitation and a human genome-wide promoter microarray (ChIP-chip) to determine the promoter occupancies of Sox2 in the MCF7 RU and RR breast cancer cell populations. We validated our findings with conventional chromatin immunoprecipitation, quantitative reverse transcription polymerase chain reaction (qPCR), and western blotting using cell lines, and also performed qPCR using patient RU and RR samples.ResultsWe found a largely mutually exclusive profile of gene promoters bound by Sox2 between RU and RR cells derived from MCF7 (1830 and 456 genes, respectively, with only 62 overlapping genes). Sox2 was bound to stem cell- and cancer-associated genes in RR cells. Using quantitative RT-PCR, we confirmed that 15 such genes, including PROM1 (CD133), BMI1, GPR49 (LGR5), and MUC15, were expressed significantly higher in RR cells. Using siRNA knockdown or enforced expression of Sox2, we found that Sox2 directly contributes to the higher expression of these genes in RR cells. Mucin-15, a novel Sox2 downstream target in BC, contributes to the mammosphere formation of BC cells. Parallel findings were observed in the RU and RR cells derived from patient samples.ConclusionsIn conclusion, our data supports the model that the Sox2 induces differential gene expression in the two distinct cell subsets in BC, and contributes to their phenotypic differences.

DDX17 (P72), a Sox2 binding partner, promotes stem-like features conferred by Sox2 in a small cell population in estrogen receptor-positive breast cancer.

We have previously demonstrated the existence of two phenotypically distinct cell subsets in estrogen receptor (ER)-positive breast cancer (BC) based on their differential response to a Sox2 reporter (SRR2), with reporter responsive (RR) cells being more tumorigenic and stem-like than reporter unresponsive (RU) cells. To delineate the molecular mechanisms underlying this phenotypic dichotomy, we tested our hypothesis that Sox2, which is a key regulator of the RR phenotype, is under the control of its binding partners. In this study, we focused on DDX17, known to be a transcription co-activator and found to be a Sox2 binding partner by liquid chromatography-mass spectrometry. Using immunoprecipitation, we confirmed the binding between DDX17 and Sox2, although this interaction was largely restricted to RR cells. While DDX17 was found in both the cytoplasm and nuclei in RU cells, it is confined to the nuclei in RR cells. siRNA knockdown of DDX17 in RR cells substantially decreased the Sox2-SRR2 binding and significantly decreased the SRR2 reporter activity without affecting the protein level of Sox2. Using ChIP-PCR, DDX17 knockdown also significantly decreased the binding of Sox2 to genomic SRR2, as well as 3 of its specific gene targets including MUC15, CCND1 and CD133. Correlating with these findings, siRNA knockdown of DDX17 significantly reduced soft agar colony formation and mammosphere formation in RR cells but not RU cells. To conclude, DDX17 is a Sox2-binding protein in ER-positive BC. In RR but not RU cells, DDX17 enhances the tumorigenic and stem-like features of Sox2 by promoting its binding to its target genes.

Cardiac-Specific Deletion of Pyruvate Dehydrogenase Impairs Glucose Oxidation Rates and Induces Diastolic Dysfunction

Obesity and type 2 diabetes (T2D) increase the risk for cardiomyopathy, which is the presence of ventricular dysfunction in the absence of underlying coronary artery disease and/or hypertension. As myocardial energy metabolism is altered during obesity/T2D (increased fatty acid oxidation and decreased glucose oxidation), we hypothesized that restricting myocardial glucose oxidation in lean mice devoid of the perturbed metabolic milieu observed in obesity/T2D would produce a cardiomyopathy phenotype, characterized via diastolic dysfunction. We tested our hypothesis via producing mice with a cardiac-specific gene knockout for pyruvate dehydrogenase (PDH, gene name Pdha1), the rate-limiting enzyme for glucose oxidation. Cardiac-specific Pdha1 deficient (Pdha1Cardiac−/−) mice were generated via crossing a tamoxifen-inducible Cre expressing mouse under the control of the alpha-myosin heavy chain (αMHC-MerCreMer) promoter with a floxed Pdha1 mouse. Energy metabolism and cardiac function were assessed via isolated working heart perfusions and ultrasound echocardiography, respectively. Tamoxifen administration produced an ~85% reduction in PDH protein expression in Pdha1Cardiac−/− mice versus their control littermates, which resulted in a marked reduction in myocardial glucose oxidation and a corresponding increase in palmitate oxidation. This myocardial metabolism profile did not impair systolic function in Pdha1Cardiac−/− mice, which had comparable left ventricular ejection fractions and fractional shortenings as their αMHC-MerCreMer control littermates, but did produce diastolic dysfunction as seen via the reduced mitral E/A ratio. Therefore, it does appear that forced restriction of glucose oxidation in the hearts of Pdha1Cardiac−/− mice is sufficient to produce a cardiomyopathy-like phenotype, independent of the perturbed metabolic milieu observed in obesity and/or T2D.

FOXO1 cysteine-612 mediates stimulatory effects of the coregulators CBP and PGC1α on FOXO1 basal transcriptional activity

ABSTRACT Hepatic production and release of metabolites, nutrients and micronutrient transporters is tightly regulated at the level of gene expression. In this regard, transcription factor FOXO1 modulates the expression of genes such as G6PC and SELENOP, encoding the catalytic subunit of glucose 6‐phosphatase and the plasma selenium transporter selenoprotein P, respectively. Here, we analyzed the role of cysteine residues in FOXO1 in controlling its activity with respect to regulation of G6PC and SELENOP in HepG2 human hepatoma cells. None of the seven FOXO1 cysteines affected FOXO1 binding to DNA or its basal subcellular distribution. Whereas overexpression of wildtype FOXO1 caused a strong induction of both G6PC and SELENOP promoter activities and mRNA levels, the induction was lowered by approx. 50% if cysteine‐deficient FOXO1 was overexpressed instead. Only the most C‐terminal of the seven FOXO1 cysteines, Cys612, was required and sufficient to ensure full FOXO1 transactivation activity. Coexpression of FOXO1 coregulators, CBP or PGC1&agr;, had a strong synergistic effect in stimulating G6PC promoter activity and expression, fully relying on the presence of FOXO1 Cys612. Similarly, a synergistic effect of FOXO1 and CBP was observed for SELENOP. In contrast, stimulation of SELENOP by PGC1&agr; was independent of FOXO1‐Cys612, due to the close proximity of a hepatocyte nuclear factor‐4&agr; binding site to the FOXO1 binding site within the SELENOP promoter, as demonstrated using mutant SELENOP promoter constructs. In summary, full basal FOXO1 transactivation activity relies on Cys612, which mediates synergistic effects of coregulators, CBP or PGC1&agr;, on FOXO1 transcriptional activity. The extent of Cys612 contribution depends on the promoter context of FOXO1 target genes. Graphical abstract Figure. No caption available. HighlightsFOXO1 stimulates expression of genes for glucose 6‐phosphatase and selenoprotein P.One cysteine residue (C612) is required for full FOXO1 activity.C612 mediates synergistic effects of coregulators, CBP or PGC1&agr;, on FOXO1.The extent of C612 contribution depends on promoter context in FOXO1 target genes.A HNF4&agr; site renders FOXO1 effects on the SELENOP promoter insensitive to C612.