Suman Maity

Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Oncogenic Properties in Triple-Negative Breast Cancer

Transmitochondrial cybrids and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple-negative breast cancer (TNBC). Analysis of cybrids and established breast cancer (BC) cell lines showed that metastatic TNBC maintains high levels of ATP through fatty acid β oxidation (FAO) and activates Src oncoprotein through autophosphorylation at Y419. Manipulation of FAO including the knocking down of carnitine palmitoyltransferase-1A (CPT1) and 2 (CPT2), the rate-limiting proteins of FAO, and analysis of patient-derived xenograft models confirmed the role of mitochondrial FAO in Src activation and metastasis. Analysis of TCGA and other independent BC clinical data further reaffirmed the role of mitochondrial FAO and CPT genes in Src regulation and their significance in BC metastasis.

Differential regulation of metabolic pathways by androgen receptor (AR) and its constitutively active splice variant, AR-V7, in prostate cancer cells

Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets.

Acceleration of the glycolytic flux by steroid receptor coactivator-2 is essential for endometrial decidualization.

Early embryo miscarriage is linked to inadequate endometrial decidualization, a cellular transformation process that enables deep blastocyst invasion into the maternal compartment. Although much of the cellular events that underpin endometrial stromal cell (ESC) decidualization are well recognized, the individual gene(s) and molecular pathways that drive the initiation and progression of this process remain elusive. Using a genetic mouse model and a primary human ESC culture model, we demonstrate that steroid receptor coactivator-2 (SRC-2) is indispensable for rapid steroid hormone-dependent proliferation of ESCs, a critical cell-division step which precedes ESC terminal differentiation into decidual cells. We reveal that SRC-2 is required for increasing the glycolytic flux in human ESCs, which enables rapid proliferation to occur during the early stages of the decidualization program. Specifically, SRC-2 increases the glycolytic flux through induction of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3), a major rate-limiting glycolytic enzyme. Similarly, acute treatment of mice with a small molecule inhibitor of PFKFB3 significantly suppressed the ability of these animals to exhibit an endometrial decidual response. Together, these data strongly support a conserved mechanism of action by which SRC-2 accelerates the glycolytic flux through PFKFB3 induction to provide the necessary bioenergy and biomass to meet the demands of a high proliferation rate observed in ESCs prior to their differentiation into decidual cells. Because deregulation of endometrial SRC-2 expression has been associated with common gynecological disorders of reproductive-age women, this signaling pathway, involving SRC-2 and PFKFB3, promises to offer new clinical approaches in the diagnosis and/or treatment of a non-receptive uterus in patients presenting idiopathic infertility, recurrent early pregnancy loss, or increased time to pregnancy.

Pathway-Centric Integrative Analysis Identifies RRM2 as a Prognostic Marker in Breast Cancer Associated with Poor Survival and Tamoxifen Resistance

Breast cancer (BCa) molecular subtypes include luminal A, luminal B, normal-like, HER-2–enriched, and basal-like tumors, among which luminal B and basal-like cancers are highly aggressive. Biochemical pathways associated with patient survival or treatment response in these more aggressive subtypes are not well understood. With the limited availability of pathologically verified clinical specimens, cell line models are routinely used for pathway-centric studies. We measured the metabolome of luminal and basal-like BCa cell lines using mass spectrometry, linked metabolites to biochemical pathways using Gene Set Analysis, and developed a novel rank-based method to select pathways on the basis of their enrichment in patient-derived omics data sets and prognostic relevance. Key mediators of the pathway were then characterized for their role in disease progression. Pyrimidine metabolism was altered in luminal versus basal BCa, whereas the combined expression of its associated genes or expression of one key gene, ribonucleotide reductase subunit M2 (RRM2) alone, associated significantly with decreased survival across all BCa subtypes, as well as in luminal patients resistant to tamoxifen. Increased RRM2 expression in tamoxifen-resistant patients was verified using tissue microarrays, whereas the metabolic products of RRM2 were higher in tamoxifen-resistant cells and in xenograft tumors. Both genetic and pharmacological inhibition of this key enzyme in tamoxifen-resistant cells significantly decreased proliferation, reduced expression of cell cycle genes, and sensitized the cells to tamoxifen treatment. Our study suggests for evaluating RRM2-associated metabolites as noninvasive markers for tamoxifen resistance and its pharmacological inhibition as a novel approach to overcome tamoxifen resistance in BCa.

CAPER Is Vital for Energy and Redox Homeostasis by Integrating Glucose-Induced Mitochondrial Functions via ERR-α-Gabpa and Stress-Induced Adaptive Responses via NF-κB-cMYC

Ever since we developed mitochondria to generate ATP, eukaryotes required intimate mito-nuclear communication. In addition, since reactive oxygen species are a cost of mitochondrial oxidative phosphorylation, this demands safeguards as protection from these harmful byproducts. Here we identified a critical transcriptional integrator which eukaryotes share to orchestrate both nutrient-induced mitochondrial energy metabolism and stress-induced nuclear responses, thereby maintaining carbon-nitrogen balance, and preserving life span and reproductive capacity. Inhibition of nutrient-induced expression of CAPER arrests nutrient-dependent cell proliferation and ATP generation and induces autophagy-mediated vacuolization. Nutrient signaling to CAPER induces mitochondrial transcription and glucose-dependent mitochondrial respiration via coactivation of nuclear receptor ERR-α-mediated Gabpa transcription. CAPER is also a coactivator for NF-κB that directly regulates c-Myc to coordinate nuclear transcriptome responses to mitochondrial stress. Finally, CAPER is responsible for anaplerotic carbon flux into TCA cycles from glycolysis, amino acids and fatty acids in order to maintain cellular energy metabolism to counter mitochondrial stress. Collectively, our studies reveal CAPER as an evolutionarily conserved ‘master’ regulatory mechanism by which eukaryotic cells control vital homeostasis for both ATP and antioxidants via CAPER-dependent coordinated control of nuclear and mitochondrial transcriptomic programs and their metabolisms. These CAPER dependent bioenergetic programs are highly conserved, as we demonstrated that they are essential to preserving life span and reproductive capacity in human cells—and even in C. elegans.

An anatomically resolved mouse brain proteome reveals Parkinson disease-relevant pathways

Here, we present a mouse brain protein atlas that covers 17 surgically distinct neuroanatomical regions of the adult mouse brain, each less than 1 mm3 in size. The protein expression levels are determined for 6,500 to 7,500 gene protein products from each region and over 12,000 gene protein products for the entire brain, documenting the physiological repertoire of mouse brain proteins in an anatomically resolved and comprehensive manner. We explored the utility of our spatially defined protein profiling methods in a mouse model of Parkinsons disease. We compared the proteome from a vulnerable region (substantia nigra pars compacta) of wild type and parkinsonian mice with that of an adjacent, less vulnerable, region (ventral tegmental area) and identified several proteins that exhibited both spatiotemporal- and genotype-restricted changes. We validated the most robustly altered proteins using an alternative profiling method and found that these modifications may highlight potential new pathways for future studies. This proteomic atlas is a valuable resource that offers a practical framework for investigating the molecular intricacies of normal brain function as well as regional vulnerability in neurological diseases. All of the mouse regional proteome profiling data are published on line at http://mbpa.bprc.ac.cn/.

Role of Cytochrome P450 (CYP)1A in Hyperoxic Lung Injury: Analysis of the Transcriptome and Proteome

Hyperoxia contributes to lung injury in experimental animals and diseases such as acute respiratory distress syndrome in humans. Cytochrome P450 (CYP)1A enzymes are protective against hyperoxic lung injury (HLI). The molecular pathways and differences in gene expression that modulate these protective effects remain largely unknown. Our objective was to characterize genotype specific differences in the transcriptome and proteome of acute hyperoxic lung injury using the omics platforms: microarray and Reverse Phase Proteomic Array. Wild type (WT), Cyp1a1−/− and Cyp1a2−/− (8–10 wk, C57BL/6J background) mice were exposed to hyperoxia (FiO2 > 0.95) for 48 hours. Comparison of transcriptome changes in hyperoxia-exposed animals (WT versus knock-out) identified 171 genes unique to Cyp1a1−/− and 119 unique to Cyp1a2−/− mice. Gene Set Enrichment Analysis revealed pathways including apoptosis, DNA repair and early estrogen response that were differentially regulated between WT, Cyp1a1−/− and Cyp1a2−/− mice. Candidate genes from these pathways were validated at the mRNA and protein level. Quantification of oxidative DNA adducts with 32P-postlabeling also revealed genotype specific differences. These findings provide novel insights into mechanisms behind the differences in susceptibility of Cyp1a1−/− and Cyp1a2−/− mice to HLI and suggest novel pathways that need to be investigated as possible therapeutic targets for acute lung injury.

Gene Expression Profiling Identifies Cell Proliferation and Inflammation as the Predominant Pathways Regulated by Aryl Hydrocarbon Receptor in Primary Human Fetal Lung Cells Exposed to Hyperoxia

Exposure to hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. We observed that aryl hydrocarbon receptor (AhR) signaling protects newborn mice and primary fetal human pulmonary microvascular endothelial cells (HPMECs) against hyperoxic injury. Additionally, a recent genome-wide transcriptome study in a newborn mouse model of BPD identified AhR as a key regulator of hyperoxia-induced gene dysregulation. Whether the AhR similarly deregulates genes in HPMEC is unknown. Therefore, the objective of this study was to characterize transcriptome level gene expression profile in AhR-sufficient and -deficient HPMEC exposed to normoxic and hyperoxic conditions. Global gene expression profiling was performed using Illumina microarray platform and selected genes were validated by real-time RT-PCR. AhR gene expression and hyperoxia independently affected the expression of 540 and 593 genes, respectively. Two-way ANOVA further identified 85 genes that were affected by an interaction between AhR expression and exposure to hyperoxia. Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology, and Reactome pathway analysis identified cell proliferation, immune function, cytokine signaling, and organ development as the major pathways affected in AhR-deficient cells. The biological processes that were significantly enriched by hyperoxia included metabolic process, stress response, signal transduction, cell cycle, and immune regulation. Cell cycle was the predominant pathway affected by the combined effect of AhR knockdown and hyperoxia. Functional analysis of cell cycle showed that AhR-deficient cells had decreased proliferation compared with AhR-sufficient cells. These findings suggest that AhR modulates hyperoxic lung injury by regulating the genes that are necessary for cell proliferation and inflammation.

Pharmacological inhibition of CaMKK2 with the selective antagonist STO-609 regresses NAFLD

Binding of calcium to its intracellular receptor calmodulin (CaM) activates a family of Ca2+/CaM-dependent protein kinases. CaMKK2 (Ca2+/CaM-dependent protein kinase kinase 2) is a central member of this kinase family as it controls the actions of a CaMK cascade involving CaMKI, CaMKIV or AMPK. CaMKK2 controls insulin signaling, metabolic homeostasis, inflammation and cancer cell growth highlighting its potential as a therapeutic target for a variety of diseases. STO-609 is a selective, small molecule inhibitor of CaMKK2. Although STO-609 has been used extensively in vitro and in cells to characterize and define new mechanistic functions of CaMKK2, only a few studies have reported the in vivo use of STO-609. We synthesized functional STO-609 and assessed its pharmacological properties through in vitro (kinase assay), ex vivo (human liver microsomes) and in vivo (mouse) model systems. We describe the metabolic processing of STO-609, its toxicity, pharmacokinetics and bioavailability in a variety of mouse tissues. Utilizing these data, we show STO-609 treatment to inhibit CaMKK2 function confers protection against non-alcoholic fatty liver disease. These data provide a valuable resource by establishing criteria for use of STO-609 to inhibit the in vivo functions of CaMKK2 and demonstrate its utility for treating metabolically-related hepatic disease.

Distinct Lipidomic Landscapes Associated with Clinical Stages of Urothelial Cancer of the Bladder.

BACKGROUND The first global lipidomic profiles associated with urothelial cancer of the bladder (UCB) and its clinical stages associated with progression were identified. OBJECTIVE To identify lipidomic signatures associated with survival and different clinical stages of UCB. DESIGN, SETTING, AND PARTICIPANTS Pathologically confirmed 165 bladder-derived tissues (126 UCB, 39 benign adjacent or normal bladder tissues). UCB tissues included Ta (n=16), T1 (n=30), T2 (n=43), T3 (n=27), and T4 (n=9); lymphovascular invasion (LVI) positive (n=52) and negative (n=69); and lymph node status N0 (n=28), N1 (n=11), N2 (n=9), N3 (n=3), and Nx (n=75). RESULTS AND LIMITATIONS UCB tissues have higher levels of phospholipids and fatty acids, and reduced levels of triglycerides compared with benign tissues. A total of 59 genes associated with altered lipids in UCB strongly correlate with patient survival in an UCB public dataset. Within UCB, there was a progressive decrease in the levels of phosphatidylserine (PS), phosphatidylethanolamines (PEs), and phosphocholines, whereas an increase in the levels of diacylglycerols (DGs) with tumor stage. Transcript and protein expression of phosphatidylserine synthase 1, which converts DGs to PSs, decreased progressively with tumor stage. Levels of DGs and lyso-PEs were significantly elevated in tumors with LVI and lymph node involvement, respectively. Lack of carcinoma in situ and treatment information is the limitation of our study. CONCLUSIONS To date, this is the first study describing the global lipidomic profiles associated with UCB and identifies lipids associated with tumor stages, LVI, and lymph node status. Our data suggest that triglycerides serve as the primary energy source in UCB, while phospholipid alterations could affect membrane structure and/or signaling associated with tumor progression. PATIENT SUMMARY Lipidomic alterations identified in this study set the stage for characterization of pathways associated with these altered lipids that, in turn, could inform the development of first-of-its-kind lipid-based noninvasive biomarkers and novel therapeutic targets for aggressive urothelial cancer of the bladder.