Depleted hexokinase1 and lack of AMPKα activation favor OXPHOS-dependent energetics in retinoblastoma tumors.

Lack of retinoblastoma (Rb) protein causes aggressive intraocular retinal tumors in children. Recently, Rb tumors have been shown to have a distinctly altered metabolic phenotype, such as reduced expression of glycolytic pathway proteins alongside altered pyruvate and fatty acid levels. In this study, we demonstrate that loss of hexokinase 1(HK1) in tumor cells rewires their metabolism allowing enhanced oxidative phosphorylation-dependent energy production. We show that rescuing HK1 or retinoblastoma protein 1 (RB1) in these Rb cells reduced cancer hallmarks such as proliferation, invasion, and spheroid formation and increased their sensitivity to chemotherapy drugs. Induction of HK1 was accompanied by a metabolic shift of the cells to glycolysis and a reduction in mitochondrial mass. Cytoplasmic HK1 bound Liver Kinase B1 and phosphorylated AMP-activated kinase-α (AMPKα Thr172), thereby reducing mitochondria-dependent energy production. We validated these findings in tumor samples from Rb patients compared to age-matched healthy retinae. HK1 or RB1 expression in Rb-/- cells led to a reduction in their respiratory capacity and glycolytic proton flux. HK1 overexpression reduced tumor burden in an intraocular tumor xenograft model. AMPKα activation by AICAR also enhanced the tumoricidal effects of the chemotherapeutic drug topotecan in vivo. Therefore, enhancing HK1 or AMPKα activity can reprogram cancer metabolism and sensitize Rb tumors to lower doses of existing treatments, a potential therapeutic modality for Rb.

Enhanced Epithelial-to-Mesenchymal Transition and Chemoresistance in Advanced Retinoblastoma Tumors Is Driven by miR-181a.

Advanced retinoblastoma (Rb) tumors display high metastatic spread to distant tissues, causing a potent threat to vision and life. Through transcriptomic profiling, we discovered key upregulated genes that belonged to the epithelial-mesenchymal transition (EMT) and chemotherapy resistance pathways in advanced Rb tumors. Through in vitro models, we further showed that Rb null tumor cells under prolonged chemo drug exposure, acquires a metastasis-like phenotype through the EMT program mediated by ZEB1 and SNAI2 and these cells further acquires chemotherapeutic resistance through cathepsin-L- and MDR1-mediated drug efflux mechanisms. Using a miRNA microarray, we identified miR-181a-5p as being significantly reduced in advanced Rb tumors, which was associated with an altered EMT and drug-resistance genes. We showed that enhancing miR-181a-5p levels in Rb null chemo-resistant sublines reduced the ZEB1 and SNAI2 levels and halted the mesenchymal transition switch, further reducing the drug resistance. We thus identified miR-181a-5p as a therapeutically exploitable target for EMT-triggered drug-resistant cancers that halted their invasion and migration and sensitized them to low-dose chemotherapy drugs.

Lack of Retinoblastoma Protein Shifts Tumor Metabolism from Glycolysis to OXPHOS and Allows the Use of Alternate Fuels.

Mutations in the RB1 locus leading to a loss of functional Rb protein cause intraocular tumors, which uniquely affect children worldwide. These tumors demonstrate rapid proliferation, which has recently been shown to be associated with an altered metabolic signature. We found that retinoblastoma tumors and in-vitro models lack Hexokinase 1 (HK1) and exhibit elevated fatty acid oxidation. We show that ectopic expression of RB1 induces HK1 protein in Rb null cells, and both RB1 and HK1 can mediate a metabolic switch from OXPHOS to glycolysis with increased pyruvate levels, reduced ATP production and reduced mitochondrial mass. Further, cells lacking Rb or HK1 can flexibly utilize glutamine and fatty acids to enhance oxidative phosphorylation-dependent ATP generation, as revealed by metabolic and biochemical assays. Thus, loss of Rb and HK1 in retinoblastoma reprograms tumor metabolic circuits to enhance the glucose-independent TCA (tricarboxylic acid) cycle and the intermediate NAD+/NADH ratios, with a subsequent increase in fatty-acid derived L-carnitine to enhance mitochondrial OXPHOS for ATP production instead of glycolysis dependence. We also demonstrate that modulation of the Rb-regulated transcription factor E2F2 does not result in any of these metabolic perturbations. In conclusion, we demonstrate RB1 or HK1 as critical regulators of the cellular bioenergetic profile and identify the altered tumor metabolism as a potential therapeutic target for cancers lacking functional Rb protein.

Integrated Analysis of Cancer Tissue and Vitreous Humor from Retinoblastoma Eyes Reveals Unique Tumor-Specific Metabolic and Cellular Pathways in Advanced and Non-Advanced Tumors.

Retinoblastoma (Rb) is a pediatric intraocular malignancy that is proposed to originate from maturing cone cell precursors in the developing retina. The molecular mechanisms underlying the biological and clinical behaviors are important to understand in order to improve the management of advanced-stage tumors. While the genetic causes of Rb are known, an integrated understanding of the gene expression and metabolic processes in tumors of human eyes is deficient. By integrating transcriptomic profiling from tumor tissues and metabolomics from tumorous eye vitreous humor samples (with healthy, age-matched pediatric retinae and vitreous samples as controls), we uncover unique functional associations between genes and metabolites. We found distinct gene expression patterns between clinically advanced and non-advanced Rb. Global metabolomic analysis of the vitreous humor of the same Rb eyes revealed distinctly altered metabolites, indicating how tumor metabolism has diverged from healthy pediatric retina. Several key enzymes that are related to cellular energy production, such as hexokinase 1, were found to be reduced in a manner corresponding to altered metabolites; notably, a reduction in pyruvate levels. Similarly, E2F2 was the most significantly elevated E2F family member in our cohort that is part of the cell cycle regulatory circuit. Ectopic expression of the wild-type RB1 gene in the Rb-null Y79 and WERI-Rb1 cells rescued hexokinase 1 expression, while E2F2 levels were repressed. In an additional set of Rb tumor samples and pediatric healthy controls, we further validated differences in the expression of HK1 and E2F2. Through an integrated omics analysis of the transcriptomics and metabolomics of Rb, we uncovered a significantly altered tumor-specific metabolic circuit that reduces its dependence on glycolytic pathways and is governed by Rb1 and HK1.

MNX1 Is Oncogenically Upregulated in African-American Prostate Cancer.

Incidence and mortality rates for prostate cancer are higher in African-American (AA) men than in European-American (EA) men, but the biologic basis for this disparity is unclear. We carried out a detailed analysis of gene expression changes in prostate cancer compared with their matched benign tissues in a cohort of AA men and compared them with existing data from EA men. In this manner, we identified MNX1 as a novel oncogene upregulated to a relatively greater degree in prostate cancer from AA men. Androgen and AKT signaling play a central role in the pathogenesis of prostate cancer and we found that both of these signaling pathways increased MNX1 expression. MNX1 in turn upregulated lipid synthesis by stimulating expression of SREBP1 and fatty acid synthetase. Our results define MNX1 as a novel targetable oncogene increased in AA prostate cancer that is associated with aggressive disease. Cancer Res; 76(21); 6290-8. ©2016 AACR.

Molecular mechanism of anticancer effect of Sclerotium rolfsii lectin in HT29 cells involves differential expression of genes associated with multiple signaling pathways: A microarray analysis.

Sclerotium rolfsii lectin (SRL) is a lectin isolated from fungus S. rolfsii and has high binding specificity toward the oncofetal Thomsen-Friedenreich carbohydrate antigen (Galß1-3GalNAc-α-O-Ser/Thr, T or TF), which is expressed in more than 90% of human cancers. Our previous studies have shown that binding of SRL to human colon, breast and ovarian cancer cells induces cell apoptosis in vitro and suppresses tumor growth in vivo. This study investigated the SRL-mediated cell signaling in human colon cancer HT29 cells by mRNA and miRNA microarrays. It was found that SRL treatment results in altered expression of several hundred molecules including mitogen-activated protein kinase (MAPK) and c-JUN-associated, apoptosis-associated and cell cycle and DNA replication-associated signaling molecules. Pathway analysis using GeneSpring 12.6.1 revealed that SRL treatment induces changes of MAPK and c-JUN-associated signaling pathways as early as 2 h while changes of cell cycle, DNA replication and apoptosis pathways were significantly affected only after 24 h. A significant change of cell miRNA expression was also observed after 12 h treatment of the cells with SRL. These changes were further validated by quantitative real time polymerase chain reaction and immunoblotting. This study thus suggests that the presence of SRL affects multiple signaling pathways in cancer cells with early effects on cell proliferation pathways associated with MAPK and c-JUN, followed by miRNA-associated cell activity and apoptosis. This provides insight information into the molecular mechanism of the anticancer activity of this fungal lectin.

Plc1p is required for proper chromatin structure and activity of the kinetochore in Saccharomyces cerevisiae by facilitating recruitment of the RSC complex.

High-fidelity chromosome segregation during mitosis requires kinetochores, protein complexes that assemble on centromeric DNA and mediate chromosome attachment to spindle microtubules. In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in alterations in chromatin structure of centromeres, reduced binding of microtubules to minichromosomes, and a higher frequency of chromosome loss. The mechanism of Plc1p's involvement in kinetochore activity was not initially obvious; however, a testable hypothesis emerged with the discovery of the role of inositol polyphosphates (InsPs), produced by a Plc1p-dependent pathway, in the regulation of chromatin-remodeling complexes. In addition, the remodels structure of chromatin (RSC) chromatin-remodeling complex was found to associate with kinetochores and to affect centromeric chromatin structure. We report here that Plc1p and InsPs are required for recruitment of the RSC complex to kinetochores, which is important for establishing proper chromatin structure of centromeres and centromere proximal regions. Mutations in PLC1 and components of the RSC complex exhibit strong genetic interactions and display synthetic growth defect, altered nuclear morphology, and higher frequency of minichromosome loss. The results thus provide a mechanistic explanation for the previously elusive role of Plc1p and InsPs in kinetochore function.

Saccharomyces cerevisiae phospholipase C regulates transcription of Msn2p-dependent stress-responsive genes.

Phosphatidylinositol phosphates are involved in signal transduction, cytoskeletal organization, and membrane trafficking. Inositol polyphosphates, produced from phosphatidylinositol phosphates by the phospholipase C-dependent pathway, regulate chromatin remodeling. We used genome-wide expression analysis to further investigate the roles of Plc1p (phosphoinositide-specific phospholipase C in Saccharomyces cerevisiae) and inositol polyphosphates in transcriptional regulation. Plc1p contributes to the regulation of approximately 2% of yeast genes in cells grown in rich medium. Most of these genes are induced by nutrient limitation and other environmental stresses and are derepressed in plc1 Delta cells. Surprisingly, genes regulated by Plc1p do not correlate with gene sets regulated by Swi/Snf or RSC chromatin remodeling complexes but show correlation with genes controlled by Msn2p. Our results suggest that the increased expression of stress-responsive genes in plc1 Delta cells is mediated by decreased cyclic AMP synthesis and protein kinase A (PKA)-mediated phosphorylation of Msn2p and increased binding of Msn2p to stress-responsive promoters. Accordingly, plc1 Delta cells display other phenotypes characteristic of cells with decreased PKA activity. Our results are consistent with a model in which Plc1p acts together with the membrane receptor Gpr1p and associated G(alpha) protein Gpa2p in a pathway separate from Ras1p/Ras2p and converging on PKA.

Plc1p is required for SAGA recruitment and derepression of Sko1p-regulated genes.

In Saccharomyces cerevisiae, many osmotically inducible genes are regulated by the Sko1p-Ssn6p-Tup1p complex. On osmotic shock, the MAP kinase Hog1p associates with this complex, phosphorylates Sko1p, and converts it into an activator that subsequently recruits Swi/Snf and SAGA complexes. We have found that phospholipase C (Plc1p encoded by PLC1) is required for derepression of Sko1p-Ssn6p-Tup1p-controlled osmoinducible genes upon osmotic shock. Although plc1Delta mutation affects the assembly of the preinitiation complex after osmotic shock, it does not affect the recruitment of Hog1p and Swi/Snf complex at these promoters. However, Plc1p facilitates osmotic shock-induced recruitment of the SAGA complex. Like plc1Delta cells, SAGA mutants are osmosensitive and display compromised expression of osmotically inducible genes. The reduced binding of SAGA to Sko1p-Ssn6p-Tup1p-repressed promoters in plc1Delta cells does not correlate with reduced histone acetylation. However, SAGA functions at these promoters to facilitate recruitment of the TATA-binding protein. The results thus provide evidence that Plc1p and inositol polyphosphates affect derepression of Sko1p-Ssn6p-Tup1p-controlled genes by a mechanism that involves recruitment of the SAGA complex and TATA-binding protein.

Stimulation of rat liver mitochondrial sn-glycerol-3-phosphate acyltransferase by polymyxin B via enhanced extraction of lysophosphatidic acid.

The purpose of this investigation was to determine how polymyxin B stimulates the activity of mitochondrial glycerophosphate acyltransferase. Polymyxin B did not change the integrity of the mitochondrial outer membrane as judged by testing the latency (>80%) of cytochrome oxidase activity. The stimulation totally disappeared when polymyxin B-treated mitochondria were washed. The FA side chain in polymyxin B was unnecessary for stimulation, as the nonapeptide was as effective as the whole antibiotic. The stimulation by polymyxin B or the nonapeptide was observed only in the presence of BSA. Cytochrome c, when added to the incubation medium instead of albumin, did not stimulate the mitochondrial enzyme, but did produce a stimulatory effect of polymyxin B on the mitochondrial acyltransferase. As reported earlier for the bacterial and microsomal acyltransferase, other polycationic compounds such as spermine and spermidine stimulated mitochondrial glycerophosphate acyltransferase. The stimulation of the mitochondrial acyltransferase by spermine and spermidine also occurred only in the presence of BSA. The analysis of the products of esterification demonstrated the presence of more lysophosphatidic acid (LPA) in the polymyxin B- and polyamine-stimulated assays in comparison to their respective control. Furthermore, in comparison to the albumin-treated control, there was 60% more LPA present in the assay supernatant fractions of polymyxin B-treated samples. Our results suggest that polymyxin B stimulates the mitochondrial glycerophosphate acyltransferase activity by enhancing the extraction of more LPA from the mitochondria to the supernatant fraction.