Protein Translation Machinery Research Articles

Global Gene Expression Analysis Reveals Genotoxic Effect of Nrf2 Pharmacological Inhibitor Brusatol in Myeloid Leukemia Cells- a Promising Novel Anticancer Agent

Combating drug resistance is a paramount task in treating acute myeloid leukemia (AML). NFE2 related factor 2 (NRF2) is a major player in inducing chemo resistance in both AML and solid tumors. We have previously reported that inhibition of NRF2 both by shRNA knockdown and pharmacological inhibition using brusatol sensitizes inherent resistant AML cell lines and patient samples to chemotherapy (Karathedath et al, Plos one, 2017). Recent studies have shown that brusatol acts as a potent protein translation inhibitor in A549 lung cancer cell line and this deleterious effect is due to the molar scale concentrations of the drug (Stokoe, et al., 2016, Harder et al, 2016).As the available reports was in solid tumor lines and at higher concentrations, we undertook this study to elucidate the mechanism of action of brusatol in our experimental setup utilizing Nano molar concentration of brusatol in AML cell lines. We compared the global gene expression changes induced by brusatol in AML cell lines resistant to Ara-C, Dnr and ATO namely THP-1 and U937.Total RNA was extracted from both cell lines with and without brusatol treatment (100nM for 6 hours) using Qiagen RNeasy mini kit, cDNA was synthesized and subjected to a One -color 8X60K Agilent micro-array analysis. Data normalization, and analysis was performed using Gene Spring GX (V 12.0) software using the 75th percentile shift. Using student t-Test, 3070 genes were identified to be differentially expressed between THP-1 control and brusatol treated while 6856 genes were identified to be differentially expressed between U937 control and brusatol treated samples. About 1242 differentially expressed genes were common between brusatol treated THP-1 and U937. Differentially expressed genes(DEGs) were clustered using hierarchical clustering based on Pearson coefficient correlation algorithm to identify significant gene expression patterns. Genes were classified based on functional category using biological analysis tool DAVID. The enriched genes in both THP-1 and U937 after brusatol treatment fell into the following; Molecular function: protein binding (1396 and 1701 genes), Biological function: regulation of transcription (350 and 502 genes), Cellular component: Mitochondrion (231 and 249 genes) and so on. KEGG analysis was performed to identify enriched pathways after brusatol treatment in DEGs. DEGs in THP1 and U937 were enriched in 33 and 57 pathways were respectively, combining the pathways revealed 20 common target pathways between THP1 and U937. Pathways involved in cellular stress response such as NFkB, TNF and MAPK pathways were selectively up-regulated in the common target pathways and genes involved in cell cycle pathway were down regulated. Select genes from these target pathways and few genes DOCK3, KLF10, NR4A3, GADD45B, KDM4B, GPR155, H2FAY, from the top 100 DEGs were validated by RQ-PCR (SyBr green) all the genes screened showed results consistent with the micro-array data.Interestingly DNA damage response genes GADD45B, KDM4B, H2AFY were in the top up-regulated genes indicating that brusatol might also cause DNA damage in leukemic cells. NR4A3 the orphan nuclear receptor is an established tumor suppressor in AML and is generally silenced in both AML cell lines and primary samples. One of the key finding from our data is that brusatol strikingly up-regulated NRA4A3 expression in both THP1 and U937. This study has shed light into other modes of action of brusatol apart from being a modulator of the protein translational machinery. Mechanism of chemo sensitization by brusatol could be tissue, time and concentration specific. Our data reveals brusatol at nano-molar concentration acts as a potent DNA damaging agent and reactivates NR4A3 expression in AML apart from being an inhibitor of NRF2. We have also reported at nano-molar concentration brusatol has minimal toxicity towards normal Peripheral blood mono-nuclear cells. Brusatol could be an ideal compound to combat drug resistance in AML. Further experimental validation is warranted to substantiate these finding of the mode of action of brusatol in chemo sensitizing AML cells. DisclosuresNo relevant conflicts of interest to declare.

Deciphering the Role of the Non-Coding Genome in Regulating Gene-Diet Interactions.

Protein encoding genes constitute a small fraction of mammalian genomes. In addition to the protein coding genes, there are other functional units within the genome that are transcribed, but not translated into protein, the so called non-coding RNAs. There are many types of non-coding RNAs that have been identified and shown to have important roles in regulating gene expression either at the transcriptional or post-transcriptional level. A number of recent studies have highlighted that dietary manipulation in mammals can influence the expression or function of a number of classes of non-coding RNAs that contribute to the protein translation machinery. The identification of protein translation as a common target for nutritional regulation underscores the need to investigate how this may mechanistically contribute to phenotypes and diseases that are modified by nutritional intervention. Finally, we describe the state of the art and the application of emerging ‘-omics’ technologies to address the regulation of protein translation in response to diet.

TRNA modifications and islet function.

Efficient and accurate protein translation is essential to producing insulin in pancreatic β-cells. Transfer RNA (tRNA) is known as the key component of the protein translational machinery. Interestingly, tRNA contains a wide variety of chemical modifications, which are posttranscriptionally catalysed by tRNA modifying enzymes. Recent advances in genome-sequencing technology have unveiled a number of genetic variations that are associated with the development of type 2 diabetes (T2D). Some of these mutations are located in the genes of tRNA modifying enzymes. Using cellular and animal models, it has been showed that dysregulation of tRNA modification impairs protein translation in pancreatic β-cells and leads to aberrant insulin production. In this review, we discuss the recent findings in the molecular functions of tRNA modifications and their involvement in the development of T2D.

A Robust and Quantitative Reporter System To Evaluate Noncanonical Amino Acid Incorporation in Yeast.

Engineering protein translation machinery to incorporate noncanonical amino acids (ncAAs) into proteins has advanced applications ranging from proteomics to single-molecule studies. As applications of ncAAs emerge, efficient ncAA incorporation is crucial to exploiting unique chemistries. We have established a quantitative reporter platform to evaluate ncAA incorporation in response to the TAG (amber) codon in yeast. This yeast display-based reporter utilizes an antibody fragment containing an amber codon at which a ncAA is incorporated when the appropriate orthogonal translation system (OTS) is present. Epitope tags at both termini allow for flow cytometry-based end point readouts of OTS efficiency and fidelity. Using this reporter, we evaluated several factors that influence amber suppression, including the amber codon position and different aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs. Interestingly, previously described aaRSs that evolved from different parent enzymes to incorporate O-methyl-l-tyrosine exhibit vastly different behavior. Escherichia coli leucyl-tRNA synthetase variants demonstrated efficient incorporation of a range of ncAAs, and we discovered unreported activities of several variants. Compared to a plate reader-based reporter, our assay yields more precise bulk-level measurements while also supporting single-cell readouts compatible with cell sorting. This platform is expected to allow quantitative elucidation of principles dictating efficient stop codon suppression and evolution of next-generation stop codon suppression systems to further enhance genetic code manipulation in eukaryotes. These efforts will improve our understanding of how the genetic code can be further evolved while expanding the range of chemical diversity available in proteins for applications ranging from fundamental epigenetics studies to engineering new classes of therapeutics.

Cytotoxic CD8+ T cells recognize and kill Plasmodium vivax-infected reticulocytes.

Plasmodium vivax causes approximately 100 million clinical malaria cases yearly1,2. The basis of protective immunity is poorly understood and thought to be mediated by antibodies3,4. Cytotoxic CD8+ T cells (CTLs) protect against other intracellular parasites by detecting parasite peptides presented by Human Leukocyte Antigen Class I (HLA-I) on host cells. CTLs kill parasite-infected mammalian cells and intracellular parasites by releasing their cytotoxic granules5,6. Perforin (PFN) delivers the antimicrobial peptide granulysin (GNLY) and death-inducing granzymes (Gzm) into the host cell, and GNLY then delivers Gzms into the parasite. CTLs were thought to have no role against Plasmodium spp. blood stages because red blood cells (RBCs) generally do not express HLA-I7. However, P. vivax infects reticulocytes (Retics) that retain the protein translation machinery. Here we show that P. vivax-infected Retics (iRetic) express HLA-I. Infected patient circulating CD8+ T cells highly express cytotoxic proteins and recognize and form immunological synapses with iRetics in an HLA-dependent manner, releasing their cytotoxic granules to kill both host cell and intracellular parasite, preventing reinvasion. iRetic and parasite killing is PFN-independent, but depends on GNLY, which generally efficiently forms pores only in microbial membranes8. We find that P. vivax depletes cholesterol from the iRetic cell membrane, rendering it GNLY-susceptible. This unexpected T cell defense might be mobilized to improve P. vivax vaccine efficacy.

Abstract 4455: Fatty acid synthase-induced S6Kinase facilitates USP11-eIF4B complex formation for sustained oncogenic translation in DLBCL

Abstract Fatty acid synthase (FASN) and protein translational machinery are two emergent therapeutic targets for cancer treatment, but their mutual dependency on each other for tumor propagation especially in diffuse B-cell large lymphoma (DLBCL) is still unknown. Therefore, we investigated the functional implication of enhanced FASN activity on protein translational machinery employing a panel of 7 DLCBL cell lines and 40 clinical samples. Inhibition of FASN activity by a specific inhibitor, C75 or shRNA mediated knockdown, suppressed de novo protein synthesis in SUDHL2, TMD8, Pfeiffer and HLY-1 (ABC-origin) cells, whereas SUDHL4, SUDHL6 and Toledo (GC-origin) cells showed minimal effect after C75 treatment or FASN knockdown. Assessing the core translational machinery, the eIF4B (eukaryotic initiation factor 4B) levels were depleted in a dose dependent manner of C75 treatment or shRNA mediated knock down of FASN in ABC-DLBCLs. Mechanistically, eIF4B undergoes ubiquitin mediated protein degradation upon FASN depletion. Depleting the expression of eIF4B significantly reduced the proliferative capacity of both FASN sensitive as well resistant DLBCL cells. Screening the interacting protein partners from available databases, we observed that USP11 interacts with and deubiquitinates eIF4B increasing its overall stability. Consistently, altering the expression of USP11 or its activity by either chemical inhibition or mutation significantly alters nascent peptide synthesis. Of functional significance, either down regulating USP11 or inhibition via small molecules significantly reduced the cell proliferation with increased apoptosis of both, FASN activity sensitive and resistant DLBCL cells. USP11 expression was also elevated in DLBCL tissues as evidenced by significantly higher staining. Significantly there is a strong correlation between USP11, FASN as well as eIF4B expression in DLBCL. Elucidating the basis for FASN not regulating GC DLBCL, we observed that sustained PI3K signalling in GC-DLBCL plays a key role in FASN dependent eIF4B stability. Detailed molecular analysis established that S6Kinase phosphorylates USP11 at Ser453, which regulates its interaction with, and stability of eIF4B thereby promoting proto-oncogene expression. These results together demonstrate that FASN activated S6Kinase induced eIF4B/USP11 axis is required for cell proliferation and survival by regulating the translation of proliferative and pro-survival mRNAs. Our findings support targeting the eIF4B/USP11 axis and provide a novel therapeutic option for patients with relapsed/refractory DLBCL. Citation Format: Bandish Kapadia, Nahid Nanaji, Kavita Bhalla, Binny Bhandary, Rena Lapidus, Afshin Beheshti, Andrew Evens, Ronald Gartenhaus. Fatty acid synthase-induced S6Kinase facilitates USP11-eIF4B complex formation for sustained oncogenic translation in DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4455.

Identification of Novel Physiological Substrates of Mycobacterium bovis BCG Protein Kinase G (PknG) by Label-free Quantitative Phosphoproteomics

Mycobacterial Ser/Thr kinases play a critical role in bacterial physiology and pathogenesis. Linking kinases to the substrates they phosphorylate in vivo, thereby elucidating their exact functions, is still a challenge. The aim of this work was to associate protein phosphorylation in mycobacteria with important subsequent macro cellular events by identifying the physiological substrates of PknG in Mycobacterium bovis BCG. The study compared the phosphoproteome dynamics during the batch growth of M. bovis BCG versus the respective PknG knock-out mutant (ΔPknG-BCG) strains. We employed TiO2 phosphopeptide enrichment techniques combined with label-free quantitative phosphoproteomics workflow on LC-MS/MS. The comprehensive analysis of label-free data identified 603 phosphopeptides on 307 phosphoproteins with high confidence. Fifty-five phosphopeptides were differentially phosphorylated, of these, 23 phosphopeptides were phosphorylated in M. bovis BCG wild-type only and not in the mutant. These were further validated through targeted mass spectrometry assays (PRMs). Kinase-peptide docking studies based on a published crystal structure of PknG in complex with GarA revealed that the majority of identified phosphosites presented docking scores close to that seen in previously described PknG substrates, GarA, and ribosomal protein L13. Six out of the 22 phosphoproteins had higher docking scores than GarA, consistent with the proteins identified here being true PknG substrates. Based on protein functional analysis of the PknG substrates identified, this study confirms that PknG plays an important regulatory role in mycobacterial metabolism, through phosphorylation of ATP binding proteins and enzymes in the TCA cycle. This work also reinforces PknG's regulation of protein translation and folding machinery.

Abstract 4137: Inhibition of protein synthesis with polyamine depletion in high-risk neuroblastoma

Abstract The MYC family of oncogenes coordinately deregulate cellular programs that link cell cycle progression with the requisite biomass and energy creation. Neuroblastoma is a MYC-driven, commonly lethal pediatric tumor, with a high proportion of tumors containing MYCN amplification. Polyamine synthesis is a pathway downstream of MYC, with polyamines functioning, in part, to support protein synthesis. ODC1 encodes ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, and we found that ODC1 is co-amplified with MYCN in a subset of high-risk neuroblastomas (~8%). Difluoromethylornithine (DFMO) is an irreversible inhibitor or ornithine decarboxylase and is an approved agent for the treatment of Trypanosomiasis. We have previously shown that DFMO inhibits tumor progression and synergizes with chemotherapy in murine mouse models of neuroblastoma, and we postulate that inhibition of protein synthesis is a predominant mechanism of anti-tumor activity. The effects of polyamine depletion on specific components of protein translational machinery have yet to be defined. Here, we show inhibition of global protein synthesis of up to 90% after treatment with various concentrations of DFMO in vitro, with a larger decrease in protein synthesis generally correlating with the presence of MYCN amplification. We found that colony formation is inhibited at DFMO exposures as low as 150 to 500 μM DFMO, which are achievable concentrations in adults based on previous clinical trials. We also show via isoelectric focusing that treatment of neuroblastoma cells in vitro with DFMO leads to a marked decrease in the proportion of hypusinated, or active, eIF5A (an elongation factor important in translation of polyproline stretches and possibly broader influence on translation initiation). To interrogate influences of polyamine depletion on the cap-dependent translation eIF4F complex, we evaluated for changes in the phosphorylation of 4E-BP1 in DFMO treatment conditions, though no changes in phosphorylation were seen compared to controls. Studies to further elucidate the protein synthetic components affected by polyamine depletion are ongoing, as are studies to assess for selective effects of DFMO on polyproline-containing proteins via eIF5A. Further elucidation of the mechanisms of DFMO activity will identify opportunities for drug synergy and provide a responder hypothesis to test in upcoming pivotal Phase 2 and 3 clinical trial of DFMO in the treatment of neuroblastoma. Citation Format: Andrea T. Flynn, Kangning Liu, Annette Vu, Michael D. Hogarty. Inhibition of protein synthesis with polyamine depletion in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4137.

Crosstalk between Hedgehog pathway and energy pathways in human adipose-derived stem cells: A deep sequencing analysis of polysome-associated RNA

Adult stem cells are considered promising candidates for cellular therapies due to their capacity to differentiate and self-renew. Differentiation leads to changes in the metabolism, structure, and gene expression patterns of cells. Hedgehog is one of the pathways that is involved in the enhancement of osteogenesis and chondrogenesis in adult stem cells, but its mechanisms are poorly understood. In this study, we treated adipose tissue-derived stem cells (ADSC) with two well-characterized drugs, purmorphamine (Hedgehog pathway activator) and cyclopamine (Hedgehog pathway inhibitor), and identified mRNAs associated with polysomes in each treatment group to determine the post transcriptional genetic networks governed by the Hedgehog pathway. Activation of the Hedgehog pathway by purmorphamine results in significant upregulation of mRNAs associated with cellular communication and signal transduction. Furthermore, our experiments show that cyclopamine acts late downregulating GLI1 expression in ADSCs but promotes the upregulation of mRNAs associated with energy pathways and metabolism at early times. Through in silico analysis, we identified some miRNAs, such as miR-355, that could regulate these mRNAs association with polysomes and thereby modulate the Hedgehog pathway. Our results suggest that activation of the Hedgehog pathway by purmorphamine also results in a negative regulation of mRNAs in the protein translation machinery.

TRIP-1 in the extracellular matrix promotes nucleation of calcium phosphate polymorphs

ABSTRACTIn search for bone and dentin extracellular matrix (ECM) proteins, transforming growth factor beta receptor II interacting protein 1 (TRIP-1) was identified as a novel protein synthesized by osteoblasts and odontoblasts and exported to the ECM. TRIP-1 is a WD-40 (WD is Tryptophan-Aspartic acid dipeptide) protein that has been well recognized for its physiological role in the endoplasmic reticulum (ER). In the ER, TRIP-1 functions as an essential subunit of eukaryotic elongation initiation factor 3 and is involved in the protein translational machinery. Recently, we reported that TRIP-1 is localized in the ECM of bone and dentin. In this study, we demonstrate that varying concentrations of TRIP-1 can participate in the nucleation of calcium phosphate polymorphs. Nucleation studies performed with high calcium and phosphate concentration demonstrated that recombinant TRIP-1 could orchestrate the formation of hydroxyapatite crystals. Nucleation experiments performed on demineralized and deproteinized dentin wafer under physiological conditions and subsequent transmission electron microscope analysis of the deposits at the end of 7 and 14 days showed that TRIP-1 promoted the deposition of calcium phosphate mineral aggregates in the gap-overlap region of type I collagen. Taken together, we provide mechanistic insight into the role of this intracellular protein in matrix mineralization.

Epitranscriptomic m6A Regulation of Axon Regeneration in the Adult Mammalian Nervous System

N6-methyladenosine (m6A) affects multiple aspects of mRNA metabolism and regulates developmental transitions by promoting mRNA decay. Little is known about the role of m6A in the adult mammalian nervous system. Here we report that sciatic nerve lesion elevates levels of m6A-tagged transcripts encoding many regeneration-associated genes and protein translation machinery components in the adult mouse dorsal root ganglion (DRG). Single-base resolution m6A-CLIP mapping further reveals a dynamic m6A landscape in the adult DRG upon injury. Loss of either m6A methyltransferase complex component Mettl14 or m6A-binding protein Ythdf1 globally attenuates injury-induced protein translation in adult DRGs and reduces functional axon regeneration in the peripheral nervous system invivo. Furthermore, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult central nervous system is attenuated upon Mettl14 knockdown. Our study reveals a critical epitranscriptomic mechanism in promoting injury-induced protein synthesis and axon regeneration in the adult mammalian nervous system.

Strategies of biochemical adaptation for hibernation in a South American marsupial, Dromiciops gliroides: 2. Control of the Akt pathway and protein translation machinery.

When faced with harsh environmental conditions, the South American marsupial, monito del monte (Dromiciops gliroides), reduces its body temperature and uses either daily torpor or multiday hibernation to survive. This study used ELISA and multiplex assays to characterize the responses to hibernation by three regulatory components of protein translation machinery [p-eIF2α(S51), p-eIF4E(S209), p-4EBP(Thr37/46)] and eight targets involved in upstream signaling control of translation [p-IGF-1R(Tyr1135/1136), PTEN(S380), p-Akt(S473), p-GSK-3α(S21), p-GSK-3β(S9), p-TSC2(S939), p-mTOR(S2448), and p70S6K(T412)]. Liver, brain and kidney were analyzed comparing control and hibernation (4days continuous torpor) conditions. In the liver, increased phosphorylation of IGF-1R, Akt, GSK-3β, TSC2, mTOR, eIF2α, and 4EBP (1.60-1.98 fold compared to control) occurred during torpor suggesting that the regulatory phosphorylation cascade and protein synthesis remained active during torpor. However, responses by brain and kidney differed; torpor resulted in increased phosphorylation of GSK-3β (2.15-4.17 fold) and TSC2 (2.03-3.65 fold), but phosphorylated Akt decreased (to 34-62% of control levels). Torpor also led to an increase in phosphorylated eIF2α (1.4 fold) content in the brain. These patterns of differential protein phosphorylation in brain and kidney were indicative of suppression of protein translation but also could suggest an increase in antioxidant and anti-apoptotic signaling during torpor. Previous studies of liver metabolism in hibernating eutherian mammals have shown that Akt kinase and its downstream signaling components play roles in facilitating hypometabolism by suppressing energy expensive anabolic processes during torpor. However, the results in this study reveal differences between eutherian and marsupial hibernators, suggesting alternative actions of liver Akt during torpor.

Interactome analysis reveals ZNF804A, a schizophrenia risk gene, as a novel component of protein translational machinery critical for embryonic neurodevelopment

Recent genome-wide association studies identified over 100 genetic loci that significantly associate with schizophrenia (SZ). A top candidate gene, ZNF804A, was robustly replicated in different populations. However, its neural functions are largely unknown. Here we show in mouse that ZFP804A, the homolog of ZNF804A, is required for normal progenitor proliferation and neuronal migration. Using a yeast two-hybrid genome-wide screen, we identified novel interacting proteins of ZNF804A. Rather than transcriptional factors, genes involved in mRNA translation are highly represented in our interactome result. ZNF804A co-fractionates with translational machinery and modulates the translational efficiency as well as the mTOR pathway. The ribosomal protein RPSA interacts with ZNF804A and rescues the migration and translational defects caused by ZNF804A knockdown. RNA immunoprecipitation–RNAseq (RIP-Seq) identified transcripts bound to ZFP804A. Consistently, ZFP804A associates with many short transcripts involved in translational and mitochondrial regulation. Moreover, among the transcripts associated with ZFP804A, a SZ risk gene, neurogranin (NRGN), is one of ZFP804A targets. Interestingly, downregulation of ZFP804A decreases NRGN expression and overexpression of NRGN can ameliorate ZFP804A-mediated migration defect. To verify the downstream targets of ZNF804A, a Duolink in situ interaction assay confirmed genes from our RIP-Seq data as the ZNF804A targets. Thus, our work uncovered a novel mechanistic link of a SZ risk gene to neurodevelopment and translational control. The interactome-driven approach here is an effective way for translating genome-wide association findings into novel biological insights of human diseases.

Degradation of protein translation machinery by amino acid starvation-induced macroautophagy

ABSTRACTMacroautophagy is regarded as a nonspecific bulk degradation process of cytoplasmic material within the lysosome. However, the process has mainly been studied by nonspecific bulk degradation assays using radiolabeling. In the present study we monitor protein turnover and degradation by global, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways in stress-induced macroautophagy.

Giant viruses with an expanded complement of translation system components.

The discovery of giant viruses blurred the sharp division between viruses and cellular life. Giant virus genomes encode proteins considered as signatures of cellular organisms, particularly translation system components, prompting hypotheses that these viruses derived from a fourth domain of cellular life. Here we report the discovery of a group of giant viruses (Klosneuviruses) in metagenomic data. Compared with other giant viruses, the Klosneuviruses encode an expanded translation machinery, including aminoacyl transfer RNA synthetases with specificities for all 20 amino acids. Notwithstanding the prevalence of translation system components, comprehensive phylogenomic analysis of these genes indicates that Klosneuviruses did not evolve from a cellular ancestor but rather are derived from a much smaller virus through extensive gain of host genes.

OS01.1 Identification of the pre-malignant cell of origin in Human Glioblastoma Multiforme

AbstractOne of the major difficulties in identifying the cell of origin for glioblastoma is the complex cellular composition of this disease. It is now hypothesized that the different subtypes of high-grade glioma may have different cell of origin and in mouse models it has been shown that the cell of origin resides within the progenitor populations. To identify the cell of origin in human GBM we decided to first dissect out the various stages of CNS stem cell differentiation in fetal and adult human brain. After a functional high throughput screen, we identified a panel of four antibodies (i.e CD15, Notch1, EGFR, and CD90) that in various combinations can separate long-term self-renewing multi-potent neural stem cells (NSCs: LinCD15Notch1EGFRCD90) from multi-potent progenitors with limited self-renewal capacity (NPC: LinCD15Notch1EGFRCD90) from non-neoplastic human fetal and adult Brain (sub-ventricular zone). FACS sorted cells were analyzed for single cell lineage multi-potentiality, lineage bias and self-renewal to trace and characterize their lineage relationships. In surgical human glioblastoma samples, however we observed in-vivo tumor initiating and in-vitro tumorsphere-forming frequency was predominant in the progenitor cells and not in the neural stem cells. Interestingly we did observe a small frequency of NSC cells forming distinct lesions in the brain, which on further analysis showed the presence of both the neural stem cells and Glioma stem cells, where as the tumor initiating enriched population (GSC: LinCD15Notch1EGFRCD90) did not show the presence of neural stem cells but only tumor initiating NPCs. limiting dilution transplantation and serial transplantation confirmed the LinCD15Notch1EGFRCD90 to have the highest frequency of self renewing cells compared to other populations. Taken together our data suggests the presence of “normal” or pre-malignant stem cells in glioblastoma, which suggests a possible mechanism of relapse. Using Transcriptome analysis and targeted sequencing of known mutations we identify the signal transduction and genomic events (PTEN point mutations vs Deletions) which distinguish pre-malignant neural stem cells from the tumor initiating glioma stem cells. We further show a significant enrichment of the protein translational machinery as cells transition from pre-malignant to tumor initiating cells. These pre-malignant cells comprise a cellular reservoir that needs to be targeted to prevent tumor relapse. SSM: Seibel Fellowship and Ludwig Cancer Center, ILW: National Institutes of Health, Ludwig Cancer Center, SHC: Stanford Child Health Research Institute, St Baldrick’s Foundation and Ludwig Cancer Center

Abstract A33: Understanding the functional role of enhanced FASN activity in regulating protein translation in DLCBL

Abstract Fatty acid synthase (FASN) and protein translational machinery are two emerging cancer therapeutic targets, but their mutual dependency on each other for tumor growth especially in diffuse B-cell large lymphoma (DLBCL) is currently unknown. Therefore, we investigated the functional consequences of enhanced FASN activity on protein translational machinery in both DLCBL cell lines as well as primary samples. Inhibition of FASN activity by a specific inhibitor, C75 or shRNA mediated knockdown, suppressed de novo protein synthesis in SUDHL2, Pfeiffer, and HLY-1 cells, whereas SUDHL4 and SUDHL6 cells displayed minimal impact. Assessing the core translational machinery, the eif4b (eukaryotic initiation factor 4b) levels were depleted in a dose dependent manner of C75 treatment or shRNA mediated knock down of FASN. Mechanistically, eif4b undergoes ubiquitin mediated protein degradation upon FASN depletion. Importantly there seems to be a direct correlation between FASN levels and eif4b expression in DLBCL samples. Depleting the expression of eif4b significantly reduced the cell proliferative capacity of both FASN sensitive as well as resistant DLBCL cells. Screening the interacting protein partners from the available databases, we observed that USP11 interacts and deubiquitinates eif4b which increases its overall stability. Furthermore altering the expression of USP11 or its activity by either chemical inhibition or mutations significantly alters nascent peptide synthesis. Significantly, either down regulating USP11 or inhibiting via small molecules significantly reduced the cell proliferation with increased apoptosis of either FASN activity sensitive or resistant DLBCL cells. USP11 expression was also elevated in DLBCL tissues. Importantly, there is a strong correlation between USP11 and FASN as well as eif4b expression in DLBCL with higher level of this proteins associated with poorer survival. Enhanced FASN activity increased the Akt kinase activity, which phosphorylates USP11 at an evolutionary conserved site. FASN stimulated Akt-meditated phosphorylation events is critical for USP11 recruitment on translational initiation machinery as well as interaction with eif4b to alter protein biosynthesis. These results demonstrate that FASN mediated eif4b/USP11 axis is required for cell proliferation and survival by regulating the translation of proliferative and prosurvival mRNAs. Targeting the eif4b/USP11 axis for therapeutic intervention may add additional treatment options for resistant/relapsed DLBCL patients. Citation Format: Bandish Kapadia, Ronald Gartenhaus. Understanding the functional role of enhanced FASN activity in regulating protein translation in DLCBL. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A33.

Quantitative proteomic analysis of HIV-1 Tat-induced dysregulation in SH-SY5Y neuroblastoma cells.

Despite affecting up to 70% of HIV-positive patients and being the leading cause of dementia in patients under 40 years, the molecular mechanisms involved in the onset of HIV-associated neurocognitive disorders (HAND) are not well understood. To address this, we performed SILAC-based quantitative proteomic analysis on HIV-Tat treated SH-SY5Y neuroblastoma cells. Isolated protein was fractionated by SDS-PAGE and analyzed by nLC-MS/MS on an Orbitrap Velos. Using MaxQuant, we identified and quantified 3077 unique protein groups, of which 407 were differentially regulated. After applying an additional standard deviation-based cutoff, 29 of these were identified as highly significantly and stably dysregulated. GO term analysis shows dysregulation in both protein translation machinery as well as cytoskeletal regulation that have both been implicated in other dementias. In addition, several key cytoskeletal regulatory proteins such as ARHGEF17, the Rho GTPase, SHROOM3, and CMRP1 are downregulated. Together, these data demonstrate that HIV-Tat can dysregulate neuronal cytoskeletal regulatory proteins that could lead to the major HAND clinical manifestation-synapse loss.

Epigenetic Regulation of the Protein Translation Machinery

Epigenetic Regulation of the Protein Translation Machinery

Abstract P1-01-08: The evolution of breast cancer circulating tumor cells mediating brain metastasis

Abstract Multiple studies concur that CTCs - the “seeds” of fatal metastasis - intravasate into the bloodstream throughout the early stages of cancer promoting generation of micro-metastatic reservoirs, some of which ultimately evolve to metastatic tumors. However, while it may take decades for CTCs to progress from cells in circulation to clinically detectable metastases, once detected, the tumor usually grows at an exponential rate. This difference in growth dynamics at two ends of the metastatic spectrum should also be reflected in the behavior of CTCs shed from these organ sites. Having a focus towards breast cancer brain-colonizing CTCs and their properties, we hypothesized that a gradual evolution of dormant CTCs into mitotically active CTCs with brain-metastatic potency is central to the progression of primary breast cancer to brain metastasis. We isolated CD44+/CD24- (stem-like) and Pan-Cytokeratin+ (epithelial) breast cancer CTCs from peripheral blood of two groups of breast cancer patients – five with brain metastases (BCBM) (MRI-detectable) and five without brain metastases (No BCBM). CTCs were isolated by multi-parametric flow cytometry and the expression of CTC-specific antigen was confirmed by immuno-cytochemistry and DEPArrayTM platform. Intra-cellular flow cytometry showed that No BCBM patients had 3-fold higher mitotically dormant (Ki67-) CTCs compared to BCBM patients. Whole genome expression arrays demonstrated low generalized transcriptional activity in CTCs suggestive of mitotic/metabolic dormancy. Comparison of CTC transcriptomes with ER+/PR+, HER2+ and triple negative breast cancers identified a unique CTC gene signature valid not only across the three molecular subtypes but also independent of the molecular subtype patients were initially diagnosed as. Pathway analyses predicted increased activation of pluripotency-related pathways along with decreased protein translational machinery and proliferative pathways in CTCs. Further, to dissect mechanisms of metastatic reactivation in the brain microenvironment, we compared the CTC transcriptome in BCBM vs No BCBM patients and identified a 126 gene signature potentiated in BCBM CTCs. Subsequent pathway analyses revealed upregulation of known CTC pathways like Notch along with novel hematopoietic and immune evasion networks. Cellular and functional annotations of cell migration and chemotaxis were significantly activated in BCBM along with pro-inflammatory (TNF, IL1β, NF-kB), immunomodulatory chemokines (CXCL8, CXCR4, CD86) and mitogenic growth factors (PDGF-BB). Lastly, lack of cell surface expression of Urokinase Plasminogen Activator Receptor (uPAR) and integrin beta-1 (int-β1) were used to identify CTCs in a state of metastatic dormancy. Whole genome sequencing showed significantly higher incidence of genomic mutations in proliferating CTCs compared to their dormant counterparts. We provide first time evidence of signaling pathways keenly implicated in breast cancer CTC biology based on comprehensive analyses of CTC transcriptomes. We discovered the existence of CTCs in a state of metabolic/mitotic dormancy and identified genomic instability as a switch for CTCs to revert to their proliferative phenotype. Citation Format: Boral D, Vishnoi M, Liu HN, Yin W, Hong DS, Scamardo AT, Marchetti D. The evolution of breast cancer circulating tumor cells mediating brain metastasis [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 P1-01-08.