Vanita Natu

An RNAi Screen Identifies TRRAP as a Regulator of Brain Tumor-Initiating Cell Differentiation

Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer associated with a very poor prognosis. Recently, the initiation and growth of GBM has been linked to brain tumor-initiating cells (BTICs), which are poorly differentiated and share features with neural stem cells (NSCs). Here we describe a kinome-wide RNA interference screen to identify factors that control the tumorigenicity of BTICs. We identified several genes whose silencing induces differentiation of BTICs derived from multiple GBM patients. In particular, knockdown of the adaptor protein TRRAP significantly increased differentiation of cultured BTICs, sensitized the cells to apoptotic stimuli, and negatively affected cell cycle progression. TRRAP knockdown also significantly suppressed tumor formation upon intracranial BTIC implantation into mice. Together, these findings support a critical role for TRRAP in maintaining a tumorigenic, stem cell-like state.

Interaction of Hormone-sensitive Lipase with Steroidogeneic Acute Regulatory Protein FACILITATION OF CHOLESTEROL TRANSFER IN ADRENAL

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis.

Insulin regulates lipoprotein lipase activity in rat adipose cells via Wortmannin- and rapamycin-sensitive pathways

Lipoprotein lipase (LPL) hydrolyzes the triacylglycerol component of circulating lipoprotein particles, mediating the uptake of fatty acids into adipose tissue and muscle. Insulin is the principal factor responsible for regulating LPL activity in adipose tissue, yet the mechanisms whereby insulin controls LPL expression are unknown. The current studies used wortmannin, a specific inhibitor of phosphatidylinositol (PI) 3-kinase, and rapamycin, a specific inhibitor of activation of phosphoprotein 70 ribosomal protein S6 kinase (p70s6k), to explore some of the components of the insulin signaling pathway controlling LPL activity in adipose cells. Preincubation of isolated rat adipose cells with wortmannin completely abrogated the stimulation of LPL activity by insulin, while preincubation with rapamycin caused approximately a 60% inhibition of insulin-stimulated LPL activity. Thus, the current studies show that the regulation of adipose tissue LPL by insulin is mediated via a wortmannin-sensitive pathway, most likely PI 3-kinase, and that a rapamycin-sensitive pathway, most likely p705s6k, constitutes an important downstream component in the insulin signaling pathway through which LPL is regulated.

Adrenal Neutral Cholesteryl Ester Hydrolase: Identification, Subcellular Distribution, and Sex Differences

Adrenals express a high level of neutral cholesteryl ester hydrolase (CEH) activity, and male rats have greater activity than females; however, the identity of the enzyme(s) responsible for this activity and the basis for the sex differences are unknown. Using mice in which hormone-sensitive lipase (HSL) was inactivated by homologous recombination (HSL −/−), neutral CEH activity was reduced more than 98% compared with controls. Female HSL −/− mice showed a reduction in stimulated corticosterone values. Mechanical separation of rat adrenals revealed less HSL in the outer than the inner cortex. Examination of subfractions of rat adrenals showed that immunoreactive HSL was prominently expressed in microsomes, with lesser amounts in the cytosol and little to no HSL in mitochondrial and nuclear fractions or the lipid droplet. Four- to 10-fold more neutral CEH activity was in the microsomal fraction than any other fraction. No sex differences in the expression or subcellular distribution of HSL protein were fou...

Taf1 regulates Pax3 protein by monoubiquitination in skeletal muscle progenitors.

Pax3 plays critical roles during developmental and postnatal myogenesis. We have previously shown that levels of Pax3 protein are regulated by monoubiquitination and proteasomal degradation during postnatal myogenesis, but none of the key regulators of the monoubiquitination process were known. Here we show that Pax3 monoubiquitination is mediated by the ubiquitin-activating/conjugating activity of Taf1, a component of the core transcriptional machinery that was recently reported to be downregulated during myogenic differentiation. We show that Taf1 binds directly to Pax3 and overexpression of Taf1 increases the level of monoubiquitinated Pax3 and its degradation by the proteasome. A decrease of Taf1 results in a decrease in Pax3 monoubiquitination, an increase in the levels of Pax3 protein, and a concomitant increase in Pax3-mediated inhibition of myogenic differentiation and myoblast migration. These results suggest that Taf1 regulates Pax3 protein levels through its ability to mediate monoubiquitination, revealing a critical interaction between two proteins that are involved in distinct aspects of myogenic differentiation. Finally, these results suggest that the components of the core transcriptional are integrally involved in the process of myogenic differentiation, acting as nodal regulators of the differentiation program.

Combinatorial extracellular matrix microenvironments promote survival and phenotype of human induced pluripotent stem cell-derived endothelial cells in hypoxia.

UNLABELLED Recent developments in cell therapy using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) hold great promise for treating ischemic cardiovascular tissues. However, poor post-transplantation viability largely limits the potential of stem cell therapy. Although the extracellular matrix (ECM) has become increasingly recognized as an important cell survival factor, conventional approaches primarily rely on single ECMs for in vivo co-delivery with cells, even though the endothelial basement membrane is comprised of a milieu of different ECMs. To address this limitation, we developed a combinatorial ECM microarray platform to simultaneously interrogate hundreds of micro-scale multi-component chemical compositions of ECMs on iPSC-EC response. After seeding iPSC-ECs onto ECM microarrays, we performed high-throughput analysis of the effects of combinatorial ECMs on iPSC-EC survival, endothelial phenotype, and nitric oxide production under conditions of hypoxia (1% O2) and reduced nutrients (1% fetal bovine serum), as is present in ischemic injury sites. Using automated image acquisition and analysis, we identified combinatorial ECMs such as collagen IV+gelatin+heparan sulfate+laminin and collagen IV+fibronectin+gelatin+heparan sulfate+laminin that significantly improved cell survival, nitric oxide production, and CD31 phenotypic expression, in comparison to single-component ECMs. These results were further validated in conventional cell culture platforms and within three-dimensional scaffolds. Furthermore, this approach revealed complex ECM interactions and non-intuitive cell behavior that otherwise could not be easily determined using conventional cell culture platforms. Together these data suggested that iPSC-EC delivery within optimal combinatorial ECMs may improve their survival and function under the condition of hypoxia with reduced nutrients. STATEMENT OF SIGNIFICANCE Human endothelial cells (ECs) derived from induced pluripotent stem cells (iPSC-ECs) are promising for treating diseases associated with reduced nutrient and oxygen supply like heart failure. However, diminished iPSC-EC survival after implantation into diseased environments limits their therapeutic potential. Since native ECs interact with numerous extracellular matrix (ECM) proteins for functional maintenance, we hypothesized that combinatorial ECMs may improve cell survival and function under conditions of reduced oxygen and nutrients. We developed a high-throughput system for simultaneous screening of iPSC-ECs cultured on multi-component ECM combinations under the condition of hypoxia and reduced serum. Using automated image acquisition and analytical algorithms, we identified combinatorial ECMs that significantly improved cell survival and function, in comparison to single ECMs. Furthermore, this approach revealed complex ECM interactions and non-intuitive cell behavior that otherwise could not be easily determined.

Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection

Genomic characterization of circulating tumor cells (CTCs) may prove useful as a surrogate for conventional tissue biopsies. This is particularly important as studies have shown different mutational profiles between CTCs and ctDNA in some tumor subtypes. However, isolating rare CTCs from whole blood has significant hurdles. Very limited DNA quantities often can’t meet NGS requirements without whole genome amplification (WGA). Moreover, white blood cells (WBC) germline contamination may confound CTC somatic mutation analyses. Thus, a good CTC enrichment platform with an efficient WGA and NGS workflow are needed. Here, Vortex label-free CTC enrichment platform was used to capture CTCs. DNA extraction was optimized, WGA evaluated and targeted NGS tested. We used metastatic colorectal cancer (CRC) as the clinical target, HCT116 as the corresponding cell line, GenomePlex® and REPLI-g as the WGA methods, GeneRead DNAseq Human CRC Panel as the 38 gene panel. The workflow was further validated on metastatic CRC patient samples, assaying both tumor and CTCs. WBCs from the same patients were included to eliminate germline contaminations. The described workflow performed well on samples with sufficient DNA, but showed bias for rare cells with limited DNA input. REPLI-g provided an unbiased amplification on fresh rare cells, enabling an accurate variant calling using the targeted NGS. Somatic variants were detected in patient CTCs and not found in age matched healthy donors. This demonstrates the feasibility of a simple workflow for clinically relevant monitoring of tumor genetics in real time and over the course of a patient’s therapy using CTCs.Liquid biopsy: Simple workflow allows DNA analysis of circulating tumor cellsA microfluidic device that isolates cancer cells circulating in a blood sample allows for real-time genetic monitoring. A team led by Elodie Sollier-Christen of Vortex Biosciences, a cancer diagnostics company in Menlo Park, California, USA, in collaboration with Professor Stefanie Jeffrey at Stanford University School of Medicine, developed a simple workflow for analyzing the genomes of rare circulating tumor cells (CTCs) found in the bloodstream after they’ve been collected through a proprietary microfluidic system. They optimized rare cell DNA extraction, compared different whole genome amplification methods, and then tested the workflow on blood samples from patients with metastatic colorectal cancer. The analysis also included white blood cells from the same patients to parse cancer-causing mutations from inherited ones. The method could aid in the translation of liquid biopsies for the clinical care of cancer patients.

Deltex2 represses MyoD expression and inhibits myogenic differentiation by acting as a negative regulator of Jmjd1c

Significance The data presented address a fundamental mechanism controlling the expression of a master regulator of cellular differentiation, MyoD, by a member of the Deltex family of proteins. We show that MyoD expression is regulated by modulation of histone methylation in its promoter region by the histone demethylase, Jjmd1c. These data provide insight into the epigenetic control of gene expression in the regulation of myogenic differentiation. The myogenic regulatory factor MyoD has been implicated as a key regulator of myogenesis, and yet there is little information regarding its upstream regulators. We found that Deltex2 inhibits myogenic differentiation in vitro, and that skeletal muscle stem cells from Deltex2 knockout mice exhibit precocious myogenic differentiation and accelerated regeneration in response to injury. Intriguingly, Deltex2 inhibits myogenesis by suppressing MyoD transcription, and the Deltex2 knockout phenotype can be rescued by a loss-of-function allele for MyoD. In addition, we obtained evidence that Deltex2 regulates MyoD expression by promoting the enrichment of histone 3 modified by dimethylation at lysine 9 at a key regulatory region of the MyoD locus. The enrichment is attributed to a Deltex2 interacting protein, Jmjd1c, whose activity is directly inhibited by Deltex2 and whose expression is required for MyoD expression in vivo and in vitro. Finally, we find that Deltex2 causes Jmjd1c monoubiquitination and inhibits its demethylase activity. Mutation of the monoubiquitination site in Jmjd1c abolishes the inhibitory effect of Deltex2 on Jmjd1c demethylase activity. These results reveal a mechanism by which a member of the Deltex family of proteins can inhibit cellular differentiation, and demonstrate a role of Deltex in the epigenetic regulation of myogenesis.

Combinatorial Extracellular Matrix Microenvironments for Probing Endothelial Differentiation of Human Pluripotent Stem Cells

Endothelial cells derived from human pluripotent stem cells are a promising cell type for enhancing angiogenesis in ischemic cardiovascular tissues. However, our understanding of microenvironmental factors that modulate the process of endothelial differentiation is limited. We examined the role of combinatorial extracellular matrix (ECM) proteins on endothelial differentiation systematically using an arrayed microscale platform. Human pluripotent stem cells were differentiated on the arrayed ECM microenvironments for 5 days. Combinatorial ECMs composed of collagen IV + heparan sulfate + laminin (CHL) or collagen IV + gelatin + heparan sulfate (CGH) demonstrated significantly higher expression of CD31, compared to single-factor ECMs. These results were corroborated by fluorescence activated cell sorting showing a 48% yield of CD31+/VE-cadherin+ cells on CHL, compared to 27% on matrigel. To elucidate the signaling mechanism, a gene expression time course revealed that VE-cadherin and FLK1 were upregulated in a dynamically similar manner as integrin subunit β3 (>50 fold). To demonstrate the functional importance of integrin β3 in promoting endothelial differentiation, the addition of neutralization antibody inhibited endothelial differentiation on CHL-modified dishes by >50%. These data suggest that optimal combinatorial ECMs enhance endothelial differentiation, compared to many single-factor ECMs, in part through an integrin β3-mediated pathway.

Abstract 1724: Genomic profiling of Vortex-enriched CTCs using whole genome amplification and multiplex PCR-based targeted next generation sequencing

Background: Genomic characterization of circulating tumor cells (CTCs) provides insights into cancer genetic changes, and might be utilized for cancer prognosis, diagnosis, as well as monitoring of therapeutic efficacy. Targeted Panel Next Generation Sequencing (NGS) enables analyzing CTC genetic variants of a focused gene panel at a relatively lower cost 1 . However, CTCs are rare, often resulting in very limited DNA quantities available that require whole genome amplification (WGA). In previous studies, we introduced the Vortex technology, a platform enabling label-free enrichment of CTCs from blood samples of colorectal cancer (CRC) patients and their use for genomic assays downstream 2 . In this study, we developed a simple and efficient NGS workflow for CTC samples collected by this technology. Method: An optimized workflow using the Qiagen GeneRead DNAseq targeted panel and Illumina MiSeq NGS was first verified on HCT116 CRC cell line before being applied on patient CTCs. For patient blood samples, CTCs were collected with the Vortex technology, immunostained (CK, Vimentin, CD45) and enumerated. Matched white blood cell (WBC) DNA was included to subtract germline background. Fresh frozen liver metastasis tissue was collected and analyzed using the same NGS workflow. DNA from CTCs was extracted and amplified using Qiagen REPLI-g single cell WGA kit. Mutation detection on the WGA amplified DNA was performed using the GeneRead DNAseq CRC targeted panel of 38 genes and MiSeq sequencing. The sequencing data were analyzed by QIAGEN NGS Data Analysis Web Portal and Ingenuity Variant Analysis software. Results: The Vortex technology was validated for the capture of CTCs from CRC patients. REPLI-g performed a uniform, unbiased amplification on fresh rare cells with a coverage of 97.7%, which enabled further targeted panel NGS. Blood from 3 CRC patients (P1, P2, P3) and 2 healthy donors (HD1, HD2) was processed with Vortex platform. Less than 1 CTCs/mL blood were found in HD1 and HD2. P1 and P2 had 66 and 20 CTCs/ mL of blood respectively, with many vimentin positive CTC clusters. P3 had 2 CTCs/mL of blood. No somatic mutation was found in healthy donors. Somatic variants were only detected in the CTCs from patient samples that were not present in matched germline WBCs. For P1, more mutations were found in the CTCs than in the liver metastasis while it was the opposite for P2 and P3. Conclusion: For each patient, variants in CTCs and germline WBCs were analyzed from one blood sample using an optimized targeted NGS workflow and compared to liver mets. Our optimized workflow, using the Qiagen REPLIg and GeneRead DNAseq Targeted Panel NGS enabled the detection of CTC mutations for 38 CRC-focused genes. The inclusion of a germline WBC control in the workflow allowed the detection of mutations from pooled CTC samples collected using the Vortex technology. Altmuller J, et al. (2014). Biol Chem. Kidess-Sigal E, et al. (2016). Oncotarget. Citation Format: Haiyan E. Liu, Melanie Triboulet, Amin Zia, Meghah Vuppalapaty, Evelyn Kidess-Sigal, John Coller, Vanita S. Natu, Vida Shokoohi, James Che, Corinne Renier, Natalie Chan, Violet Hanft, Elodie Sollier-Christen, Stefanie S. Jeffrey. Genomic profiling of Vortex-enriched CTCs using whole genome amplification and multiplex PCR-based targeted next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1724. doi:10.1158/1538-7445.AM2017-1724