Publications by authors named "Ramanuj DasGupta"

53 Publications

Single-Cell RNA-seq Reveals Angiotensin-Converting Enzyme 2 and Transmembrane Serine Protease 2 Expression in TROP2 Liver Progenitor Cells: Implications in Coronavirus Disease 2019-Associated Liver Dysfunction.

Front Med (Lausanne) 2021 22;8:603374. Epub 2021 Apr 22.

Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore.

The recent coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2. COVID-19 was first reported in China (December 2019) and is now prevalent across the globe. Entry of severe acute respiratory syndrome coronavirus 2 into mammalian cells requires the binding of viral Spike (S) proteins to the angiotensin-converting enzyme 2 receptor. Once entered, the S protein is primed by a specialized serine protease, transmembrane serine protease 2 in the host cell. Importantly, besides the respiratory symptoms that are consistent with other common respiratory virus infections when patients become viremic, a significant number of COVID-19 patients also develop liver comorbidities. We explored whether a specific target cell-type in the mammalian liver could be implicated in disease pathophysiology other than the general deleterious response to cytokine storms. Here, we used single-cell RNA-seq to survey the human liver and identified potentially implicated liver cell-type for viral ingress. We analyzed ~300,000 single cells across five different (i.e., human fetal, healthy, cirrhotic, tumor, and adjacent normal) liver tissue types. This study reports on the co-expression of angiotensin-converting enzyme 2 and transmembrane serine protease 2 in a TROP2 liver progenitor population. Importantly, we detected enrichment of this cell population in the cirrhotic liver when compared with tumor tissue. These results indicated that in COVID-19-associated liver dysfunction and cell death, a viral infection of TROP2 progenitors in the liver might significantly impair liver regeneration in patients with liver cirrhosis.
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http://dx.doi.org/10.3389/fmed.2021.603374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100026PMC
April 2021

DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death.

Nat Commun 2021 04 16;12(1):2284. Epub 2021 Apr 16.

Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Drug resistance is a major obstacle to the treatment of most human tumors. In this study, we find that dual-specificity phosphatase 16 (DUSP16) regulates resistance to chemotherapy in nasopharyngeal carcinoma, colorectal cancer, gastric and breast cancer. Cancer cells expressing higher DUSP16 are intrinsically more resistant to chemotherapy-induced cell death than cells with lower DUSP16 expression. Overexpression of DUSP16 in cancer cells leads to increased resistance to cell death upon chemotherapy treatment. In contrast, knockdown of DUSP16 in cancer cells increases their sensitivity to treatment. Mechanistically, DUSP16 inhibits JNK and p38 activation, thereby reducing BAX accumulation in mitochondria to reduce apoptosis. Analysis of patient survival in head & neck cancer and breast cancer patient cohorts supports DUSP16 as a marker for sensitivity to chemotherapy and therapeutic outcome. This study therefore identifies DUSP16 as a prognostic marker for the efficacy of chemotherapy, and as a therapeutic target for overcoming chemoresistance in cancer.
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http://dx.doi.org/10.1038/s41467-021-22638-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052345PMC
April 2021

miR-582-5p Is a Tumor Suppressor microRNA Targeting the Hippo-YAP/TAZ Signaling Pathway in Non-Small Cell Lung Cancer.

Cancers (Basel) 2021 Feb 11;13(4). Epub 2021 Feb 11.

Laboratory of Precision Oncology and Cancer Evolution, Genome Institute of Singapore, A*STAR, Singapore 138672, Singapore.

The Hippo-YAP/TAZ signaling pathway is an evolutionarily conserved signaling pathway involved in a broad spectrum of biological processes, including tumorigenesis. Whilst aberrant Hippo-YAP/TAZ signaling is frequently reported in various cancers, the genetic alterations of this pathway are relatively rare, suggesting regulation at the post-transcriptional level. MicroRNAs play key role in tumorigenesis by regulating gene expression post-transcriptionally. Amongst the cancer-relevant microRNAs, miR-582-5p suppresses cell growth and tumorigenesis by inhibiting the expression of oncogenes, including AKT3, MAP3K2 and NOTCH1. Given the oncogenic role of YAP/TAZ in solid tumors, we scrutinized the possible interplay between miR-582-5p and Hippo-YAP/TAZ signaling. Correlation analysis in NSCLC cells revealed a positive relationship between the expression of mature miR-582-5p and the proportion of phosphorylated YAP/TAZ. Intriguingly, YAP/TAZ knockdown reduced the expression of mature miR-582-5p but increased that of primary miR-582. Overexpression of miR-582-5p resulted in increased phosphorylation of YAP/TAZ with a concomitant reduction in cell proliferation and enhanced apoptosis. Mechanistically, we find that miR-582-5p targets actin regulators NCKAP1 and PIP5K1C, which may be responsible for the observed alteration in F-actin, known to modulate YAP/TAZ. We postulate that regulation of the actin cytoskeleton by miR-582-5p may attenuate YAP/TAZ activity. Altogether, this study reveals a novel mechanism of YAP/TAZ regulation by miR-582-5p in a cytoskeleton-dependent manner and suggests a negative feedback loop, highlighting the therapeutic potential of restoring miR-582-5p expression in treating NSCLC.
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http://dx.doi.org/10.3390/cancers13040756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918774PMC
February 2021

Pan-cancer analysis of ligand-receptor crosstalk in the tumor microenvironment.

Cancer Res 2021 Feb 5. Epub 2021 Feb 5.

Computational and Systems Biology, Genome Institute of Singapore

Signaling between cancer and nonmalignant (stromal) cells in the tumor microenvironment (TME) is key to tumor progression. Here we deconvoluted bulk tumor transcriptomes to infer crosstalk between ligands and receptors on cancer and stromal cells in the TME of 20 solid tumor types. This approach recovered known transcriptional hallmarks of cancer and stromal cells and was concordant with single-cell, in-situ hybridization and immunohistochemistry data. Inferred autocrine cancer cell interactions varied between tissues but often converged on Ephrin, BMP, and FGFR-signaling pathways. Analysis of immune checkpoints nominated interactions with high levels of cancer-to-immune crosstalk across distinct tumor types. Strikingly, PD-L1 was found to be highly expressed in stromal rather than cancer cells. Overall, our study presents a new resource for hypothesis generation and exploration of crosstalk in the TME.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2352DOI Listing
February 2021

A chemical genetic screen identifies Aurora kinases as a therapeutic target in EGFR T790M negative, gefitinib-resistant head and neck squamous cell carcinoma (HNSCC).

EBioMedicine 2021 Feb 30;64:103220. Epub 2021 Jan 30.

Laboratory of Precision Oncology and Cancer Evolution, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Genome #02-01, Singapore 138672, Singapore. Electronic address:

Background: Overexpression of epidermal growth factor receptor (EGFR), and downstream pathway activation appears to be a common oncogenic driver in the majority of head and neck squamous cell cancers (HNSCCs); yet targeting EGFR for the treatment of HNSCC has met with limited success. Apart from the anti-EGFR antibody cetuximab, no small molecule EGFR/tyrosine kinase inhibitors (TKIs) have progressed to routine clinical use. The aim of this study was to determine factors contributing to the lack of response to TKIs and identify alternative therapeutic vulnerabilities.

Methods: Genomic and transcriptomic sequencing, high-throughput compound screens, overexpression and siRNA knockdown, western blot, in vivo xenograft studies.

Findings: We derived three pairs of isogenic gefitinib (TKI)-sensitive and resistant patient-derived HNSCC cell lines. Genomic sequencing of gefitinib-resistant cell lines identified a lack of activating and resistance-associated EGFR mutations. Instead, transcriptomic sequencing showed upregulated EMT gene signature in the gefitinib-resistant cells with a corresponding increase in their migratory phenotype. Additionally, the resistant cell displayed reduced growth rate. Surprisingly, while gefitinib-resistant cells were independent of EGFR for survival, they nonetheless displayed activation of downstream ERK and AKT signalling. High-throughput screening (HTS) of druggable, small molecule libraries revealed that the gefitinib-resistant cells were particularly sensitive to inhibitors of genes involved in cell cycle and mitosis, such as Aurora kinase inhibitors (AKIs), cyclin-dependent kinase (CDK) inhibitors, and microtubule inhibitors. Notably our results showed that in the EGFR inhibited state, Aurora kinases are essential for cell survival.

Interpretation: Our study demonstrates that in the absence of activating EGFR mutations, HNSCCs may gain resistance to gefitinib through decreased cell proliferation, which makes them exceptionally vulnerable to cell-cycle inhibitors.

Funding: Agency for Science, Technology, and Research (A*STAR), National Medical Research Council (NMRC), and the National Institutes of Health (NIH)/National Cancer Institute (NCI).
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http://dx.doi.org/10.1016/j.ebiom.2021.103220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851772PMC
February 2021

tRNA-derived fragments (tRFs): establishing their turf in post-transcriptional gene regulation.

Cell Mol Life Sci 2021 Mar 2;78(6):2607-2619. Epub 2021 Jan 2.

Institute for Stem Cell Science and Regenerative Medicine, Bangalore, India.

Transfer RNA (tRNA)-derived fragments (tRFs) are an emerging class of conserved small non-coding RNAs that play important roles in post-transcriptional gene regulation. High-throughput sequencing of multiple biological samples have identified heterogeneous species of tRFs with distinct functionalities. These small RNAs have garnered a lot of scientific attention due to their ubiquitous expression and versatility in regulating various biological processes. In this review, we highlight our current understanding of tRF biogenesis and their regulatory functions. We summarize the diverse modes of biogenesis through which tRFs are generated and discuss the mechanism through which different tRF species regulate gene expression and the biological implications. Finally, we conceptualize research areas that require focus to strengthen our understanding of the biogenesis and function of tRFs.
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http://dx.doi.org/10.1007/s00018-020-03720-7DOI Listing
March 2021

Two high-yield complementary methods to sort cell populations by their 2D or 3D migration speed.

Mol Biol Cell 2020 12 21;31(25):2779-2790. Epub 2020 Oct 21.

Mechanobiology Institute, National University of Singapore, Singapore 117411.

The potential to migrate is one of the most fundamental functions for various epithelial, mesenchymal, and immune cells. Image analysis of motile cell populations, both primary and cultured, typically reveals an intercellular variability in migration speeds. However, cell migration chromatography, the sorting of large populations of cells based on their migratory characteristics, cannot be easily performed. The lack of such methods has hindered our understanding of the direct correlation between the capacity to migrate and other cellular properties. Here, we report two novel, easily implementable and readily scalable methods to sort millions of live migratory cancer and immune cells based on their spontaneous migration in two-dimensional and three-dimensional microenvironments, respectively. Correlative downstream transcriptomic, molecular and functional tests reveal marked differences between the fast and slow subpopulations in patient-derived cancer cells. We further employ our method to reveal that sorting the most migratory cytotoxic T lymphocytes yields a pool of cells with enhanced cytotoxicity against cancer cells. This phenotypic assay opens new avenues for the precise characterization of the mechanisms underlying hither to unexplained heterogeneities in migratory phenotypes within a cell population, and for the targeted enrichment of the most potent migratory leukocytes in immunotherapies.
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http://dx.doi.org/10.1091/mbc.E20-07-0466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851856PMC
December 2020

Onco-fetal Reprogramming of Endothelial Cells Drives Immunosuppressive Macrophages in Hepatocellular Carcinoma.

Cell 2020 10 24;183(2):377-394.e21. Epub 2020 Sep 24.

Genome Institute of Singapore, A(∗)STAR, 60 Biopolis Street, Genome, #02-01, Singapore 138672, Singapore. Electronic address:

We employed scRNA sequencing to extensively characterize the cellular landscape of human liver from development to disease. Analysis of ∼212,000 cells representing human fetal, hepatocellular carcinoma (HCC), and mouse liver revealed remarkable fetal-like reprogramming of the tumor microenvironment. Specifically, the HCC ecosystem displayed features reminiscent of fetal development, including re-emergence of fetal-associated endothelial cells (PLVAP/VEGFR2) and fetal-like (FOLR2) tumor-associated macrophages. In a cross-species comparative analysis, we discovered remarkable similarity between mouse embryonic, fetal-liver, and tumor macrophages. Spatial transcriptomics further revealed a shared onco-fetal ecosystem between fetal liver and HCC. Furthermore, gene regulatory analysis, spatial transcriptomics, and in vitro functional assays implicated VEGF and NOTCH signaling in maintaining onco-fetal ecosystem. Taken together, we report a shared immunosuppressive onco-fetal ecosystem in fetal liver and HCC. Our results unravel a previously unexplored onco-fetal reprogramming of the tumor ecosystem, provide novel targets for therapeutic interventions in HCC, and open avenues for identifying similar paradigms in other cancers and disease.
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http://dx.doi.org/10.1016/j.cell.2020.08.040DOI Listing
October 2020

Single-cell analysis of EphA clustering phenotypes to probe cancer cell heterogeneity.

Commun Biol 2020 08 6;3(1):429. Epub 2020 Aug 6.

National University of Singapore, Singapore, Singapore.

The Eph family of receptor tyrosine kinases is crucial for assembly and maintenance of healthy tissues. Dysfunction in Eph signaling is causally associated with cancer progression. In breast cancer cells, dysregulated Eph signaling has been linked to alterations in receptor clustering abilities. Here, we implemented a single-cell assay and a scoring scheme to systematically probe the spatial organization of activated EphA receptors in multiple carcinoma cells. We show that cancer cells retain EphA clustering phenotype over several generations, and the degree of clustering reported for migration potential both at population and single-cell levels. Finally, using patient-derived cancer lines, we probed the evolution of EphA signalling in cell populations that underwent metastatic transformation and acquisition of drug resistance. Taken together, our scalable approach provides a reliable scoring scheme for EphA clustering that is consistent over multiple carcinomas and can assay heterogeneity of cancer cell populations in a cost- and time-effective manner.
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http://dx.doi.org/10.1038/s42003-020-01136-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7411022PMC
August 2020

Dynamic expression of tRNA-derived small RNAs define cellular states.

EMBO Rep 2019 07 12;20(7):e47789. Epub 2019 Jun 12.

Precision Oncology, Genome Institute of Singapore, Singapore City, Singapore.

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation-dependent enrichment of 5'-tsRNAs. We report the identification of a set of 5'-tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5'-tsRNAs revealed a switch in their association with "effector" RNPs and "target" mRNAs in different cell states. We demonstrate that specific 5'-tsRNAs can preferentially interact with the RNA-binding protein, Igf2bp1, in the RA-induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency-promoting factor, c-Myc, thus providing a mechanistic basis for how 5'-tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5'-tsRNAs in defining distinct cell states.
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http://dx.doi.org/10.15252/embr.201947789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6607006PMC
July 2019

Self-aligning Tetris-Like (TILE) modular microfluidic platform for mimicking multi-organ interactions.

Lab Chip 2019 06;19(13):2178-2191

Department of Biomedical Engineering, National University of Singapore, 4, Engineering Drive 3, E4-04-10, 117583, Singapore.

Multi-organ perfusion systems offer the unique opportunity to mimic different physiological systemic interactions. However, existing multi-organ culture platforms have limited flexibility in specifying the culture conditions, device architectures, and fluidic connectivity simultaneously. Here, we report a modular microfluidic platform that addresses this limitation by enabling easy conversion of existing microfluidic devices into tissue and fluid control modules with self-aligning magnetic interconnects. This enables a 'stick-n-play' approach to assemble planar perfusion circuits that are amenable to both bioimaging-based and analytical measurements. A myriad of tissue culture and flow control TILE modules were successfully constructed with backward compatibility. Finally, we demonstrate applications in constructing recirculating multi-organ systems to emulate liver-mediated bioactivation of nutraceuticals and prodrugs to modulate their therapeutic efficacies in the context of atherosclerosis and cancer. This platform greatly facilitates the integration of existing organs-on-chip models to provide an intuitive and flexible way for users to configure different multi-organ perfusion systems.
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http://dx.doi.org/10.1039/c9lc00160cDOI Listing
June 2019

Tracking tumor evolution one-cell-at-a-time.

Mol Cell Oncol 2019 2;6(3):1590089. Epub 2019 Apr 2.

Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, Singapore, Singapore.

Accumulating evidence suggests that intra-tumor heterogeneity (ITH) within tumor sub-populations is a major contributor to therapy resistance, with genomically distinct clonal populations exhibiting different therapeutic vulnerabilities. Here we employed single-cell transcriptomics in patient-derived primary cells to understand the impact of phenotypic intra-tumor heterogeneity on drug-induced tumor evolution in oral squamous cell carcinoma.
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http://dx.doi.org/10.1080/23723556.2019.1590089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512908PMC
April 2019

The chromatin structuring protein HMGA2 influences human subtelomere stability and cancer chemosensitivity.

PLoS One 2019 8;14(5):e0215696. Epub 2019 May 8.

School of Biological Sciences, Nanyang Technological University, Singapore.

The transient build-up of DNA supercoiling during the translocation of replication forks threatens genome stability and is controlled by DNA topoisomerases (TOPs). This crucial process has been exploited with TOP poisons for cancer chemotherapy. However, pinpointing cellular determinants of the best clinical response to TOP poisons still remains enigmatic. Here, we present an integrated approach and demonstrate that endogenous and exogenous expression of the oncofetal high-mobility group AT-hook 2 (HMGA2) protein exhibited broad protection against the formation of hydroxyurea-induced DNA breaks in various cancer cells, thus corroborating our previously proposed model in which HMGA2 functions as a replication fork chaperone that forms a protective DNA scaffold at or close to stalled replication forks. We now further demonstrate that high levels of HMGA2 also protected cancer cells against DNA breaks triggered by the clinically important TOP1 poison irinotecan. This protection is most likely due to the recently identified DNA supercoil constraining function of HMGA2 in combination with exclusion of TOP1 from binding to supercoiled substrate DNA. In contrast, low to moderate HMGA2 protein levels surprisingly potentiated the formation of irinotecan-induced genotoxic covalent TOP1-DNA cleavage complexes. Our data from cell-based and several in vitro assays indicate that, mechanistically, this potentiating role involves enhanced drug-target interactions mediated by HMGA2 in ternary complexes with supercoiled DNA. Subtelomeric regions were found to be extraordinarily vulnerable to these genotoxic challenges induced by TOP1 poisoning, pointing at strong DNA topological barriers located at human telomeres. These findings were corroborated by an increased irinotecan sensitivity of patient-derived xenografts of colorectal cancers exhibiting low to moderate HMGA2 levels. Collectively, we uncovered a therapeutically important control mechanism of transient changes in chromosomal DNA topology that ultimately leads to enhanced human subtelomere stability.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0215696PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505889PMC
January 2020

Longitudinal single-cell RNA sequencing of patient-derived primary cells reveals drug-induced infidelity in stem cell hierarchy.

Nat Commun 2018 11 22;9(1):4931. Epub 2018 Nov 22.

Genome Institute of Singapore, Cancer Therapeutics & Stratified Oncology 5, 60 Biopolis Street, #02-01 Genome, Singapore, 138672, Singapore.

Chemo-resistance is one of the major causes of cancer-related deaths. Here we used single-cell transcriptomics to investigate divergent modes of chemo-resistance in tumor cells. We observed that higher degree of phenotypic intra-tumor heterogeneity (ITH) favors selection of pre-existing drug-resistant cells, whereas phenotypically homogeneous cells engage covert epigenetic mechanisms to trans-differentiate under drug-selection. This adaptation was driven by selection-induced gain of H3K27ac marks on bivalently poised resistance-associated chromatin, and therefore not expressed in the treatment-naïve setting. Mechanistic interrogation of this phenomenon revealed that drug-induced adaptation was acquired upon the loss of stem factor SOX2, and a concomitant gain of SOX9. Strikingly we observed an enrichment of SOX9 at drug-induced H3K27ac sites, suggesting that tumor evolution could be driven by stem cell-switch-mediated epigenetic plasticity. Importantly, JQ1 mediated inhibition of BRD4 could reverse drug-induced adaptation. These results provide mechanistic insights into the modes of therapy-induced cellular plasticity and underscore the use of epigenetic inhibitors in targeting tumor evolution.
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http://dx.doi.org/10.1038/s41467-018-07261-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250721PMC
November 2018

Ultrafast Single-Cell Level Enzymatic Tumor Profiling.

Anal Chem 2019 01 2;91(2):1277-1285. Epub 2018 Nov 2.

Department of Biomedical Engineering , National University of Singapore , 4 Engineering Drive 3, 04-08 , Singapore , Singapore.

In the context of tumor analysis, the implementation of precision medicine requires on-time clinical measurements, which requires rapid large-scale single-cell screening that obtains cell population distributions and functions in tumors to determine disease progression for therapeutics. In this study, a high-throughput screening (HTS) platform integrating optical fluorescence detectors and a computational method was developed as a droplet-based microfluidic flow cytometer (Droplet-μFC) to comprehensively analyze multiple proteolytic activities of a patient-derived tumor (with ∼0.5-2 M cells) at single-cell resolution within 2 h. The data-driven analytical method identified distinct cell types and status through protease profiling with high precision. Multiple protease activities of single cells harvested from a tumor were thus determined with a throughput of ∼100 cells per second. This platform was used to screen protease activities of a wide range of cell types, forming a library. With the development of advanced computational clustering and cell mapping, rapid quantitative tumor profiling with a comprehensive description of cell population distributions and functions could be obtained for clinical treatments.
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http://dx.doi.org/10.1021/acs.analchem.8b02576DOI Listing
January 2019

The transcription factor Lef1 switches partners from β-catenin to Smad3 during muscle stem cell quiescence.

Sci Signal 2018 07 24;11(540). Epub 2018 Jul 24.

Centre for Cellular and Molecular Biology, Hyderabad 500007, India.

Skeletal muscle stem cells (MuSCs), also known as satellite cells, persist in adult mammals by entering a state of quiescence (G) during the early postnatal period. Quiescence is reversed during damage-induced regeneration and re-established after regeneration. Entry of cultured myoblasts into G is associated with a specific, reversible induction of Wnt target genes, thus implicating members of the Tcf and Lef1 (Tcf/Lef) transcription factor family, which mediate transcriptional responses to Wnt signaling, in the initiation of quiescence. We found that the canonical Wnt effector β-catenin, which cooperates with Tcf/Lef, was dispensable for myoblasts to enter quiescence. Using pharmacological and genetic approaches in cultured C2C12 myoblasts and in MuSCs, we demonstrated that Tcf/Lef activity during quiescence depended not on β-catenin but on the transforming growth factor-β (TGF-β) effector and transcriptional coactivator Smad3, which colocalized with Lef1 at canonical Wnt-responsive elements and directly interacted with Lef1 specifically in G Depletion of Smad3, but not β-catenin, reduced Lef1 occupancy at target promoters, Tcf/Lef target gene expression, and self-renewal of myoblasts. In vivo, MuSCs underwent a switch from β-catenin-Lef1 to Smad3-Lef1 interactions during the postnatal switch from proliferation to quiescence, with β-catenin-Lef1 interactions recurring during damage-induced reactivation. Our findings suggest that the interplay of Wnt-Tcf/Lef and TGF-β-Smad3 signaling activates canonical Wnt target promoters in a manner that depends on β-catenin during myoblast proliferation but is independent of β-catenin during MuSC quiescence.
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http://dx.doi.org/10.1126/scisignal.aan3000DOI Listing
July 2018

Identification and characterization of a novel Sso7d scaffold-based binder against Notch1.

Sci Rep 2017 09 20;7(1):12021. Epub 2017 Sep 20.

New York University Langone Medical center, Perlmutter Cancer Center, Deparment of Biochemistry and Molecular Pharmacology, 522 1st Ave., Smilow Research Building, Rm 1206, New York, NY, 10016, USA.

Notch signaling has important functions in regulating cell growth and development, misregulation of which has been implicated in various cancers. Monoclonal antibodies (mAbs) targeting Notch protein activity have already moved into clinical trials. However due to the limitations associated with cost and productivity of mAbs, there has been a surge in the development of complementary approaches that are based on non-antibody scaffolds. Non-antibody scaffolds are small proteins that are stable and can be engineered to develop high-affinity binders against specific targets of interest. Here we describe the isolation and characterization of a novel Notch1-binding protein, N9, obtained by screening of a combinatorial library based on the ultra-stable Sso7d scaffold. N9 targets the extracellular EGF-like repeats (ELR) 11-13 in Notch1, and therefore serves as a competitive inhibitor for Notch ligands to decrease expression of Notch target genes. We demonstrate that N9 recognizes surface expression of Notch1 on the plasma membrane and binds preferentially to cell lines misexpressing Notch1. Although N9 was selected against Notch1, we also observe cross-reactivity against other Notch receptors, including Notch2/3. Finally, we demonstrate that N9 inhibits proliferation and generation of tumorspheres in Notch expressing cancer cell lines, suggesting its potential as a therapeutic agent in Notch-associated malignancies.
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http://dx.doi.org/10.1038/s41598-017-12246-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607287PMC
September 2017

Long noncoding RNA EGFR-AS1 mediates epidermal growth factor receptor addiction and modulates treatment response in squamous cell carcinoma.

Nat Med 2017 Oct 18;23(10):1167-1175. Epub 2017 Sep 18.

Cancer Therapeutics Research Laboratory, National Cancer Centre Singapore, Singapore.

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.
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http://dx.doi.org/10.1038/nm.4401DOI Listing
October 2017

Phenotype-driven precision oncology as a guide for clinical decisions one patient at a time.

Nat Commun 2017 09 5;8(1):435. Epub 2017 Sep 5.

Genome Institute of Singapore, A*STAR, Cancer Therapeutics & Stratified Oncology, PerkinElmer-GIS Centre for Precision Oncology, 60 Biopolis Street, #02-01 Genome, Singapore, 138672, Singapore.

Genomics-driven cancer therapeutics has gained prominence in personalized cancer treatment. However, its utility in indications lacking biomarker-driven treatment strategies remains limited. Here we present a "phenotype-driven precision-oncology" approach, based on the notion that biological response to perturbations, chemical or genetic, in ex vivo patient-individualized models can serve as predictive biomarkers for therapeutic response in the clinic. We generated a library of "screenable" patient-derived primary cultures (PDCs) for head and neck squamous cell carcinomas that reproducibly predicted treatment response in matched patient-derived-xenograft models. Importantly, PDCs could guide clinical practice and predict tumour progression in two n = 1 co-clinical trials. Comprehensive "-omics" interrogation of PDCs derived from one of these models revealed YAP1 as a putative biomarker for treatment response and survival in ~24% of oral squamous cell carcinoma. We envision that scaling of the proposed PDC approach could uncover biomarkers for therapeutic stratification and guide real-time therapeutic decisions in the future.Treatment response in patient-derived models may serve as a biomarker for response in the clinic. Here, the authors use paired patient-derived mouse xenografts and patient-derived primary culture models from head and neck squamous cell carcinomas, including metastasis, as models for high-throughput screening of anti-cancer drugs.
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http://dx.doi.org/10.1038/s41467-017-00451-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585361PMC
September 2017

A 3D printed microfluidic perfusion device for multicellular spheroid cultures.

Biofabrication 2017 Sep 11;9(4):045005. Epub 2017 Sep 11.

Department of Biomedical Engineering, National University of Singapore, 4, Engineering Drive 3, E4-04-10, Singapore 117583, Singapore.

The advent of 3D printing technologies promises to make microfluidic organ-on-chip technologies more accessible for the biological research community. To date, hydrogel-encapsulated cells have been successfully incorporated into 3D printed microfluidic devices. However, there is currently no 3D printed microfluidic device that can support multicellular spheroid culture, which facilitates extensive cell-cell contacts important for recapitulating many multicellular functional biological structures. Here, we report a first instance of fabricating a 3D printed microfluidic cell culture device capable of directly immobilizing and maintaining the viability and functionality of 3D multicellular spheroids. We evaluated the feasibility of two common 3D printing technologies i.e. stereolithography (SLA) and PolyJet printing, and found that SLA could prototype a device comprising of cell immobilizing micro-structures that were housed within a microfluidic network with higher fidelity. We have also implemented a pump-free perfusion system, relying on gravity-driven flow to perform medium perfusion in order to reduce the complexity and footprint of the device setup, thereby improving its adaptability into a standard biological laboratory. Finally, we demonstrated the biological performance of the 3D printed device by performing pump-free perfusion cultures of patient-derived parental and metastatic oral squamous cell carcinoma tumor and liver cell (HepG2) spheroids with good cell viability and functionality. This paper presents a proof-of-concept in simplifying and integrating the prototyping and operation of a microfluidic spheroid culture device, which will facilitate its applications in various drug efficacy, metabolism and toxicity studies.
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http://dx.doi.org/10.1088/1758-5090/aa8858DOI Listing
September 2017

Disrupting Interactions Between β-Catenin and Activating TCFs Reconstitutes Ground State Pluripotency in Mouse Embryonic Stem Cells.

Stem Cells 2017 08 15;35(8):1924-1933. Epub 2017 Jun 15.

Cancer Therapeutics and Stratified Oncology.

The 2i-media, composed of two small molecule inhibitors (PD0325901 and CHIR99021) against MEK and GSK3-kinases, respectively, is known to establish naïve ground state pluripotency in mouse embryonic stem cells (mESCs). These inhibitors block MEK-mediated differentiation, while driving β-catenin dependent de-repression of pluripotency promoting targets. However, accumulating evidence suggest that β-catenin's association with activating TCFs (TCF7 and TCF7L2) can induce expression of several lineage-specific prodifferentiation genes. We posited that CHIR-induced upregulation of β-catenin levels could therefore compromise the stability of the naïve state in long-term cultures. Here, we investigated whether replacing CHIR with iCRT3, a small molecule that abrogates β-catenin-TCF interaction, can still retain ground state pluripotency in mESCs. Our data suggests that iCRT3 + PD mediated coinhibition of MEK and β-catenin/TCF-dependent transcriptional activity over multiple passages significantly reduces expression of differentiation markers, as compared to 2i. Furthermore, the ability to efficiently contribute toward chimera generation and germline transmission suggests that the inhibition of β-catenin's TCF-dependent transcriptional activity, independent of its protein expression level, retains the naïve ground state pluripotency in mESCs. Additionally, growth medium containing iCRT3 + PD can provide an alternative to 2i as a stable culture method. Stem Cells 2017;35:1924-1933.
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http://dx.doi.org/10.1002/stem.2647DOI Listing
August 2017

TCF7L1 Modulates Colorectal Cancer Growth by Inhibiting Expression of the Tumor-Suppressor Gene EPHB3.

Sci Rep 2016 06 23;6:28299. Epub 2016 Jun 23.

New York University Langone Medical Center, Perlmutter Cancer Center, Department of Stem Cell Biology, 522 1st Ave., Smilow Research Building, Rm 1206 New York, NY, 10016, USA.

Dysregulation of the Wnt pathway leading to accumulation of β-catenin (CTNNB1) is a hallmark of colorectal cancer (CRC). Nuclear CTNNB1 acts as a transcriptional coactivator with TCF/LEF transcription factors, promoting expression of a broad set of target genes, some of which promote tumor growth. However, it remains poorly understood how CTNNB1 interacts with different transcription factors in different contexts to promote different outcomes. While some CTNNB1 target genes are oncogenic, others regulate differentiation. Here, we found that TCF7L1, a Wnt pathway repressor, buffers CTNNB1/TCF target gene expression to promote CRC growth. Loss of TCF7L1 impaired growth and colony formation of HCT116 CRC cells and reduced tumor growth in a mouse xenograft model. We identified a group of CTNNB1/TCF target genes that are activated in the absence of TCF7L1, including EPHB3, a marker of Paneth cell differentiation that has also been implicated as a tumor suppressor in CRC. Knockdown of EPHB3 partially restores growth and normal cell cycle progression of TCF7L1-Null cells. These findings suggest that while CTNNB1 accumulation is critical for CRC progression, activation of specific Wnt target genes in certain contexts may in fact inhibit tumor growth.
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http://dx.doi.org/10.1038/srep28299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917863PMC
June 2016

Colorectal cancer atlas: An integrative resource for genomic and proteomic annotations from colorectal cancer cell lines and tissues.

Nucleic Acids Res 2016 Jan 22;44(D1):D969-74. Epub 2015 Oct 22.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia

In order to advance our understanding of colorectal cancer (CRC) development and progression, biomedical researchers have generated large amounts of OMICS data from CRC patient samples and representative cell lines. However, these data are deposited in various repositories or in supplementary tables. A database which integrates data from heterogeneous resources and enables analysis of the multidimensional data sets, specifically pertaining to CRC is currently lacking. Here, we have developed Colorectal Cancer Atlas (http://www.colonatlas.org), an integrated web-based resource that catalogues the genomic and proteomic annotations identified in CRC tissues and cell lines. The data catalogued to-date include sequence variations as well as quantitative and non-quantitative protein expression data. The database enables the analysis of these data in the context of signaling pathways, protein-protein interactions, Gene Ontology terms, protein domains and post-translational modifications. Currently, Colorectal Cancer Atlas contains data for >13 711 CRC tissues, >165 CRC cell lines, 62 251 protein identifications, >8.3 million MS/MS spectra, >18 410 genes with sequence variations (404 278 entries) and 351 pathways with sequence variants. Overall, Colorectal Cancer Atlas has been designed to serve as a central resource to facilitate research in CRC.
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http://dx.doi.org/10.1093/nar/gkv1097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702801PMC
January 2016

Inhibition of β-catenin-TCF1 interaction delays differentiation of mouse embryonic stem cells.

J Cell Biol 2015 Oct;211(1):39-51

Department of Biochemistry and Molecular Pharmacology, New York University Cancer Institute, New York University Langone Medical Center, New York, NY 10016 Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, Singapore 138672.

The ability of mouse embryonic stem cells (mESCs) to self-renew or differentiate into various cell lineages is regulated by signaling pathways and a core pluripotency transcriptional network (PTN) comprising Nanog, Oct4, and Sox2. The Wnt/β-catenin pathway promotes pluripotency by alleviating T cell factor TCF3-mediated repression of the PTN. However, it has remained unclear how β-catenin's function as a transcriptional activator with TCF1 influences mESC fate. Here, we show that TCF1-mediated transcription is up-regulated in differentiating mESCs and that chemical inhibition of β-catenin/TCF1 interaction improves long-term self-renewal and enhances functional pluripotency. Genetic loss of TCF1 inhibited differentiation by delaying exit from pluripotency and conferred a transcriptional profile strikingly reminiscent of self-renewing mESCs with high Nanog expression. Together, our data suggest that β-catenin's function in regulating mESCs is highly context specific and that its interaction with TCF1 promotes differentiation, further highlighting the need for understanding how its individual protein-protein interactions drive stem cell fate.
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http://dx.doi.org/10.1083/jcb.201503017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4602028PMC
October 2015

Wnt inhibition leads to improved chemosensitivity in paediatric acute lymphoblastic leukaemia.

Br J Haematol 2014 Oct 4;167(1):87-99. Epub 2014 Jul 4.

NYU Cancer Institute, NYU Langone Medical Center, New York, NY.

While childhood acute lymphoblastic leukaemia (ALL) is now highly curable, the dismal prognosis for children who relapse warrants novel therapeutic approaches. Previously, using an integrated genomic analysis of matched diagnosis-relapse paired samples, we identified overactivation of the Wnt pathway as a possible mechanism of recurrence. To validate these findings and document whether Wnt inhibition may sensitize cells to chemotherapy, we analysed the expression of activated β-catenin (and its downstream target BIRC5) using multiparameter phosphoflow cytometry and tested the efficacy of a recently developed Wnt inhibitor, iCRT14, in ALL cell lines and patient samples. We observed increased activation of β-catenin at relapse in 6/10 patients. Furthermore, treatment of leukaemic cell lines with iCRT14 led to significant downregulation of Wnt target genes and combination with traditional chemotherapeutic drugs resulted in a synergistic decrease in viability as well as a significant increase in apoptotic cell death. Finally, pre-treatment of purified blasts from patients with relapsed leukaemia with the Wnt inhibitor followed by exposure to prednisolone, restored chemosensitivity in these cells. Our results demonstrate that overactivation of the Wnt pathway may contribute to chemoresistance in relapsed childhood ALL and that Wnt-inhibition may be a promising therapeutic approach.
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http://dx.doi.org/10.1111/bjh.13011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207443PMC
October 2014

Wnt coreceptor Lrp5 is a driver of idiopathic pulmonary fibrosis.

Am J Respir Crit Care Med 2014 Jul;190(2):185-95

1 Division of Pulmonary and Critical Care Medicine and.

Rationale: Wnt/β-catenin signaling has been implicated in lung fibrosis, but how this occurs and whether expression changes in Wnt pathway components predict disease progression is unknown.

Objectives: To determine whether the Wnt coreceptor Lrp5 drives pulmonary fibrosis in mice and is predictive of disease severity in humans.

Methods: We examined mice with impaired Wnt signaling caused by loss of the Wnt coreceptor Lrp5 in models of lung fibrosis induced by bleomycin or an adenovirus encoding an active form of transforming growth factor (TGF)-β. We also analyzed gene expression in peripheral blood mononuclear cells (PBMC) from patients with idiopathic pulmonary fibrosis (IPF).

Measurements And Main Results: In patients with IPF, analysis of peripheral blood mononuclear cells revealed that elevation of positive regulators, Lrp5 and 6, was independently associated with disease progression. LRP5 was also associated with disease severity at presentation in an additional cohort of patients with IPF. Lrp5 null mice were protected against bleomycin-induced pulmonary fibrosis, an effect that was phenocopied by direct inhibition of β-catenin signaling by the small molecular inhibitor of β-catenin responsive transcription. Transplantation of Lrp5 null bone marrow cells into wild-type mice did not limit fibrosis. Instead, Lrp5 loss was associated with reduced TGF-β production by alveolar type 2 cells and leukocytes. Consistent with a role of Lrp5 in the activation of TGF-β, Lrp5 null mice were not protected against lung fibrosis induced by TGF-β.

Conclusions: We show that the Wnt coreceptor, Lrp5, is a genetic driver of lung fibrosis in mice and a marker of disease progression and severity in humans with IPF. Evidence that TGF-β signaling can override a loss in Lrp5 has implications for patient selection and timing of Wnt pathway inhibitors in lung fibrosis.
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http://dx.doi.org/10.1164/rccm.201401-0079OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226053PMC
July 2014

Luciferase reporter assay in Drosophila and mammalian tissue culture cells.

Curr Protoc Chem Biol 2014 Mar 14;6(1):7-23. Epub 2014 Mar 14.

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, NYU Cancer Institute, New York, New York.

Luciferase reporter gene assays are one of the most common methods for monitoring gene activity. Because of their sensitivity, dynamic range, and lack of endogenous activity, luciferase assays have been particularly useful for functional genomics in cell-based assays, such as RNAi screening. This unit describes delivery of two luciferase reporters with other nucleic acids (siRNA/dsRNA), measurement of the dual luciferase activities, and analysis of data generated. The systematic query of gene function (RNAi) combined with the advances in luminescent technology have made it possible to design powerful whole genome screens to address diverse and significant biological questions.
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http://dx.doi.org/10.1002/9780470559277.ch130149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4059354PMC
March 2014