Prabha Sampath
Email Address
bchps@nus.edu.sg
Organizational Units
DUKE-NUS MEDICAL SCHOOL
faculty
YONG LOO LIN SCH OF MEDICINE
faculty
BIOCHEMISTRY
dept
11 results
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Publication HOXD-AS1 is a novel lncRNA encoded in HOXD cluster and a marker of neuroblastoma progression revealed via integrative analysis of noncoding transcriptome(2014) Yarmishyn, A.A; Batagov, A.O; Tan, J.Z; Sundaram, G.M; Sampath, P; Kuznetsov, V.A; Kurochkin, I.V; BIOCHEMISTRYBackground: Long noncoding RNAs (lncRNAs) constitute a major, but poorly characterized part of human transcriptome. Recent evidence indicates that many lncRNAs are involved in cancer and can be used as predictive and prognostic biomarkers. Significant fraction of lncRNAs is represented on widely used microarray platforms, however they have usually been ignored in cancer studies. Results: We developed a computational pipeline to annotate lncRNAs on popular Affymetrix U133 microarrays, creating a resource allowing measurement of expression of 1581 lncRNAs. This resource can be utilized to interrogate existing microarray datasets for various lncRNA studies. We found that these lncRNAs fall into three distinct classes according to their statistical distribution by length. Remarkably, these three classes of lncRNAs were co-localized with protein coding genes exhibiting distinct gene ontology groups. This annotation was applied to microarray analysis which identified a 159 lncRNA signature that discriminates between localized and metastatic stages of neuroblastoma. Analysis of an independent patient cohort revealed that this signature differentiates also relapsing from non-relapsing primary tumors. This is the first example of the signature developed via the analysis of expression of lncRNAs solely. One of these lncRNAs, termed HOXD-AS1, is encoded in HOXD cluster. HOXD-AS1 is evolutionary conserved among hominids and has all bona fide features of a gene. Studying retinoid acid (RA) response of SH-SY5Y cell line, a model of human metastatic neuroblastoma, we found that HOXD-AS1 is a subject to morphogenic regulation, is activated by PI3K/Akt pathway and itself is involved in control of RA-induced cell differentiation. Knock-down experiments revealed that HOXD-AS1 controls expression levels of clinically significant protein-coding genes involved in angiogenesis and inflammation, the hallmarks of metastatic cancer. Conclusions: Our findings greatly extend the number of noncoding RNAs functionally implicated in tumor development and patient treatment and highlight their role as potential prognostic biomarkers of neuroblastomas. © 2014 Yarmishyn et al.Publication Translational control of ceruloplasmin gene expression: Beyond the IRE(Society of Biology of Chile, 2006) Mazumder, B; Sampath, P; Fox, P.L; BIOCHEMISTRYTranslational control is a common regulatory mechanism for the expression of iron-related proteins. For example, three enzymes involved in erythrocyte development are regulated by three different control mechanisms: globin synthesis is modulated by heme-regulated translational inhibitor; erythroid 5-aminolevulinate synthase translation is inhibited by binding of the iron regulatory protein to the iron response element in the 5?-untranslated region (UTR); and 15-lipoxygenase is regulated by specific proteins binding to the 3?-UTR. Ceruloplasmin (Cp) is a multi-functional, copper protein made primarily by the liver and by activated macrophages. Cp has important roles in iron homeostasis and in inflammation. Its role in iron metabolism was originally proposed because of its ferroxidase activity and because of its ability to stimulate iron loading into apo-transferrin and iron efflux from liver. We have shown that Cp mRNA is induced by interferon (IFN)-y in U937 monocytic cells, but synthesis of Cp protein is halted by translational silencing. The silencing mechanism requires binding of a cytosolic inhibitor complex, IFN-Gamma-Activated Inhibitor of Translation (GAIT), to a specific GAIT element in the Cp 3?-UTR. Here, we describe our studies that define and characterize the GAIT element and elucidate the specific trans-acting proteins that bind the GAIT element. Our experiments describe a new mechanism of translational control of an iron-related protein and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation. © 2006 Sociedad de Biología Chile.Publication EGF hijacks miR-198/FSTL1 wound-healing switch and steers a two-pronged pathway toward metastasis(Rockefeller University Press, 2017) Sundaram, G.M; Ismail, H.M; Bashir, M; Muhuri, M; Vaz, C; Nama, S; Ow, G.S; Vladimirovna, I.A; Ramalingam, R; Burke, B; Tanavde, V; Kuznetsov, V; Birgitte Lane, E; Sampath, P; BIOCHEMISTRYEpithelial carcinomas are well known to activate a prolonged wound-healing program that promotes malignant transformation. Wound closure requires the activation of keratinocyte migration via a dual-state molecular switch. This switch involves production of either the anti-migratory microRNA miR-198 or the pro-migratory follistatin-like 1 (FSTL1) protein from a single transcript; miR-198 expression in healthy skin is down-regulated in favor of FSTL1 upon wounding, which enhances keratinocyte migration and promotes re-epithelialization. Here, we reveal a defective molecular switch in head and neck squamous cell carcinoma (HNS CC ). This defect shuts off miR-198 expression in favor of sustained FSTL1 translation, driving metastasis through dual parallel pathways involving DIA PH1 and FSTL1. DIA PH1, a miR-198 target, enhances directional migration through sequestration of Arpin, a competitive inhibitor of Arp2/3 complex. FSTL1 blocks Wnt7a-mediated repression of extracellular signal-regulated kinase phosphorylation, enabling production of MMP9, which degrades the extracellular matrix and facilitates metastasis. The prognostic significance of the FSTL1-DIA PH1 gene pair makes it an attractive target for therapeutic intervention. © 2017 Sundaram et al.Publication Hippo/MST blocks breast cancer by downregulating WBP2 oncogene expression via miRNA processor Dicer(Springer Nature, 2020) Lim, S.K.; Tabatabaeian, H.; Lu, S.Y.; Kang, S.-A.; Sundaram, G.M.; Sampath, P.; Chan, S.W.; Hong, W.J.; Lim, Y.P.; CANCER SCIENCE INSTITUTE OF SINGAPORE; BIOCHEMISTRYWBP2 transcription coactivator is an emerging oncoprotein and a key node of convergence between EGF and Wnt signaling pathways. Understanding how WBP2 is regulated has important implications for cancer therapy. WBP2 is tightly controlled by post-translational modifications, including phosphorylation and ubiquitination, leading to changes in subcellular localization, protein–protein interactions, and protein turnover. As the function of WBP2 is intricately linked to YAP and TAZ, we hypothesize that WBP2 is negatively regulated by the Hippo tumor suppressor pathway. Indeed, MST is demonstrated to negatively regulate WBP2 expression in a kinase-dependent but LATS-independent manner. This was observed in the majority of the breast cancer cell lines tested. The effect of MST was enhanced by SAV and concomitant with the inhibition of the transcription co-activation, in vitro and in vivo tumorigenesis activities of WBP2, resulting in good prognosis in xenografts. Downregulation of WBP2 by MST involved miRNA but not proteasomal or lysosomal degradation. Our data support the existence of a novel MST-Dicer signaling axis, which in turn regulates both WBP2 CDS- and UTR-targeting miRNAs expression, including miR-23a. MiR-23a targets the 3?UTR of WBP2 mRNA directly. Significant inverse relationships between WBP2 and MST or miR23a expression levels in clinical specimens were observed. In conclusion, WBP2 is a target of the Hippo/MST kinase; MST is identified as yet another rheostat in the regulation of WBP2 and its oncogenic function. The findings have implications in targeted therapeutics and precision medicine for breast cancer. © 2020, The Author(s).Publication MicroRNA-138 is a Prognostic Biomarker for Triple-Negative Breast Cancer and Promotes Tumorigenesis via TUSC2 repression(Nature Publishing Group, 2019) Nama, S.; Muhuri, M.; Di Pascale, F.; Quah, S.; Aswad, L.; Fullwood, M.; Sampath, P.; CANCER SCIENCE INSTITUTE OF SINGAPORE; BIOCHEMISTRYBreast cancer manifests as a spectrum of subtypes with distinct molecular signatures, and different responses to treatment. Of these subtypes, triple-negative breast cancer (TNBC) has the worst prognoses and limited therapeutic options. Here we report aberrant expression of microRNA-138 (miR-138) in TNBC. Increased miR-138 expression is highly specific to this subtype, correlates with poor prognosis in patients, and is functionally relevant to cancer progression. Our findings establish miR-138 as a specific diagnostic and prognostic biomarker for TNBC. OncomiR-138 is pro-survival; sequence-specific miR-138 inhibition blocks proliferation, promotes apoptosis and inhibits tumour growth in-vivo. miR-138 directly targets a suite of pro-apoptotic and tumour suppressive genes, including tumour suppressor candidate 2 (TUSC2). miR-138 silences TUSC2 by binding to a unique 5?-UTR target-site, which overlaps with the translation start-site of the transcript. Over-expression of TUSC2 mimics the phenotype of miR-138 knockdown and functional rescue experiments confirm that TUSC2 is a direct downstream target of miR-138. Our report of miR-138 as an oncogenic driver in TNBC, positions it as a viable target for oligonucleotide therapeutics and we envision the potential value of using antimiR-138 as an adjuvant therapy to alleviate this therapeutically intractable cancer. © 2019, The Author(s).Publication Kindlin-3 interacts with the ribosome and regulates c-Myc expression required for proliferation of chronic myeloid leukemia cells(Nature Publishing Group, 2015) Qu, J; Ero, R; Feng, C; Ong, L.-T; Tan, H.-F; Lee, H.-S; Ismail, M.H; Bu, W.-T; Nama, S; Sampath, P; Gao, Y.-G; Tan, S.-M; BIOCHEMISTRYKindlins are FERM-containing cytoplasmic proteins that regulate integrin-mediated cell-cell and cell-extracellular matrix (ECM) attachments. Kindlin-3 is expressed in hematopoietic cells, platelets, and endothelial cells. Studies have shown that kindlin-3 stabilizes cell adhesion mediated by ß1, ß2, and ß3 integrins. Apart from integrin cytoplasmic tails, kindlins are known to interact with other cytoplasmic proteins. Here we demonstrate that kindlin-3 can associate with ribosome via the receptor for activated-C kinase 1 (RACK1) scaffold protein based on immunoprecipitation, ribosome binding, and proximity ligation assays. We show that kindlin-3 regulates c-Myc protein expression in the human chronic myeloid leukemia cell line K562. Cell proliferation was reduced following siRNA reduction of kindlin-3 expression and a significant reduction in tumor mass was observed in xenograft experiments. Mechanistically, kindlin-3 is involved in integrin ?5ß1-Akt-mTOR-p70S6K signaling; however, its regulation of c-Myc protein expression could be independent of this signaling axis.Publication HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis(Nature Publishing Group, 2018) Yan, T; Ooi, W.F; Qamra, A; Cheung, A; Ma, D; Sundaram, G.M; Xu, C; Xing, M; Poon, L; Wang, J; Loh, Y.P; Ho, J.H.J; Ng, J.J.Q; Ramlee, M.K; Aswad, L; Rozen, S.G; Ghosh, S; Bard, F.A; Sampath, P; Tergaonkar, V; Davies, J.O.J; Hughes, J.R; Goh, E; Bi, X; Fullwood, M.J; Tan, P; Li, S; DEAN'S OFFICE (DUKE-NUS MEDICAL SCHOOL); CANCER SCIENCE INSTITUTE OF SINGAPORE; DUKE-NUS MEDICAL SCHOOLThe repression of telomerase activity during cellular differentiation promotes replicative aging and functions as a physiological barrier for tumorigenesis in long-lived mammals, including humans. However, the underlying mechanisms remain largely unclear. Here we describe how miR-615-3p represses hTERT expression. mir-615-3p is located in an intron of the HOXC5 gene, a member of the highly conserved homeobox family of transcription factors controlling embryogenesis and development. Unexpectedly, we found that HoxC5 also represses hTERT expression by disrupting the long-range interaction between hTERT promoter and its distal enhancer. The 3?UTR of hTERT and its upstream enhancer region are well conserved in long-lived primates. Both mir-615-3p and HOXC5 are activated upon differentiation, which constitute a feed-forward loop that coordinates transcriptional and post-Transcriptional repression of hTERT during cellular differentiation. Deregulation of HOXC5 and mir-615-3p expression may contribute to the activation of hTERT in human cancers. © 2017 The Author(s).Publication C/EBPβ mediates RNA polymerase III-driven transcription of oncomiR-138 in malignant gliomas(Oxford University Press, 2018) Di Pascale, F.; Nama, S.; Muhuri, M.; Quah, S.; Ismail, H.M.; Chan, X.H.D.; Sundaram, G.M.; Ramalingam, R.; Burke, B.; Sampath, P.; BIOCHEMISTRYMicroRNA-138 (miR-138) is a pro-survival oncomiR for glioma stem cells. In malignant gliomas, dysregulated expression of microRNAs, such as miR-138, promotes Tumour initiation and progression. Here, we identify the ancillary role of the CCAAT/enhancer binding protein ? (C/EBP?) as a transcriptional activator of miR-138. We demonstrate that a short 158 bp DNA sequence encoding the precursor of miR-138-2 is essential and sufficient for transcription of miR-138. This short sequence includes the A-box and B-box elements characteristic of RNA Polymerase III (Pol III) promoters, and is also directly bound by C/EBP? via an embedded 'C/EBP? responsive element' (CRE). CRE and the Pol III B-box element overlap, suggesting that C/EBP? and transcription factor 3C (TFIIIC) interact at the miR-138-2 locus. We propose that this interaction is essential for the recruitment of the RNA Pol III initiation complex and associated transcription of the oncomiR, miR-138 in malignant gliomas. © The Author(s) 2017.Publication Genetic and Epigenetic Profiling Reveals EZH2-mediated Down Regulation of OCT-4 Involves NR2F2 during Cardiac Differentiation of Human Embryonic Stem Cells(Nature Publishing Group, 2017) Pursani, V; Pethe, P; Bashir, M; Sampath, P; Tanavde, V; Bhartiya, D; BIOCHEMISTRYHuman embryonic (hES) stem cells are widely used as an in vitro model to understand global genetic and epigenetic changes that occur during early embryonic development. In-house derived hES cells (KIND1) were subjected to directed differentiation into cardiovascular progenitors (D12) and beating cardiomyocytes (D20). Transcriptome profiling of undifferentiated (D0) and differentiated (D12 and 20) cells was undertaken by microarray analysis. ChIP and sequential ChIP were employed to study role of transcription factor NR2F2 during hES cells differentiation. Microarray profiling showed that an alteration of about 1400 and 1900 transcripts occurred on D12 and D20 respectively compared to D0 whereas only 19 genes were altered between D12 and D20. This was found associated with corresponding expression pattern of chromatin remodelers, histone modifiers, miRNAs and lncRNAs marking the formation of progenitors and cardiomyocytes on D12 and D20 respectively. ChIP sequencing and sequential ChIP revealed the binding of NR2F2 with polycomb group member EZH2 and pluripotent factor OCT4 indicating its crucial involvement in cardiac differentiation. The study provides a detailed insight into genetic and epigenetic changes associated with hES cells differentiation into cardiac cells and a role for NR2F2 is deciphered for the first time to down-regulate OCT-4 via EZH2 during cardiac differentiation. © 2017 The Author(s).Publication Contrasting expression patterns of coding and noncoding parts of the human genome upon oxidative stress(2015) Giannakakis, A; Zhang, J; Jenjaroenpun, P; Nama, S; Zainolabidin, N; Aau, M.Y; Yarmishyn, A.A; Vaz, C; Ivshina, A.V; Grinchuk, O.V; Voorhoeve, M; Vardy, L.A; Sampath, P; Kuznetsov, V.A; Kurochkin, I.V; Guccione, E; PHYSIOLOGY; DUKE-NUS MEDICAL SCHOOL; BIOCHEMISTRYOxidative stress (OS) is caused by an imbalance between pro- and anti-oxidant reactions leading to accumulation of reactive oxygen species within cells. We here investigate the effect of OS on the transcriptome of human fibroblasts. OS causes a rapid and transient global induction of transcription characterized by pausing of RNA polymerase II (PolII) in both directions, at specific promoters, within 30 minutes of the OS response. In contrast to protein-coding genes, which are commonly down-regulated, this novel divergent, PolII pausing-phenomenon leads to the generation of thousands of long noncoding RNAs (lncRNAs) with promoter-associated antisense lncRNAs transcripts (si-paancRNAs) representing the major group of stress-induced transcripts. OS causes transient dynamics of si-lncRNAs in nucleus and cytosol, leading to their accumulation at polysomes, in contrast to mRNAs, which get depleted from polysomes. We propose that si-lncRNAs represent a novel component of the transcriptional stress that is known to determine the outcome of immediate-early and later cellular stress responses and we provide insights on the fate of those novel mature lncRNA transcripts by showing that their association with polysomal complexes is significantly increased in OS.