Gopinath M. Sundaram

‘See-saw’ expression of microRNA-198 and FSTL1 from a single transcript in wound healing

Post-transcriptional switches are flexible effectors of dynamic changes in gene expression. Here we report a new post-transcriptional switch that dictates the spatiotemporal and mutually exclusive expression of two alternative gene products from a single transcript. Expression of primate-specific exonic microRNA-198 (miR-198), located in the 3′-untranslated region of follistatin-like 1 (FSTL1) messenger RNA, switches to expression of the linked open reading frame of FSTL1 upon wounding in a human ex vivo organ culture system. We show that binding of a KH-type splicing regulatory protein (KSRP, also known as KHSRP) to the primary transcript determines the fate of the transcript and is essential for the processing of miR-198: transforming growth factor-β signalling switches off miR-198 expression by downregulating KSRP, and promotes FSTL1 protein expression. We also show that FSTL1 expression promotes keratinocyte migration, whereas miR-198 expression has the opposite effect by targeting and inhibiting DIAPH1, PLAU and LAMC2. A clear inverse correlation between the expression pattern of FSTL1 (pro-migratory) and miR-198 (anti-migratory) highlights the importance of this regulatory switch in controlling context-specific gene expression to orchestrate wound re-epithelialization. The deleterious effect of failure of this switch is apparent in non-healing chronic diabetic ulcers, in which expression of miR-198 persists, FSTL1 is absent, and keratinocyte migration, re-epithelialization and wound healing all fail to occur.

Targeting glioma stem cells by functional inhibition of a prosurvival oncomiR-138 in malignant gliomas.

Malignant gliomas are the most aggressive forms of brain tumors, associated with high rates of morbidity and mortality. Recurrence and tumorigenesis are attributed to a subpopulation of tumor-initiating glioma stem cells (GSCs) that are intrinsically resistant to therapy. Initiation and progression of gliomas have been linked to alterations in microRNA expression. Here, we report the identification of microRNA-138 (miR-138) as a molecular signature of GSCs and demonstrate a vital role for miR-138 in promoting growth and survival of bona fide tumor-initiating cells with self-renewal potential. Sequence-specific functional inhibition of miR-138 prevents tumorsphere formation in vitro and impedes tumorigenesis in vivo. We delineate the components of the miR-138 regulatory network by loss-of-function analysis to identify specific regulators of apoptosis. Finally, the higher expression of miR-138 in GSCs compared to non-neoplastic tissue and association with tumor recurrence and survival highlights the clinical significance of miR-138 as a prognostic biomarker and a therapeutic target for treatment of malignant gliomas.

HOXD-AS1 is a novel lncRNA encoded in HOXD cluster and a marker of neuroblastoma progression revealed via integrative analysis of noncoding transcriptome.

BackgroundLong 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.ResultsWe 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.ConclusionsOur 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.

EGF hijacks miR-198/FSTL1 wound-healing switch and steers a two-pronged pathway toward metastasis

Epithelial 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 (HNSCC). This defect shuts off miR-198 expression in favor of sustained FSTL1 translation, driving metastasis through dual parallel pathways involving DIAPH1 and FSTL1. DIAPH1, 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-DIAPH1 gene pair makes it an attractive target for therapeutic intervention.

HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis.

The 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.The expression of telomerase catalytic subunit hTERT is frequently upregulated in many cancers. Here, the authors show HoxC5 and miR-615-3p can negatively regulate hTERT to impede tumorigenesis by targeting the newly evolved cis-regulatory genomic elements of hTERT.

C/EBPβ mediates RNA polymerase III-driven transcription of oncomiR-138 in malignant gliomas

Abstract MicroRNA-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 β (C/EBPβ) 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/EBPβ via an embedded ‘C/EBPβ responsive element’ (CRE). CRE and the Pol III B-box element overlap, suggesting that C/EBPβ 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.

Cancer: the dark side of wound healing

Complex multicellular organisms have evolved sophisticated mechanisms to rapidly resolve epithelial injuries. Epithelial integrity is critical to maintaining internal homeostasis. An epithelial breach represents the potential for pathogen ingress and fluid loss, both of which may have severe consequences if not limited. The mammalian wound healing response involves a finely tuned, self‐limiting series of cellular and molecular events orchestrated by the transient activation of specific signalling pathways. Accurate regulation of these events is essential; failure to initiate key steps at the right time delays healing and leads to chronic wounds, while aberrant initiation of wound healing processes may produce cell behaviours that promote cancer progression. In this review, we discuss how wound healing pathways co‐opted in cancer lose their stringent regulation and become compromised in their reversibility. We hypothesize on how the commandeering of wound healing ‘master regulators’ is involved in this process, and also highlight the implications of these findings in the treatment of both chronic wounds and cancer.

Carcinoma Cells Reprogram a Wound-healing Switch to Promote Metastasis

ABSTRACT Wound healing is a dynamic event where barrier disruption is transient and miR-198/FSTL1 molecular switch orchestrate wound re-epithelialization. However, epithelial carcinomas maintain a prolonged wound-healing phase to promote malignant transformation. Delineating the molecular mechanism we demonstrate, how epidermal growth factor (EGF) hijacks the wound-healing switch to promote metastasis of carcinoma.

NF-κB Role and Potential Drug Targets in Gastrointestinal Cancer

A multifactorial disease cancer arises due to mutation in the gene encoding particular transcription factor or proteins. Globally cancer is one of the diseases which is responsible for maximum mortality annually. Transcription factor plays an important role in cell physiology, and any alteration in this transcription factor may lead to diseases like cancers. NF-κB is a transcription factor which has immense homeostasis role in cell physiology and in several diseases. NF-κB is actively expressed in many cancers and helps in initiations, cell proliferations, and metastasis of different cancers. The present chapter discusses the role of NF-κB in cancer promotion and different drug targets, targeting NF-κB pathway for the treatment of cancers.

Regulation of context-specific gene expression by posttranscriptional switches.

Post-transcriptional control of mRNA is a vital step in the regulation of gene expression. Highly specific combinations of RNA-binding–proteins (RBPs) and microRNAs conjointly contribute to the complexity of post-transcriptional control of mRNAs. Post-transcriptional switches regulated by RBPs control context-specific expression of two alternative gene products from a single transcript.