Arindam Chaudhury

Myosin II isoform switching mediates invasiveness after TGF-β–induced epithelial–mesenchymal transition

Despite functional significance of nonmuscle myosin II in cell migration and invasion, its role in epithelial–mesenchymal transition (EMT) or TGF-β signaling is unknown. Analysis of normal mammary gland expression revealed that myosin IIC is expressed in luminal cells, whereas myosin IIB expression is up-regulated in myoepithelial cells that have more mesenchymal characteristics. Furthermore, TGF-β induction of EMT in nontransformed murine mammary gland epithelial cells results in an isoform switch from myosin IIC to myosin IIB and increased phosphorylation of myosin heavy chain (MHC) IIA on target sites known to regulate filament dynamics (S1916, S1943). These expression and phosphorylation changes are downstream of heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of TGF-β signaling. E1 knockdown drives cells into a migratory, invasive mesenchymal state and concomitantly up-regulates MHC IIB expression and MHC IIA phosphorylation. Abrogation of myosin IIB expression in the E1 knockdown cells has no effect on 2D migration but significantly reduced transmigration and macrophage-stimulated collagen invasion. These studies indicate that transition between myosin IIC/myosin IIB expression is a critical feature of EMT that contributes to increases in invasive behavior.

MTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases

Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases.

Establishment of a TGFβ-Induced Post-Transcriptional EMT Gene Signature

A major challenge in the clinical management of human cancers is to accurately stratify patients according to risk and likelihood of a favorable response. Stratification is confounded by significant phenotypic heterogeneity in some tumor types, often without obvious criteria for subdivision. Despite intensive transcriptional array analyses, the identity and validation of cancer specific ‘signature genes’ remains elusive, partially because the transcriptome does not mirror the proteome. The simplification associated with transcriptomic profiling does not take into consideration changes in the relative expression among transcripts that arise due to post-transcriptional regulatory events. We have previously shown that TGFβ post-transcriptionally regulates epithelial-mesenchymal transition (EMT) by causing increased expression of two transcripts, Dab2 and ILEI, by modulating hnRNP E1 phosphorylation. Using a genome-wide combinatorial approach involving expression profiling and RIP-Chip analysis, we have identified a cohort of translationally regulated mRNAs that are induced during TGFβ-mediated EMT. Coordinated translational regulation by hnRNP E1 constitutes a post-transcriptional regulon inhibiting the expression of related EMT-facilitating genes, thus enabling the cell to rapidly and coordinately regulate multiple EMT-facilitating genes.

NUDT21-spanning CNVs lead to neuropsychiatric disease and altered MeCP2 abundance via alternative polyadenylation

The brain is sensitive to the dose of MeCP2 such that small fluctuations in protein quantity lead to neuropsychiatric disease. Despite the importance of MeCP2 levels to brain function, little is known about its regulation. In this study, we report eleven individuals with neuropsychiatric disease and copy-number variations spanning NUDT21, which encodes a subunit of pre-mRNA cleavage factor Im. Investigations of MECP2 mRNA and protein abundance in patient-derived lymphoblastoid cells from one NUDT21 deletion and three duplication cases show that NUDT21 regulates MeCP2 protein quantity. Elevated NUDT21 increases usage of the distal polyadenylation site in the MECP2 3′ UTR, resulting in an enrichment of inefficiently translated long mRNA isoforms. Furthermore, normalization of NUDT21 via siRNA-mediated knockdown in duplication patient lymphoblasts restores MeCP2 to normal levels. Ultimately, we identify NUDT21 as a novel candidate for intellectual disability and neuropsychiatric disease, and elucidate a mechanism of pathogenesis by MeCP2 dysregulation via altered alternative polyadenylation. DOI: http://dx.doi.org/10.7554/eLife.10782.001

CELF1 is a central node in post-transcriptional regulatory programmes underlying EMT

The importance of translational regulation in tumour biology is increasingly appreciated. Here, we leverage polyribosomal profiling to prospectively define translational regulatory programs underlying epithelial-to-mesenchymal transition (EMT) in breast epithelial cells. We identify a group of ten translationally regulated drivers of EMT sharing a common GU-rich cis-element within the 3′-untranslated region (3′-UTR) of their mRNA. These cis-elements, necessary for the regulatory activity imparted by these 3′-UTRs, are directly bound by the CELF1 protein, which itself is regulated post-translationally during the EMT program. CELF1 is necessary and sufficient for both mesenchymal transition and metastatic colonization, and CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues. Our data present an 11-component genetic pathway, invisible to transcriptional profiling approaches, in which the CELF1 protein functions as a central node controlling translational activation of genes driving EMT and ultimately tumour progression.

NADPH oxidase promotes Parkinsonian phenotypes by impairing autophagic flux in an mTORC1-independent fashion in a cellular model of Parkinson's disease.

Oxidative stress and aberrant accumulation of misfolded proteins in the cytosol are key pathological features associated with Parkinson’s disease (PD). NADPH oxidase (Nox2) is upregulated in the pathogenesis of PD; however, the underlying mechanism(s) of Nox2-mediated oxidative stress in PD pathogenesis are still unknown. Using a rotenone-inducible cellular model of PD, we observed that a short exposure to rotenone (0.5 μM) resulted in impaired autophagic flux through activation of a Nox2 dependent Src/PI3K/Akt axis, with a consequent disruption of a Beclin1-VPS34 interaction that was independent of mTORC1 activity. Sustained exposure to rotenone at a higher dose (10 μM) decreased mTORC1 activity; however, autophagic flux was still impaired due to dysregulation of lysosomal activity with subsequent induction of the apoptotic machinery. Cumulatively, our results highlight a complex pathogenic mechanism for PD where short- and long-term oxidative stress alters different signaling pathways, ultimately resulting in anomalous autophagic activity and disease phenotype. Inhibition of Nox2-dependent oxidative stress attenuated the impaired autophagy and cell death, highlighting the importance and therapeutic potential of these pathways for treating patients with PD.

A piggyBac-based reporter system for scalable in vitro and in vivo analysis of 3′ untranslated region-mediated gene regulation

Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3′ untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcripts 3′ UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cells post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3′ UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome—a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3′ UTR of interest. The pBUTR (piggyBac-based 3′ UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting.

A powerful statistical approach for large-scale differential transcription analysis.

Next generation sequencing (NGS) is increasingly being used for transcriptome-wide analysis of differential gene expression. The NGS data are multidimensional count data. Therefore, most of the statistical methods developed well for microarray data analysis are not applicable to transcriptomic data. For this reason, a variety of new statistical methods based on count data of transcript reads have been correspondingly proposed. But due to high cost and limitation of biological resources, current NGS data are still generated from a few replicate libraries. Some of these existing methods do not always have desirable performances on count data. We here developed a very powerful and robust statistical method based on beta and binomial distributions. Our method (mBeta t-test) is specifically applicable to sequence count data from small samples. Both simulated and real transcriptomic data showed mBeta t-test significantly outperformed the existing top statistical methods chosen in all 12 given scenarios and performed with high efficiency and high stability. The differentially expressed genes found by our method from real transcriptomic data were validated by qPCR experiments. Our method shows high power in finding truly differential expression, conservatively estimating FDR and high stability in RNA sequence count data derived from small samples. Our method can also be extended to genome-wide detection of differential splicing events.

Abstract B58: CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells

The importance of translational regulation in tumorigenesis and metastasis is increasingly appreciated. We have leveraged polyribosomal profiling to prospectively and functionally define translational regulatory programs underlying the epithelial to mesenchymal transition (EMT), one of the early manifestations of metastasis, in breast epithelial cells. Our approach identified scores of mRNAs both enriched and depleted from polyribosomal fractions in mesenchymal cells as compared to their epithelial counterparts. Computational analysis of the 3′ untranslated regions (UTRs) of mRNAs enriched in polysomal fractions derived from the mesenchymal state revealed enrichment of a distinct GU-rich cis-element within these mRNA transcripts. Strikingly, while relative representation of these GU-rich-containing transcripts at the level of bulk mRNA were generally decreased in mesenchymal cells, the protein encoded by each of these mRNAs was dramatically increased. 3′ UTRs from affected transcripts conferred similar regulation upon a fluorescent reporter gene dependent upon the presence of the GU-rich element within the 3′ UTR sequence. Several of the translationally upregulated transcripts were necessary and/or sufficient for the EMT program. We next identified the CELF1 protein as a common regulator of these mRNA transcripts, demonstrating direct binding of the CELF1 protein to affected transcripts dependent upon the presence of the GU-rich element within their 3′ UTRs. CELF1 was itself necessary and sufficient for EMT in several distinct in vitro models as well as for metastatic colonization in vivo. Analysis of publicly available transcriptomic data revealed no increase in the relative expression of CELF19s regulatory targets or CELF1 itself in human breast cancer. We demonstrate that the CELF1 gene product is itself post-translationally regulated during EMT, and that CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues and correlates with disease progression. Cumulatively, our data present a distinctive model of gene regulation, invisible to traditional transcriptional profiling, in which the CELF1 protein serves as a central node controlling translational activation of the EMT program and ultimately tumor metastasis in human breast cancer. Citation Format: Arindam Chaudhury, Natee Kongchan, Joseph M. Fachini, Lukas M. Simon, Tao Wang, Sufeng Mao, Daniel G. Rosen, Michael M. Ittmann, Susan G. Hilsenbeck, Chad A. Shaw, Joel R. Neilson. CELF1 is a central node in post-transcriptional regulatory programs underlying EMT and metastasis in breast epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B58.