Ping Wee

Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is commonly upregulated in cancers such as in non-small-cell lung cancer, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer. Various mechanisms mediate the upregulation of EGFR activity, including common mutations and truncations to its extracellular domain, such as in the EGFRvIII truncations, as well as to its kinase domain, such as the L858R and T790M mutations, or the exon 19 truncation. These EGFR aberrations over-activate downstream pro-oncogenic signaling pathways, including the RAS-RAF-MEK-ERK MAPK and AKT-PI3K-mTOR pathways. These pathways then activate many biological outputs that are beneficial to cancer cell proliferation, including their chronic initiation and progression through the cell cycle. Here, we review the molecular mechanisms that regulate EGFR signal transduction, including the EGFR structure and its mutations, ligand binding and EGFR dimerization, as well as the signaling pathways that lead to G1 cell cycle progression. We focus on the induction of CYCLIN D expression, CDK4/6 activation, and the repression of cyclin-dependent kinase inhibitor proteins (CDKi) by EGFR signaling pathways. We also discuss the successes and challenges of EGFR-targeted therapies, and the potential for their use in combination with CDK4/6 inhibitors.

Differential Regulation of Transcription Factors by Location-Specific EGF Receptor Signaling via a Spatio-Temporal Interplay of ERK Activation

It is well established that EGFR signals from both the plasma membrane (PM) and endosome (EN). However, very little is known about whether and how the EGFR signals at the PM and EN to differentially regulate various signaling pathways and the physiological outcomes. In this communication, we established a system that allowed the specific activations of EGFR at different cell locations: PM and EN. PM activation of EGFR is achieved by activation of endocytosis-deficient mutant EGFR1010LL/AA stably expressed in CHO cells (CHO-LL/AA cell). EN activation of EGFR is achieved by activating the wild type EGFR stably expressed in CHO cells (CHO-EGFR cell) after its internalization into EN with a previously reported protocol. We showed that both EGFR activations at PM and EN activated ERK to a similar level, but differentially stimulated transcriptional factors c-jun and c-fos. We further showed that EGFR activations at PM and EN resulted in differential spatio-temporal dynamics of phosphorylated ERK which caused the differential activation of two downstream substrates ELK1 and RSK. Finally we showed that EGFR signaling from PM and EN led to different physiological outcomes. CHO-LL/AA cells that only generate PM EGFR signals have a larger cell size and slower proliferation rate than CHO-EGFR cells. We conclude that location-specific EGFR activation differentially regulates cell functions through a spatio-temporal interplay of ERK activation.

EGF stimulates the activation of EGF receptors and the selective activation of major signaling pathways during mitosis

Mitosis and epidermal growth factor (EGF) receptor (EGFR) are both targets for cancer therapy. The role of EGFR signaling in mitosis has been rarely studied and poorly understood. The limited studies indicate that the activation of EGFR and downstream signaling pathways is mostly inhibited during mitosis. However, we recently showed that EGFR is phosphorylated in response to EGF stimulation in mitosis. Here we studied EGF-induced EGFR activation and the activation of major signaling pathways downstream of EGFR during mitosis. We showed that EGFR was strongly activated by EGF during mitosis as all the five major tyrosine residues including Y992, Y1045, Y1068, Y1086, and Y1173 were phosphorylated to a level similar to that in the interphase. We further showed that the activated EGFR is able to selectively activate some downstream signaling pathways while avoiding others. Activated EGFR is able to activate PI3K and AKT2, but not AKT1, which may be responsible for the observed effects of EGF against nocodazole-induced cell death. Activated EGFR is also able to activate c-Src, c-Cbl and PLC-γ1 during mitosis. However, activated EGFR is unable to activate ERK1/2 and their downstream substrates RSK and Elk-1. While it activated Ras, EGFR failed to fully activate Raf-1 in mitosis due to the lack of phosphorylation at Y341 and the lack of dephosphorylation at pS259. We conclude that contrary to the dogma, EGFR is activated by EGF during mitosis. Moreover, EGFR-mediated cell signaling is regulated differently from the interphase to specifically serve the needs of the cell in mitosis.

Involvement of Polycomb Repressive Complex 2 in Maturation of Induced Pluripotent Stem Cells during Reprogramming of Mouse and Human Fibroblasts.

Induced pluripotent stem cells (iPSCs) provide a reliable source for the study of regenerative medicine, drug discovery, and developmental biology. Despite extensive studies on the reprogramming of mouse and human fibroblasts into iPSCs, the efficiency of reprogramming is still low. Here, we used a bioinformatics and systems biology approach to study the two gene regulatory waves governing the reprogramming of mouse and human fibroblasts into iPSCs. Our results revealed that the maturation phase of reprogramming was regulated by a more complex regulatory network of transcription factors compared to the initiation phase. Interestingly, in addition to pluripotency factors, the polycomb repressive complex 2 (PRC2) members Ezh2, Eed, Jarid2, Mtf2, and Suz12 are crucially recruited during the maturation phase of reprogramming. Moreover, we found that during the maturation phase of reprogramming, pluripotency factors, via the expression and induction of PRC2 complex members, could silence the lineage-specific gene expression program and maintain a ground state of pluripotency in human and mouse naïve iPSCs. The findings obtained here provide us a better understanding of the gene regulatory network (GRN) that governs reprogramming, and the maintenance of the naïve state of iPSCs.

Meta-Analysis of Transcriptome Regulation During Induction to Cardiac Myocyte Fate from Mouse and Human Fibroblasts.

Ectopic expression of a defined set of transcription factors (TFs) can directly convert fibroblasts into a cardiac myocyte cell fate. Beside inefficiency in generating induced cardiomyocytes (iCMs), the molecular mechanisms that regulate this process remained to be well defined. The main purpose of this study was to provide better insight on the transcriptome regulation and to introduce a new strategy for candidating TFs for the transdifferentiation process. Eight mouse and three human high quality microarray data sets were analyzed to find differentially expressed genes (DEGs), which we integrated with TF‐binding sites and protein–protein interactions to construct gene regulatory and protein–protein interaction networks. Topological and biological analyses of constructed gene networks revealed the main regulators and most affected biological processes. The DEGs could be categorized into two distinct groups, first, up‐regulated genes that are mainly involved in cardiac‐specific processes and second, down‐regulated genes that are mainly involved in fibroblast‐specific functions. Gata4, Mef2a, Tbx5, Tead4 TFs were identified as main regulators of cardiac‐specific gene expression program; and Trp53, E2f1, Myc, Sfpi1, Lmo2, and Meis1 were identified as TFs which mainly regulate the expression of fibroblast‐specific genes. Furthermore, we compared gene expression profiles and identified TFs between mouse and human to find the similarities and differences. In summary, our strategy of meta‐analyzing the data of high‐throughput techniques by computational approaches, besides revealing the mechanisms involved in the regulation of the gene expression program, also suggests a new approach for increasing the efficiency of the direct reprogramming of fibroblasts into iCMs. J. Cell. Physiol. 232: 2053–2062, 2017.

Role of Hepatic-Specific Transcription Factors and Polycomb Repressive Complex 2 during Induction of Fibroblasts to Hepatic Fate.

Direct reprogramming using defined sets of transcription factors (TFs) is a recent strategy for generating induced hepatocytes (iHeps) from fibroblasts for use in regenerative medicine and drug development. Comprehensive studies detailing the regulatory role of TFs during this reprogramming process could help increase its efficiency. This study aimed to find the TFs with the greatest influences on the generation of iHeps from fibroblasts, and to further understand their roles in the regulation of the gene expression program. Here, we used systems biology approaches to analyze high quality expression data sets in combination with TF-binding sites data and protein-protein interactions data during the direct reprogramming of fibroblasts to iHeps. Our results revealed two main patterns for differentially expressed genes (DEGs): up-regulated genes were categorized as hepatic-specific pattern, and down-regulated genes were categorized as mesoderm- and fibroblast-specific pattern. Interestingly, hepatic-specific genes co-expressed and were regulated by hepatic-specific TFs, specifically Hnf4a and Foxa2. Conversely, the mesoderm- and fibroblast-specific pattern was mainly silenced by polycomb repressive complex 2 (PRC2) members, including Suz12, Mtf2, Ezh2, and Jarid2. Independent analysis of both the gene and core regulatory network of DE-TFs showed significant roles for Hnf4a, Foxa2, and PRC2 members in the regulation of the gene expression program and in biological processes during the direct conversion process. Altogether, using systems biology approaches, we clarified the role of Hnf4a and Foxa2 as hepatic-specific TFs, and for the first time, introduced the PRC2 complex as the main regulator that favors the direct reprogramming process in cooperation with hepatic-specific factors.

Regulating the transcriptomes that mediate the conversion of fibroblasts to various nervous system neural cell types

Our understanding of the mechanism of cell fate transition during the direct reprogramming of fibroblasts into various central nervous system (CNS) neural cell types has been limited by the lack of a comprehensive analysis on generated cells, independently and in comparison with other CNS neural cell types. Here, we applied an integrative approach on 18 independent high throughput expression data sets to gain insight into the regulation of the transcriptome during the conversion of fibroblasts into induced neural stem cells, induced neurons (iNs), induced astrocytes, and induced oligodendrocyte progenitor cells (iOPCs). We found common down‐regulated genes to be mostly related to fibroblast‐specific functions, and suggest their potential as markers for screening of the silencing of the fibroblast‐specific program. For example, Tagln was significantly down‐regulated across all considered data sets. In addition, we identified specific profiles of up‐regulated genes for each CNS neural cell types, which could be potential markers for maturation and efficiency screenings. Furthermore, we identified the main TFs involved in the regulation of the gene expression program during direct reprogramming. For example, in the generation of iNs from fibroblasts, the Rest TF was the main regulator of this reprogramming. In summary, our computational approach for meta‐analyzing independent expression data sets provides significant details regarding the molecular mechanisms underlying the regulation of the gene expression program, and also suggests potentially useful candidate genes for screening down‐regulation of fibroblast gene expression profile, maturation, and efficiency, as well as candidate TFs for increasing the efficiency of the reprogramming process.

Common gene expression patterns in the generation of induced neurons from fibroblasts

In the current study, we analyzed ten gene expression data sets including RNA-sequencing and microarray experiment data during the direct reprogramming of mouse and human fibroblasts to induced neurons and found common gene expression pattern across all data sets for this conversion.

Concerns on “Dissection of Regulatory Elements During Direct Conversion of Somatic Cells Into Neurons” Paper: CONCERNS ON DISSECTION OF REGULATORY ELEMENTS

Direct Conversion of Somatic Cells Into Neurons” Paper Moein Yaqubi,* Abdulshakour Mohammadnia ,** and Ping Wee Department of Psychiatry, Sackler Program for Epigenetics and Psychobiology at McGill University, Ludmer Centre for Neuroinformatics and Mental Health, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada Department of Human Genetics, Faculty of Medicine, McGill University, Montreal, Quebec, Canada Department of Medical Genetics and Signal Transduction Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada

Comprehensive transcriptome mining of the direct conversion of mesodermal cells

The direct reprogramming of somatic cells is a promising approach for regenerative medicine, especially in the production of mesoderm layer-derived cells. Meta-analysis studies provide precise insight into the undergoing processes and help increase the efficiency of reprogramming. Here, using 27 high-throughput expression data sets, we analyzed the direct reprogramming of mesodermal cells in humans and mice. Fibroblast-derived cells showed a common expression pattern of up- and down-regulated genes that were mainly involved in the suppression of the fibroblast-specific gene expression program, and may be used as markers of the initiation of reprogramming. Furthermore, we found a specific gene expression profile for each fibroblast-derived cell studied, and each gene set appeared to play specific functional roles in its cell type, suggesting their use as markers for their mature state. Furthermore, using data from protein-DNA interactions, we identified the main transcription factors (TFs) involved in the conversion process and ranked them based on their importance in their gene regulatory networks. In summary, our meta-analysis approach provides new insights on the direct conversion of mesodermal somatic cells, introduces a list of genes as markers for initiation and maturation, and identifies TFs for which manipulating their expression may increase the efficiency of direct conversion.