SummaryRegulatory T (Treg) cells are critical in regulating the immune response. In vitro induced Treg (iTreg) cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1) as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function.
Edited by Eric R. Fearon P. A. Jänne has received consulting fees from AstraZeneca, Boehringer Ingelheim, Pfizer, Merrimack Pharmaceuticals, Roche/Genentech, Chugai Pharmaceuticals, ACEA Biosciences, and Ariad Pharmaceuticals and sponsored research funding from Astellas Pharmaceuticals, AstraZeneca, Daiichi Sankyo, and PUMA and receives post-marketing royalties on DFCI-owned intellectual property on EGFR mutations licensed to Lab Corp. K. Elenius has a research agreement with Boehringer Ingelheim and ownership interest in Abomics, Orion, and Roche. This article was selected as one of our Editors' Picks. This article contains Figs. S1-S10.
SummaryThe RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs) and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.
BackgroundReceptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for expression or activity in the ischemic heart.MethodsRTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass, was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays.ResultsIn addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation.ConclusionsSeveral novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury.Electronic supplementary materialThe online version of this article (10.1186/s12872-018-0933-y) contains supplementary material, which is available to authorized users.
Background Prognostic markers specific to a particular cancer type can assist in the evaluation of survival probability of patients and help clinicians to assess the available treatment modalities. Methods Gene expression data was analyzed from three independent colon cancer microarray gene expression data sets ( N = 1052). Survival analysis was performed for the three data sets, stratified by the expression level of the LINE-1 type transposase domain containing 1 ( L1TD1 ). Correlation analysis was performed to investigate the role of the interactome of L1TD1 in colon cancer patients. Results We found L1TD1 as a novel positive prognostic marker for colon cancer. Increased expression of L1TD1 associated with longer disease-free survival in all the three data sets. Our results were in contrast to a previous study on medulloblastoma, where high expression of L1TD1 was linked with poor prognosis. Notably, in medulloblastoma L1TD1 was co-expressed with its interaction partners, whereas our analysis revealed lack of co-expression of L1TD1 with its interaction partners in colon cancer. Conclusions Our results identify increased expression of L1TD1 as a prognostic marker predicting longer disease-free survival in colon cancer patients. Electronic supplementary material The online version of this article (10.1186/s12885-019-5952-2) contains supplementary material, which is available to authorized users.
Although targeted therapies can be effective for a subgroup of patients, identification of individuals who benefit from the treatments is challenging. At the same time, the predictive significance of the majority of the thousands of mutations observed in the cancer tissues remains unknown. Here, we describe the identification of novel predictive biomarkers for ERBB-targeted tyrosine kinase inhibitors (TKIs) by leveraging the genetic and drug screening data available in the public cell line databases: Cancer Cell Line Encyclopedia, Genomics of Drug Sensitivity in Cancer, and Cancer Therapeutics Response Portal. We assessed the potential of 412 ERBB mutations in 296 cell lines to predict responses to 10 different ERBB-targeted TKIs. Seventy-six ERBB mutations were identified that were associated with ERBB TKI sensitivity comparable with non–small cell lung cancer cell lines harboring the well-established predictive EGFR L858R mutation or exon 19 deletions. Fourteen (18.4%) of these mutations were classified as oncogenic by the cBioPortal database, whereas 62 (81.6%) were regarded as novel potentially predictive mutations. Of the nine functionally validated novel mutations, EGFR Y1069C and ERBB2 E936K were transforming in Ba/F3 cells and demonstrated enhanced signaling activity. Mechanistically, the EGFR Y1069C mutation disrupted the binding of the ubiquitin ligase c-CBL to EGFR, whereas the ERBB2 E936K mutation selectively enhanced the activity of ERBB heterodimers. These findings indicate that integrating data from publicly available cell line databases can be used to identify novel, predictive nonhotspot mutations, potentially expanding the patient population benefiting from existing cancer therapies.
Therapeutic protocols including EGFR antibodies in the context of oxaliplatin-based regimens have variable clinical effect in colorectal cancer. Here, we tested the effect of the EGFR antibody cetuximab in different sequential combinations with oxaliplatin on the growth of colorectal cancer cells in vitro and in vivo. Cetuximab reduced the efficacy of oxaliplatin when administered before oxaliplatin but provided additive effect when administered after oxaliplatin regardless of the KRAS or BRAF mutation status of the cells. Systemic gene expression and protein phosphorylation screens revealed alternatively activated pathways regulating apoptosis, cell cycle and DNA damage response. Functional assays indicated that cetuximab-induced arrest of the cells into the G1 phase of the cell cycle was associated with reduced responsiveness of the cells to subsequent treatment with oxaliplatin. In contrast, oxaliplatin-enhanced responsiveness to subsequent treatment with cetuximab was associated with increased apoptosis, inhibition of STAT3 activity and increased EGFR down-regulation. This preclinical study indicates that optimizing the sequence of administration may enhance the antitumor effect of combination therapy with EGFR antibodies and oxaliplatin.
Introduction: Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites across different samples. The discovery of most of the currently known driver mutations has been facilitated by their accumulation in mutation hotspots within their respective genes. However, a vast majority of mutations in cancer tissues are rare and their functional significance remains unknown. Several lines of in vitro and clinical evidence also indicate that there is a significant number of, as yet unidentified, activating driver mutations which could serve as predictive markers in oncology. Here, we performed an unbiased functional screen to identify potential activating mutations of ERBB4, a frequently mutated member of the epidermal growth factor receptor family. Method: To identify functional driver mutations of ERBB4, the previously published pipeline, in vitro screen for activating mutations (iSCREAM) [1], was utilized. iSCREAM is a functional genetics screen based on the expression of random cDNA variants and the ability of driver mutations to promote cellular growth in vitro. The expression library encoding randomly mutated ERBB4 variants was retrovirally introduced into murine lymphoid Ba/F3 cells, that normally require interleukin-3 (IL-3) for survival but can exploit ectopic expression of activated variants of oncogenic kinases to compensate for the deficiency of exogenous IL-3. When expressed under an appropriate promoter, the wild-type ERBB4 receptor failed to promote IL-3-independent survival in the presence of the neuregulin-1 ligand, while the cells expressing activating mutations readily proliferated. The identity and frequency of the activating ERBB4 mutations were subsequently determined from the proliferating cell pool using targeted next-generation sequencing. Results: Ten candidate activating mutations were identified out of the over 7000 random ERBB4 missense or nonsense mutations present in the original library. The candidate activating mutations were individually characterized using functional assays both in vitro and in vivo, as well as by structural analyses. The activating mutation were sensitive to clinically used pan-ERBB tyrosine kinase inhibitors afatinib, dacomitinib and neratinib. Conclusions: A subset of ERBB4 missense mutations are activating and sensitive to tyrosine kinase inhibitor drugs. Citation Format: Deepankar Chakroborty, Veera K. Ojala, Anna M. Knittle, Kari J. Kurppa, Klaus Elenius. An unbiased in vitro screen for activating ERBB4 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 153.
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