Cell-type-specific alternative polyadenylation as a therapeutic biomarker in lung cancer progression
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Lung cancer stands as the leading cause of global cancer-related mortality, resulting in an approximate annual toll of 1.6 million lives.1Siegel R.L. Miller K.D. Wagle N.S. Jemal A. Cancer statistics, 2023.CA A Cancer J. Clin. 2023; 73: 17-48Crossref PubMed Scopus (1119) Google Scholar Within this context, non-small cell lung cancer (NSCLC) comprises about 85% of cases, encompassing a range of histological subtypes.2Petrella F. Rizzo S. Attili I. Passaro A. Zilli T. Martucci F. Bonomo L. Del Grande F. Casiraghi M. De Marinis F. Spaggiari L. Stage III Non-Small-Cell Lung Cancer: An Overview of Treatment Options.Curr. Oncol. 2023; 30: 3160-3175Crossref PubMed Scopus (2) Google Scholar Given the intricate genetic modifications underlying the emergence and progression of NSCLC, extensive gene expression profiling has pinpointed a multitude of dysregulated genes.3Rotow J. Bivona T.G. Understanding and targeting resistance mechanisms in NSCLC.Nat. Rev. Cancer. 2017; 17: 637-658Crossref PubMed Scopus (571) Google Scholar These discoveries have significantly informed clinical prognostications and predictions of therapeutic responsiveness. An array of mechanisms, including exon deletion, copy-number variation, and epigenetic modification, have been linked to the disruption of gene expression profiles, as demonstrated by numerous studies.4Xue Y. Hou S. Ji H. Han X. Evolution from genetics to phenotype: reinterpretation of NSCLC plasticity, heterogeneity, and drug resistance.Protein Cell. 2017; 8: 178-190Crossref PubMed Scopus (16) Google Scholar Nonetheless, the comprehensive understanding of the driving forces behind gene expression dysregulation in NSCLC remains an ongoing pursuit. Alternative polyadenylation (APA) has been identified as one of the drivers of aberrant gene expression. Extensive APA events were found in multiple cancer types, which could regulate the expression of oncogenes and tumor suppressors. Accumulated evidence indicates that many APA events are cancer-type and cell-state specific. In a recent issue of Molecular Therapy – Nucleic Acids, Huang et al. describe the identification of heterogeneity of alternative polyadenylation events that occurs in multiple cell types by using bioinformatics pipeline and cell line experiments.5Huang K. Zhang Y. Shi X. Yin Z. Zhao W. Huang L. Wang F. Zhou X. Cell-type specific alternative polyadenylation promotes oncogenic gene expression in non-small cell lung cancer progression.Mol. Ther. Nucleic Acids. 2023; 33: 816-831Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar In the current study, Huang et al. collected single-cell RNA sequencing data from previously published studies. Then, the authors systematically analyzed APA events in over 40,000 cells, identifying broadly dysregulated APA events across seven distinct cell types. To ascertain the potential implications of these APA events, the authors pinpointed the loss of microRNA (miRNA)-binding sites resulting from shortened 3′ UTRs, as well as the influence of APA-mediated miRNA regulation. Furthermore, the authors discerned the functional significance of APA-associated genes and validated their roles in cancer cell migration and metastasis. In a bid to explore potential interactions with APA-related genes, the authors conducted drug sensitivity tests on lung cancer cell lines using the Genomics of Drug Sensitivity in Cancer (GDSC) database (Figure 1). According to the analyses, Huang et al. identified widespread APA events in NSCLC across various cell types. Among these events, many are specific to certain cell types and regulate the expression of oncogenes. For instance, the authors noted significant upregulation of eight genes in four distinct cell types in NSCLC samples: SPARC, RGS5, and CD59 in cancer-associated fibroblasts (CAFs), IL1RN in myeloid cells, TMBIM6 in alveolar cells, and RPL22, DERL1, and HM13 in B cells. Notably, the expression of SPARC displayed a robust correlation with patient prognosis. Subsequently, the authors explored the patterns of miRNA-binding site loss induced by APA events in these seven cell types. Prior research had already highlighted the loss of multiple miRNA-binding sites due to APA events. In line with these findings, the authors revealed the widespread occurrence of miRNA-binding site loss in genes affected by APA events. As an example, both bioinformatic analysis and experiments demonstrated the loss of the miR-203a-3p.1-binding site due to APA events in SPARC. It was also shown that miR-203a-3p.1 significantly suppressed the expression of SPARC. Furthermore, the evasion of miR-203a-3p.1 led to the inhibition of tumor-suppressor genes SOCS3 and ETS2. Additionally, the authors uncovered interactions between SPARC and genes regulated by epithelial-mesenchymal transition (EMT), such as COL1A1, TGFBR2, and VCAM1. These interactions are strongly correlated with cancer progression and metastasis. The study then confirmed the role of SPARC in lung cancer proliferation and migration through experiments involving SPARC knockdown cell lines. Drug response analysis also revealed that patients with high SPARC expression levels displayed greater sensitivity to cisplatin treatment compared with the low-SPARC-expression group. Therefore, the analysis suggested that patients with high SPARC expression might derive more benefits from cisplatin treatment. The innovation of cell-type-specific APA represents a significant advancement in the field of NSCLC. Currently, our understanding of dynamic APA regulation in different cell types remains rudimentary. The heterogeneity of APA in distinct cell types may yield valuable insights into the mechanisms of carcinogenesis, progression, and metastasis. While SPARC has been extensively studied in various cancer cell lines, its role in tumorigenesis remains controversial due to its highly cell-type-specific functions.6Arnold S.A. Brekken R.A. SPARC: a matricellular regulator of tumorigenesis.J. Cell Commun. Signal. 2009; 3: 255-273Crossref PubMed Scopus (131) Google Scholar A previous study demonstrated that the low expression of SPARC in lung cancer cells results from its aberrant methylation.7Suzuki M. Hao C. Takahashi T. Shigematsu H. Shivapurkar N. Sathyanarayana U.G. Iizasa T. Fujisawa T. Hiroshima K. Gazdar A.F. Aberrant methylation of SPARC in human lung cancers.Br. J. Cancer. 2005; 92: 942-948Crossref PubMed Scopus (62) Google Scholar However, the cell-type-specific function of SPARC in NSCLC remains unknown. This study not only broadens our understanding of the underlying biological mechanisms of NSCLC but also identifies potential targets for predicting the response to cisplatin treatment in patients with NSCLC. Future research should aim to optimize the therapeutic potential of SPARC in CAFs, uncover its response to other therapies, and comprehensively assess its efficacy as a drug target in mouse models and preclinical trials. In conclusion, this study provides novel insights into the mechanisms underpinning drug resistance in patients with NSCLC. All authors actively contributed to the writing of this commentary. The authors declare no competing interests.Polyadenylation is a 3' end RNA processing event that contributes to the regulation of gene expression by affecting turnover, stability, export, and translation of mRNA. Approximately 53 % of all human genes have multiple polyadenylation sites (PAS). Regulation at the level of alternative polyadenylation has begun to be investigated, but still requires much more elucidation. Bioinformatics has identified unique alternative polyadenylation of a polyadenylation factor mRNA, poly (A) binding protein nuclear 1 (PABPN1). The mRNAs of this polyadenylation factor contains multiple polyadenylation sites. Two polyadenylation signals were found for the PABPN1 mRNA. The proximal PAS is predicted from bioinformatics to be the strong polyadenylation site. The distal PAS was suggested to be a weaker site. However, we found the distal PAS contains putative novel auxiliary upstream polyadenylation elements. There are two G‐rich elements found in the upstream region of PABPN1 mRNA that are very highly conserved, suggestive of important function. Mutations of these two G‐rich elements seem to exhibit opposing effects on polyadenylation. Previous studies from other labs have shown that G‐rich elements located downstream of polyadenylation sites enhance cleavage, and affect polyadenylation signal usage, but remains unexplored when upstream. Research supported by NIH and the Foundation of UMDNJ grants.
Post-transcriptional modification
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Post-transcriptional modification
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Almost all eukaryotic mRNAs possess 3' ends with a polyadenylate (poly(A)) tail. This poly(A) tail is not encoded in the genome but is added by the process of polyadenylation. Polyadenylation is a two-step process, and this process is accomplished by multisubunit protein factors. Here, we comprehensively compare the protein machinery responsible for polyadenylation of mRNAs across many evolutionary divergent species, and we have found these protein factors to be remarkably conserved in nature. These data suggest that polyadenylation of mRNAs is an ancient process.
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Messenger RNA polyadenylation is one of the key post-transcriptional events in eukaryotic cells. A large number of genes in mammalian species can undergo alternative polyadenylation, which leads to mRNAs with variable 3′ ends. As the 3′ end of mRNAs often contains cis elements important for mRNA stability, mRNA localization and translation, the implications of the regulation of polyadenylation can be multifold. Alternative polyadenylation is controlled by cis elements and trans factors, and is believed to occur in a tissue- or disease-specific manner. Given the availability of many databases devoted to other aspects of mRNA metabolism, such as transcriptional initiation and splicing, systematic information on polyadenylation, including alternative polyadenylation and its regulation, is noticeably lacking. Here, we present a database named polyA_DB, through which we strive to provide several types of information regarding polyadenylation in mammalian species: (i) polyadenylation sites and their locations with respect to the genomic structure of genes; (ii) cis elements surrounding polyadenylation sites; (iii) comparison of polyadenylation configuration between orthologous genes; and (iv) tissue/organ information for alternative polyadenylation sites. Currently, polyA_DB contains 45 565 polyadenylation sites for 25 097 human and mouse genes, representing the most comprehensive polyadenylation database till date. The database is accessible via the website ( http://polya.umdnj.edu/polyadb ).
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Alternative initiation, splicing, and polyadenylation are key mechanisms used by many organisms to generate diversity among mature mRNA transcripts originating from the same transcription unit. While previous computational analyses of alternative polyadenylation have focused on polyadenylation activities within or downstream of the normal 3′-terminal exons, we present the results of the first genome-wide analysis of patterns of alternative polyadenylation in the human, mouse, and rat genomes occurring over the entire transcribed regions of mRNAs using 3′-ESTs with poly(A) tails aligned to genomic sequences. Four distinct classes of patterns of alternative polyadenylation result from this analysis: tandem poly(A) sites, composite exons, hidden exons, and truncated exons. We estimate that at least 49% (human), 31% (mouse), and 28% (rat) of polyadenylated transcription units have alternative polyadenylation. A portion of these alternative polyadenylation events result in new protein isoforms.
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In animal and viral pre-mRNAS, the process of polyadenylation is mediated through several cis-acting poly (A) signals present upstream and downstream from poly (A) sites. The situation regarding polyadenylation of higher plant pre-mRNAS, however, has remained obscure so far. In this paper, a search for putative poly (A) signals is made by considering the published data from 46 plant genomic DNA sequences. Certain domains in the 3′ untranslated regions from nuclear genes of higher plants were compiled and occurrence of sequence motifs such as AATAAA, CAYTG, YGTGTTYY and YAYTG was scored in relation to poly (A) sites. Moreover, consensus sequences for important regions in the 3′ untranslated sequences and poly (A) signals were also deduced from the data. It was inferred that sequence motifs similar to poly (A) signals exist around poly (A) sites but some of them are in entirely different spatial relationship than observed in other eukaryotes. This indicates their probable non-involvement in the process of polyadenylation in higher plants necessitating a functional analysis approach to define the plant specific poly (A) signals.
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RNA polyadenylation is an important step in the messenger RNA (mRNA) maturation process, and the first step is recognizing the polyadenylation signal (PAS). The PAS type and distribution is a key determinant of post-transcriptional mRNA modification and gene expression. However, little is known about PAS usage and alternative polyadenylation (APA) regulation in livestock species. Recently, sequencing technology has enabled the generation of a large amount of sequencing data revealing variation in poly(A) signals and APA regulation in Sus scrofa. We identified 62,491 polyadenylation signals in Sus scrofa using expressed sequence tag (EST) sequences combined with RNA-seq analysis. The composition and usage frequency of polyadenylation signal in Sus scrofa is similar with that of human and mouse. The most highly conserved polyadenylation signals are AAUAAA and AUUAAA, used for over 63.35% of genes. In addition, we also analyzed the U/GU-rich downstream sequence (DSE) element, located downstream of the cleavage site. Our results indicate that APA regulation was widely occurred in Sus scrofa, as in other organisms. Our result was useful for the accurate annotation of RNA 3' ends in Sus scrofa and the analysis of polyadenylation signal usage in Sus scrofa would give the new insights into the mechanisms of transcriptional regulation.
Post-transcriptional modification
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