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17 pages, 2572 KiB

Open AccessArticle

pncCCND1_B Engages an Inhibitory Protein Network to Downregulate CCND1 Expression upon DNA Damage

by Ramona Palombo and Maria Paola Paronetto

Cancers 2022, 14(6), 1537; https://doi.org/10.3390/cancers14061537 - 17 Mar 2022

Cited by 4 | Viewed by 2061

Abstract

Promoter-associated noncoding RNAs (pancRNAs) represent a class of noncoding transcripts driven from the promoter region of protein-coding or non-coding genes that operate as cis-acting elements to regulate the expression of the host gene. PancRNAs act by altering the chromatin structure and recruiting transcription [...] Read more.

Promoter-associated noncoding RNAs (pancRNAs) represent a class of noncoding transcripts driven from the promoter region of protein-coding or non-coding genes that operate as cis-acting elements to regulate the expression of the host gene. PancRNAs act by altering the chromatin structure and recruiting transcription regulators. PncCCND1_B is driven by the promoter region of CCND1 and regulates CCND1 expression in Ewing sarcoma through recruitment of a multi-molecular complex composed of the RNA binding protein Sam68 and the DNA/RNA helicase DHX9. In this study, we investigated the regulation of CCND1 expression in Ewing sarcoma cells upon exposure to chemotherapeutic drugs. Pan-inhibitor screening indicated that etoposide, a drug used for Ewing sarcoma treatment, promotes transcription of pncCCND1_B and repression of CCND1 expression. RNA immunoprecipitation experiments showed increased binding of Sam68 to the pncCCND1_B after treatment, despite the significant reduction in DHX9 protein. This effect was associated with the formation of DNA:RNA duplexes at the CCND1 promoter. Furthermore, Sam68 interacted with HDAC1 in etoposide treated cells, thus contributing to chromatin remodeling and epigenetic changes. Interestingly, inhibition of the ATM signaling pathway by KU 55,933 treatment was sufficient to inhibit etoposide-induced Sam68-HDAC1 interaction without rescuing DHX9 expression. In these conditions, the DNA:RNA hybrids persist, thus contributing to the local chromatin inactivation at the CCND1 promoter region. Altogether, our results show an active role of Sam68 in DNA damage signaling and chromatin remodeling on the CCND1 gene by fine-tuning transitions of epigenetic complexes on the CCND1 promoter. Full article

(This article belongs to the Special Issue Alternative and Aberrant Splicing in Cancer: How Can We Fix It?)

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17 pages, 3368 KiB

Open AccessArticle

Identification and Characterization of Immunogene-Related Alternative Splicing Patterns and Tumor Microenvironment Infiltration Patterns in Breast Cancer

by Shuang Guo, Xinyue Wang, Hanxiao Zhou, Yue Gao, Peng Wang, Hui Zhi, Yue Sun, Yangyang Hao, Jing Gan, Yakun Zhang, Jie Sun, Wen Zheng, Xiaoxi Zhao, Yun Xiao and Shangwei Ning

Cancers 2022, 14(3), 595; https://doi.org/10.3390/cancers14030595 - 25 Jan 2022

Cited by 2 | Viewed by 2409

Abstract

Alternative splicing (AS) plays a crucial role in tumor development and tumor microenvironment (TME) formation. However, our current knowledge about AS, especially immunogene-related alternative splicing (IGAS) patterns in cancers, remains limited. Herein, we identified and characterized post-transcriptional mechanisms of breast cancer based on [...] Read more.

Alternative splicing (AS) plays a crucial role in tumor development and tumor microenvironment (TME) formation. However, our current knowledge about AS, especially immunogene-related alternative splicing (IGAS) patterns in cancers, remains limited. Herein, we identified and characterized post-transcriptional mechanisms of breast cancer based on IGAS, TME, prognosis, and immuno/chemotherapy. We screened the differentially spliced IGAS events and constructed the IGAS prognostic model (p-values < 0.001, AUC = 0.939), which could be used as an independent prognostic factor. Besides, the AS regulatory network suggested a complex cooperative or competitive relationship between splicing factors and IGAS events, which explained the diversity of splice isoforms. In addition, more than half of the immune cells displayed varying degrees of infiltration in the IGAS risk groups, and the prognostic characteristics of IGAS demonstrated a remarkable and consistent trend correlation with the infiltration levels of immune cell types. The IGAS risk groups showed substantial differences in the sensitivity of immunotherapy and chemotherapy. Finally, IGAS clusters defined by unsupervised cluster analysis had distinct prognostic patterns, suggesting an essential heterogeneity of IGAS events. Significant differences in immune infiltration and unique prognostic capacity of immune cells were also detected in each IGAS cluster. In conclusion, our comprehensive analysis remarkably enhanced the understanding of IGAS patterns and TME in breast cancer, which may help clarify the underlying mechanisms of IGAS in neoplasia and provide clues to molecular mechanisms of oncogenesis and progression. Full article

(This article belongs to the Special Issue Alternative and Aberrant Splicing in Cancer: How Can We Fix It?)

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21 pages, 5384 KiB

Open AccessArticle

The Estrogen Receptor α Signaling Pathway Controls Alternative Splicing in the Absence of Ligands in Breast Cancer Cells

by Jamal Elhasnaoui, Giulio Ferrero, Valentina Miano, Santina Cutrupi and Michele De Bortoli

Cancers 2021, 13(24), 6261; https://doi.org/10.3390/cancers13246261 - 13 Dec 2021

Cited by 8 | Viewed by 2695

Abstract

Background: The transcriptional activity of estrogen receptor α (ERα) in breast cancer (BC) is extensively characterized. Our group has previously shown that ERα controls the expression of a number of genes in its unliganded form (apoERα), among which a large group of RNA-binding [...] Read more.

Background: The transcriptional activity of estrogen receptor α (ERα) in breast cancer (BC) is extensively characterized. Our group has previously shown that ERα controls the expression of a number of genes in its unliganded form (apoERα), among which a large group of RNA-binding proteins (RBPs) encode genes, suggesting its role in the control of co- and post-transcriptional events. Methods: apoERα-mediated RNA processing events were characterized by the analysis of transcript usage and alternative splicing changes in an RNA-sequencing dataset from MCF-7 cells after siRNA-induced ERα downregulation. Results: ApoERα depletion induced an expression change of 681 RBPs, including 84 splicing factors involved in translation, ribonucleoprotein complex assembly, and 3′end processing. ApoERα depletion results in 758 isoform switching events with effects on 3′end length and the splicing of alternative cassette exons. The functional enrichment of these events shows that post-transcriptional regulation is part of the mechanisms by which apoERα controls epithelial-to-mesenchymal transition and BC cell proliferation. In primary BCs, the inclusion levels of the experimentally identified alternatively spliced exons are associated with overall and disease-free survival. Conclusion: Our data supports the role of apoERα in maintaining the luminal phenotype of BC cells by extensively regulating gene expression at the alternative splicing level. Full article

(This article belongs to the Special Issue Alternative and Aberrant Splicing in Cancer: How Can We Fix It?)

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18 pages, 4028 KiB

Open AccessArticle

Global Alternative Splicing Defects in Human Breast Cancer Cells

by Jagyeong Oh, Davide Pradella, Yoonseong Kim, Changwei Shao, Hairi Li, Namjeong Choi, Jiyeon Ha, Anna Di Matteo, Xiang-Dong Fu, Xuexiu Zheng, Claudia Ghigna and Haihong Shen

Cancers 2021, 13(12), 3071; https://doi.org/10.3390/cancers13123071 - 20 Jun 2021

Cited by 5 | Viewed by 2841

Abstract

Breast cancer is the most frequently occurred cancer type and the second cause of death in women worldwide. Alternative splicing (AS) is the process that generates more than one mRNA isoform from a single gene, and it plays a major role in expanding [...] Read more.

Breast cancer is the most frequently occurred cancer type and the second cause of death in women worldwide. Alternative splicing (AS) is the process that generates more than one mRNA isoform from a single gene, and it plays a major role in expanding the human protein diversity. Aberrant AS contributes to breast cancer metastasis and resistance to chemotherapeutic interventions. Therefore, identifying cancer-specific isoforms is the prerequisite for therapeutic interventions intended to correct aberrantly expressed AS events. Here, we performed RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) in breast cancer cells, to identify global breast cancer-specific AS defects. By RT-PCR validation, we demonstrate the high accuracy of RASL-seq results. In addition, we analyzed identified AS events using the Cancer Genome Atlas (TCGA) database in a large number of non-pathological and breast tumor specimens and validated them in normal and breast cancer samples. Interestingly, aberrantly regulated AS cassette exons in cancer tissues do not encode for known functional domains but instead encode for amino acids constituting regions of intrinsically disordered protein portions characterized by high flexibility and prone to be subjected to post-translational modifications. Collectively, our results reveal novel AS errors occurring in human breast cancer, potentially affecting breast cancer-related biological processes. Full article

(This article belongs to the Special Issue Alternative and Aberrant Splicing in Cancer: How Can We Fix It?)

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12 pages, 1576 KiB

Open AccessArticle

Opposite Roles of Tra2β and SRSF9 in the v10 Exon Splicing of CD44

by Jagyeong Oh, Yongchao Liu, Namjeong Choi, Jiyeon Ha, Davide Pradella, Claudia Ghigna, Xuexiu Zheng and Haihong Shen

Cancers 2020, 12(11), 3195; https://doi.org/10.3390/cancers12113195 - 30 Oct 2020

Cited by 8 | Viewed by 2095

Abstract

CD44 is a transmembrane glycoprotein involved in cell–cell and cell–matrix interactions. Several CD44 protein isoforms are generated in human through alternative splicing regulation of nine variable exons encoding for the extracellular juxta-membrane region. While the CD44 splicing variants have been described to be [...] Read more.

CD44 is a transmembrane glycoprotein involved in cell–cell and cell–matrix interactions. Several CD44 protein isoforms are generated in human through alternative splicing regulation of nine variable exons encoding for the extracellular juxta-membrane region. While the CD44 splicing variants have been described to be involved in cancer progression and development, the regulatory mechanism(s) underlying their production remain unclear. Here, we identify Tra2β and SRSF9 as proteins with opposite roles in regulating CD44 exon v10 splicing. While Tra2β promotes v10 inclusion, SRSF9 inhibits its inclusion. Mechanistically, we found that both proteins are able to target v10 exon, with GAAGAAG sequence being the binding site for Tra2β and AAGAC that for SRSF9. Collectively, our data add a novel layer of complexity to the sequential series of events involved in the regulation of CD44 splicing. Full article

(This article belongs to the Special Issue Alternative and Aberrant Splicing in Cancer: How Can We Fix It?)

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Review

Jump to: Research

19 pages, 4701 KiB

Open AccessReview

Intronic Polyadenylation in Acquired Cancer Drug Resistance Circumvented by Utilizing CRISPR/Cas9 with Homology-Directed Repair: The Tale of Human DNA Topoisomerase IIα

by Terry S. Elton, Victor A. Hernandez, Jessika Carvajal-Moreno, Xinyi Wang, Deborah Ipinmoroti and Jack C. Yalowich

Cancers 2022, 14(13), 3148; https://doi.org/10.3390/cancers14133148 - 27 Jun 2022

Cited by 3 | Viewed by 2615

Abstract

Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, progression, and cancer chemoresistance by contributing to transcriptome/proteome alterations. DNA topoisomerase IIα (170 kDa, TOP2α/170) is an established clinical target for anticancer agents whose efficacy is compromised by drug resistance often associated [...] Read more.

Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, progression, and cancer chemoresistance by contributing to transcriptome/proteome alterations. DNA topoisomerase IIα (170 kDa, TOP2α/170) is an established clinical target for anticancer agents whose efficacy is compromised by drug resistance often associated with a reduction of nuclear TOP2α/170 levels. In leukemia cell lines with acquired resistance to TOP2α-targeted drugs and reduced TOP2α/170 expression, variant TOP2α mRNA transcripts have been reported due to IPA that resulted in the translation of C-terminal truncated isoforms with altered nuclear-cytoplasmic distribution or heterodimerization with wild-type TOP2α/170. This review provides an overview of the various mechanisms regulating pre-mRNA processing and alternative polyadenylation, as well as the utilization of CRISPR/Cas9 specific gene editing through homology directed repair (HDR) to decrease IPA when splice sites are intrinsically weak or potentially mutated. The specific case of TOP2α exon 19/intron 19 splice site editing is discussed in etoposide-resistant human leukemia K562 cells as a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. This example supports the importance of aberrant IPA in acquired drug resistance to TOP2α-targeted drugs. In addition, these results demonstrate the therapeutic potential of CRISPR/Cas9/HDR to impact drug resistance associated with aberrant splicing/polyadenylation. Full article

(This article belongs to the Special Issue Alternative and Aberrant Splicing in Cancer: How Can We Fix It?)

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15 pages, 2282 KiB

Open AccessReview

A Quality Control Mechanism of Splice Site Selection Abrogated under Stress and in Cancer

by Maram Arafat and Ruth Sperling

Cancers 2022, 14(7), 1750; https://doi.org/10.3390/cancers14071750 - 30 Mar 2022

Cited by 3 | Viewed by 2275

Abstract

Latent 5’ splice sites, highly abundant in human introns, are not normally used. This led to the proposal of a quality control mechanism, Suppression of Splicing (SOS), which protects cells from splicing at the numerous intronic latent sites, and whose activation can generate [...] Read more.

Latent 5’ splice sites, highly abundant in human introns, are not normally used. This led to the proposal of a quality control mechanism, Suppression of Splicing (SOS), which protects cells from splicing at the numerous intronic latent sites, and whose activation can generate nonsense mRNAs. SOS was shown to be independent of Nonsense-Mediated mRNA Decay (NMD). Efforts to decipher the SOS mechanism revealed a pivotal role for initiator-tRNA, independent of protein translation. Recently, nucleolin (a multifunctional protein) was found to directly and specifically bind the initiator-tRNA in the nucleus and was shown to be a protein component of SOS, enabling an updated model of the SOS mechanism. Importantly, SOS is abrogated under stress and in cancer (e.g., in breast cancer cells and gliomas), generating thousands of nonsense mRNAs due to activation of latent splicing. The resulting affected human genes cover a variety of functional groups, including genes involved in cell proliferation and differentiation. Furthermore, in oligodendroglioma, the extent of activation of latent splicing increases with the severity of the cancer. Interesting examples are genes expressing aberrant nonsense mRNAs in both breast cancer and glioma, due to latent splicing activation. These findings highlight the unexplored potential of such aberrant isoforms as novel targets for cancer diagnosis and therapies. Full article

(This article belongs to the Special Issue Alternative and Aberrant Splicing in Cancer: How Can We Fix It?)

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22 pages, 1644 KiB

Open AccessReview

Alternative Splicing in Cancer and Immune Cells

by Antoine Bernard, Romain Boidot and Frédérique Végran

Cancers 2022, 14(7), 1726; https://doi.org/10.3390/cancers14071726 - 28 Mar 2022

Cited by 15 | Viewed by 4455

Abstract

Splicing is a phenomenon enabling the excision of introns from pre-mRNA to give rise to mature mRNA. All the 20,000 genes of the human genome are concerned by this mechanism. Nevertheless, it is estimated that the proteome is composed of more than 100,000 [...] Read more.

Splicing is a phenomenon enabling the excision of introns from pre-mRNA to give rise to mature mRNA. All the 20,000 genes of the human genome are concerned by this mechanism. Nevertheless, it is estimated that the proteome is composed of more than 100,000 proteins. How to go from 20,000 genes to more than 100,000 proteins? Alternative splicing (AS) is in charge of this diversity of proteins. AS which is found in most of the cells of an organism, participates in normal cells and in particular in immune cells, in the regulation of cellular behavior. In cancer, AS is highly dysregulated and involved in almost all of the hallmarks that characterize tumor cells. In view of the close link that exists between tumors and the immune system, we present in this review the literature relating to alternative splicing and immunotherapy. We also provide a global but not exhaustive view of AS in the immune system and tumor cells linked to the events that can lead to AS dysregulation in tumors. Full article

(This article belongs to the Special Issue Alternative and Aberrant Splicing in Cancer: How Can We Fix It?)

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