ABSTRACT
Chromatin modifiers are emerging as major determinants of many types of cancers, including Anaplastic Large Cell Lymphomas (ALCL), a family of highly heterogeneous T-cell lymphomas for which therapeutic options are still limited. HELLS is a multifunctional chromatin remodeling protein that affects genomic instability by participating in the DNA damage response. Although the transcriptional function of HELLS has been suggested, no clues on how HELLS controls transcription are currently available. In this study, by integrating different multi-omics and functional approaches, we characterized the transcriptional landscape of HELLS in ALCL. We explored the clinical impact of its transcriptional program in a large cohort of 44 patients with ALCL. We demonstrated that HELLS, loaded at the level of intronic regions of target promoters, facilitates RNA Polymerase II (RNAPII) progression along the gene bodies by reducing the persistence of co-transcriptional R-loops and promoting DNA damage resolution. Importantly, selective knockdown of HELLS sensitizes ALCL cells to different chemotherapeutic agents, showing a synergistic effect. Collectively, our work unveils the role of HELLS in acting as a gatekeeper of ALCL genome stability providing a rationale for drug design.
Subject(s)
DNA Damage , R-Loop Structures , RNA Polymerase II , Transcription, Genetic , Humans , RNA Polymerase II/metabolism , Cell Line, Tumor , Genomic Instability/genetics , Lymphoma, Large-Cell, Anaplastic/genetics , Lymphoma, Large-Cell, Anaplastic/pathology , Lymphoma, Large-Cell, Anaplastic/metabolism , Gene Expression Regulation, Neoplastic , DNA Helicases/genetics , DNA Helicases/metabolism , Promoter Regions, Genetic , Lymphoma, T-Cell/genetics , Lymphoma, T-Cell/metabolism , Lymphoma, T-Cell/pathologyABSTRACT
Long non-coding RNA (lncRNA) are emerging as powerful and versatile regulators of transcriptional programs and distinctive biomarkers of progression of T-cell lymphoma. Their role in the aggressive anaplastic lymphoma kinase-negative (ALK-) subtype of anaplastic large cell lymphoma (ALCL) has been elucidated only in part. Starting from our previously identified ALCL-associated lncRNA signature and performing digital gene expression profiling of a retrospective cohort of ALCL, we defined an 11 lncRNA signature able to discriminate among ALCL subtypes. We selected a not previously characterized lncRNA, MTAAT, with preferential expression in ALK- ALCL, for molecular and functional studies. We demonstrated that lncRNA MTAAT contributes to an aberrant mitochondrial turnover restraining mitophagy and promoting cellular proliferation. Functionally, lncRNA MTAAT acts as a repressor of a set of genes related to mitochondrial quality control via chromatin reorganization. Collectively, our work demonstrates the transcriptional role of lncRNA MTAAT in orchestrating a complex transcriptional program sustaining the progression of ALK- ALCL.
Subject(s)
Lymphoma, Large-Cell, Anaplastic , Lymphoma, T-Cell, Peripheral , RNA, Long Noncoding , Humans , Receptor Protein-Tyrosine Kinases/genetics , Anaplastic Lymphoma Kinase/genetics , RNA, Long Noncoding/genetics , Mitophagy/genetics , Retrospective Studies , Lymphoma, Large-Cell, Anaplastic/pathologyABSTRACT
Nonsense mutations cause several genetic diseases such as cystic fibrosis, Duchenne muscular dystrophy, ß-thalassemia, and Shwachman-Diamond syndrome. These mutations induce the formation of a premature termination codon (PTC) inside the mRNA sequence, resulting in the synthesis of truncated polypeptides. Nonsense suppression therapy mediated by translational readthrough-inducing drugs (TRIDs) is a promising approach to correct these genetic defects. TRIDs generate a ribosome miscoding of the PTC named "translational readthrough" and restore the synthesis of full-length and potentially functional proteins. The new oxadiazole-core TRIDs NV848, NV914, and NV930 (NV) showed translational readthrough activity in nonsense-related in vitro systems. In this work, the possible off-target effect of NV molecules on natural termination codons (NTCs) was investigated. Two different in vitro approaches were used to assess if the NV molecule treatment induces NTC readthrough: (1) a study of the translational-induced p53 molecular weight and functionality; (2) the evaluation of two housekeeping proteins' (Cys-C and ß2M) molecular weights. Our results showed that the treatment with NV848, NV914, or NV930 did not induce any translation alterations in both experimental systems. The data suggested that NV molecules have a specific action for the PTCs and an undetectable effect on the NTCs.
Subject(s)
Genes, Essential , Tumor Suppressor Protein p53 , Codon, Terminator , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Protein Biosynthesis , Codon, NonsenseABSTRACT
[This corrects the article DOI: 10.3389/fonc.2023.1257622.].
ABSTRACT
Antisense RNAs (asRNAs) represent an underappreciated yet crucial layer of gene expression regulation. Generally thought to modulate their sense genes in cis through sequence complementarity or their act of transcription, asRNAs can also regulate different molecular targets in trans, in the nucleus or in the cytoplasm. Here, we performed an in-depth molecular characterization of NFYC Antisense 1 (NFYC-AS1), the asRNA transcribed head-to-head to NFYC subunit of the proliferation-associated NF-Y transcription factor. Our results show that NFYC-AS1 is a prevalently nuclear asRNA peaking early in the cell cycle. Comparative genomics suggests a narrow phylogenetic distribution, with a probable origin in the common ancestor of mammalian lineages. NFYC-AS1 is overexpressed pancancer, preferentially in association with RB1 mutations. Knockdown of NFYC-AS1 by antisense oligonucleotides impairs cell growth in lung squamous cell carcinoma and small cell lung cancer cells, a phenotype recapitulated by CRISPR/Cas9-deletion of its transcription start site. Surprisingly, expression of the sense gene is affected only when endogenous transcription of NFYC-AS1 is manipulated. This suggests that regulation of cell proliferation is at least in part independent of the in cis transcription-mediated effect on NFYC and is possibly exerted by RNA-dependent in trans effects converging on the regulation of G2/M cell cycle phase genes. Accordingly, NFYC-AS1-depleted cells are stuck in mitosis, indicating defects in mitotic progression. Overall, NFYC-AS1 emerged as a cell cycle-regulating asRNA with dual action, holding therapeutic potential in different cancer types, including the very aggressive RB1-mutated tumors.
Subject(s)
Lung Neoplasms , RNA, Long Noncoding , Animals , Humans , Phylogeny , Gene Expression Regulation, Neoplastic , RNA, Antisense/genetics , Cell Cycle/genetics , Cell Proliferation/genetics , Lung Neoplasms/genetics , RNA, Long Noncoding/genetics , Cell Line, Tumor , Cell Movement , Mammals/genetics , CCAAT-Binding Factor/geneticsABSTRACT
Malignant pleural mesothelioma is a rare and lethal cancer caused by exposure to asbestos. The highly inflammatory environment caused by fibers accumulation forces cells to undergo profound adaptation to gain survival advantages. Prioritizing the synthesis of essential transcripts is an efficient mechanism coordinated by multiple molecules, including long non-coding RNAs. Enhancing the knowledge about these mechanisms is an essential weapon in combating mesothelioma. Linc00941 correlates to bad prognosis in various cancers, but it is reported to partake in distinct and apparently irreconcilable processes. In this work, we report that linc00941 supports the survival and aggressiveness of mesothelioma cells by influencing protein synthesis and ribosome biogenesis. Linc00941 binds to the translation initiation factor eIF4G, promoting the selective protein synthesis of cMYC, which, in turn, enhances the expression of key genes involved in translation. We analyzed a retrospective cohort of 97 mesothelioma patients' samples from our institution, revealing that linc00941 expression strongly correlates with reduced survival probability. This discovery clarifies linc00941's role in mesothelioma and proposes a unified mechanism of action for this lncRNA involving the selective translation of essential oncogenes, reconciling the discrepancies about its function.
Subject(s)
Eukaryotic Initiation Factor-4G , Gene Expression Regulation, Neoplastic , Lung Neoplasms , Mesothelioma, Malignant , Mesothelioma , Protein Biosynthesis , Proto-Oncogene Proteins c-myc , RNA, Long Noncoding , Humans , Mesothelioma, Malignant/genetics , Mesothelioma, Malignant/pathology , Mesothelioma, Malignant/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Eukaryotic Initiation Factor-4G/genetics , Eukaryotic Initiation Factor-4G/metabolism , Mesothelioma/genetics , Mesothelioma/pathology , Mesothelioma/metabolism , Cell Line, Tumor , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Pleural Neoplasms/genetics , Pleural Neoplasms/pathology , Pleural Neoplasms/metabolism , Ribosomes/metabolism , Ribosomes/genetics , Retrospective Studies , Prognosis , Cell ProliferationABSTRACT
The extracellular matrix (ECM) modulates cell behavior, shape, and viability as well as mechanical properties. In recent years, ECM disregulation and aberrant remodeling has gained considerable attention in cancer targeting and prevention since it may stimulate tumorigenesis and metastasis. Here, we developed an in vitro model that aims at mimicking the in vivo tumor microenvironment by recapitulating the interactions between osteosarcoma (OS) cells and ECM with respect to cancer progression. We long-term cultured 3D OS spheroids made of metastatic or non-metastatic OS cells mixed with mesenchymal stromal cells (MSCs); confirmed the deposition of ECM proteins such as Type I collagen, Type III collagen, and fibronectin by the stromal component at the interface between tumor cells and MSCs; and found that ECM secretion is inhibited by a neutralizing anti-IL-6 antibody, suggesting a new role of this cytokine in OS ECM deposition. Most importantly, we showed that the cytotoxic effect of doxorubicin is reduced by the presence of Type I collagen. We thus conclude that ECM protein deposition is crucial for modelling and studying drug response. Our results also suggest that targeting ECM proteins might improve the outcome of a subset of chemoresistant tumors.
ABSTRACT
Mortality from vmelanoma is associated with metastatic disease, but the mechanisms leading to spreading of the cancer cells remain obscure. Spatial profiling revealed that melanoma is characterized by a high degree of heterogeneity, which is established by the ability of melanoma cells to switch between different phenotypical stages. This plasticity, likely a heritage from embryonic pathways, accounts for a relevant part of the metastatic potential of these lesions, and requires the rapid and efficient reorganization of the transcriptional landscape of melanoma cells. A large part of the non-coding genome cooperates to control gene expression, specifically through the activity of enhancers (ENHs). In this study, we aimed to identify ex vivo the network of active ENHs and to outline their cooperative interactions in supporting transcriptional adaptation during melanoma metastatic progression. We conducted a genome-wide analysis to map active ENHs distribution in a retrospective cohort of 39 melanoma patients, comparing the profiles obtained in primary (N = 19) and metastatic (N = 20) melanoma lesions. Unsupervised clustering showed that the profile for acetylated histone H3 at lysine 27 (H3K27ac) efficiently segregates lesions into three different clusters corresponding to progressive stages of the disease. We reconstructed the map of super-ENHs (SEs) and cooperative ENHs that associate with metastatic progression in melanoma, which showed that cooperation among regulatory elements is a mandatory requirement for transcriptional plasticity. We also showed that these elements carry out specialized and non-redundant functions, and indicated the existence of a hierarchical organization, with SEs on top as masterminds of the entire transcriptional program and classical ENHs as executors. By providing an innovative vision of how the chromatin landscape of melanoma works during metastatic spreading, our data also point out the need to integrate functional profiling in the analysis of cancer lesions to increase definition and improve interpretation of tumor heterogeneity.
Subject(s)
Melanoma , Humans , Melanoma/genetics , Melanoma/metabolism , Retrospective Studies , Histones/metabolism , ChromatinABSTRACT
BACKGROUND: Anaplastic Thyroid Cancer (ATC) is an undifferentiated and aggressive tumor that often originates from well-Differentiated Thyroid Carcinoma (DTC) through a trans-differentiation process. Epithelial-to-Mesenchymal Transition (EMT) is recognized as one of the major players of this process. OVOL2 is a transcription factor (TF) that promotes epithelial differentiation and restrains EMT during embryonic development. OVOL2 loss in some types of cancers is linked to aggressiveness and poor prognosis. Here, we aim to clarify the unexplored role of OVOL2 in ATC. METHODS: Gene expression analysis in thyroid cancer patients and cell lines showed that OVOL2 is mainly associated with epithelial features and its expression is deeply impaired in ATC. To assess OVOL2 function, we established an OVOL2-overexpression model in ATC cell lines and evaluated its effects by analyzing gene expression, proliferation, invasion and migration abilities, cell cycle, specific protein localization through immunofluorescence staining. RNA-seq profiling showed that OVOL2 controls a complex network of genes converging on cell cycle and mitosis regulation and Chromatin Immunoprecipitation identified new OVOL2 target genes. RESULTS: Coherently with its reported function, OVOL2 re-expression restrained EMT and aggressiveness in ATC cells. Unexpectedly, we observed that it caused G2/M block, a consequent reduction in cell proliferation and an increase in cell death. This phenotype was associated to generalized abnormalities in the mitotic spindle structure and cytoskeletal organization. By RNA-seq experiments, we showed that many pathways related to cytoskeleton and migration, cell cycle and mitosis are profoundly affected by OVOL2 expression, in particular the RHO-GTPase pathway resulted as the most interesting. We demonstrated that RHO GTPase pathway is the central hub of OVOL2-mediated program in ATC and that OVOL2 transcriptionally inhibits RhoU and RhoJ. Silencing of RhoU recapitulated the OVOL2-driven phenotype pointing to this protein as a crucial target of OVOL2 in ATC. CONCLUSIONS: Collectively, these data describe the role of OVOL2 in ATC and uncover a novel function of this TF in inhibiting the RHO GTPase pathway interlacing its effects on EMT, cytoskeleton dynamics and mitosis.
Subject(s)
Thyroid Carcinoma, Anaplastic , Thyroid Neoplasms , Epithelial-Mesenchymal Transition/genetics , Female , Humans , Mitosis , Pregnancy , Thyroid Carcinoma, Anaplastic/genetics , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathology , Transcription Factors/genetics , rho GTP-Binding Proteins/geneticsABSTRACT
Malignant pleural mesothelioma (MPM) is a rare and incurable cancer, which incidence is increasing in many countries. MPM escapes the classical genetic model of cancer evolution, lacking a distinctive genetic fingerprint. Omics profiling revealed extensive heterogeneity failing to identify major vulnerabilities and restraining development of MPM-oriented therapies. Here, we performed a multilayered analysis based on a functional genome-wide CRISPR/Cas9 screening integrated with patients molecular and clinical data, to identify new non-genetic vulnerabilities of MPM. We identified a core of 18 functionally-related genes as essential for MPM cells. The chromatin reader KAP1 emerged as a dependency of MPM. We showed that KAP1 supports cell growth by orchestrating the expression of a G2/M-specific program, ensuring mitosis correct execution. Targeting KAP1 transcriptional function, by using CDK9 inhibitors resulted in a dramatic loss of MPM cells viability and shutdown of the KAP1-mediated program. Validation analysis on two independent MPM-patients sets, including a consecutive, retrospective cohort of 97 MPM, confirmed KAP1 as new non-genetic dependency of MPM and proved the association of its dependent gene program with reduced patients' survival probability. Overall these data: provided new insights into the biology of MPM delineating KAP1 and its target genes as building blocks of its clinical aggressiveness.
ABSTRACT
The control of gene expression at a transcriptional level requires a widespread landscape of regulatory elements. Central to these regulatory circuits are enhancers (ENHs), which are defined as cis-acting DNA elements able to increase the transcription of a target gene in a distance- and orientation-independent manner. ENHs are not independent functional elements but work in a complex and dynamic cooperative network, constituting the building blocks of multimodular domains of gene expression regulation. The information from each of these elements converges on the target promoter, contributing to improving the precision and sharpness of gene modulation. ENHs' interplay varies in its nature and extent, ranging from an additive to redundant effect depending on contexts. Moving from super-enhancers that drive the high expression levels of identity genes, to shadow-enhancers, whose redundant functions contribute to buffering the variation in gene expression, this review aims to describe the different modalities of ENHs' interaction and their role in the regulation of complex biological processes like cancer development.
ABSTRACT
Background: Papillary thyroid cancers (PTCs) are common, usually indolent malignancies. Still, a small but significant percentage of patients have aggressive tumors and develop distant metastases leading to death. Currently, it is not possible to discriminate aggressive lesions due to lack of prognostic markers. Long noncoding RNAs (lncRNAs), which are selectively expressed in a context-dependent manner, are expected to represent a new landscape to search for molecular discriminants. Transforming growth factor ß (TGFß) is a multifunctional cytokine that fosters epithelial-to-mesenchymal transition and metastatic spreading. In PTCs, it triggers the expression of the metastatic marker Cadherin 6 (CDH6). Here, we investigated the TGFß-dependent lncRNAs that may cooperate to potentiate PTC aggressiveness. Methods: We used a genome-wide approach to map enhancer (ENH)-associated lncRNAs under TGFß control. Linc00941 was selected and validated using functional in vitro assays. A combined approach using bioinformatic analyses of the thyroid cancer (THCA)-the cancer genome atlas (TCGA) dataset and RNA-seq analysis was used to identify the processes in which linc00941 was involved in and the genes under its regulation. Correlation with clinical data was performed to evaluate the potential of this lncRNA and its targets as prognostic markers in THCA. Results: Linc00941 was identified as transcribed starting from one of the TGFß-induced ENHs. Linc00941 expression was significantly higher in aggressive cancer both in the TCGA dataset and in a separate validation cohort from our institution. Loss of function assays for linc00941 showed that it promotes response to stimuli and invasiveness while restraining proliferation in PTC cells, a typical phenotype of metastatic cells. From the integration of TCGA data and linc00941 knockdown RNA-seq profiling, we identified 77 genes under the regulation of this lncRNA. Among these, we found the prometastatic gene CDH6. Linc00941 knockdown partially recapitulates the effects observed upon CDH6 silencing, promoting cell cytoskeleton and membrane adhesions rearrangements and autophagy. The combined expression of CDH6 and linc00941 is a distinctive feature of highly aggressive PTC lesions. Conclusions: Our data provide new insights into the biology driving metastasis in PTCs and highlight how lncRNAs cooperate with coding transcripts to sustain these processes.