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Cancer cells entail metabolic adaptation and microenvironmental remodeling to survive and progress. Both calcium (Ca2+) flux and Ca2+-dependent signaling play a crucial role in this process, although the underlying mechanism has yet to be elucidated. Through RNA screening, we identified one long noncoding RNA (lncRNA) named CamK-A (lncRNA for calcium-dependent kinase activation) in tumorigenesis. CamK-A is highly expressed in multiple human cancers and involved in cancer microenvironment remodeling via activation of Ca2+-triggered signaling. Mechanistically, CamK-A activates Ca2+/calmodulin-dependent kinase PNCK, which in turn phosphorylates IκBα and triggers calcium-dependent nuclear factor κB (NF-κB) activation. This regulation results in the tumor microenvironment remodeling, including macrophage recruitment, angiogenesis, and tumor progression. Notably, our human-patient-derived xenograft (PDX) model studies demonstrate that targeting CamK-A robustly impaired cancer development. Clinically, CamK-A expression coordinates with the activation of CaMK-NF-κB axis, and its high expression indicates poor patient survival rate, suggesting its role as a potential biomarker and therapeutic target.
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Carcinogênese/genética , Neoplasias/genética , RNA Longo não Codificante/genética , Microambiente Tumoral/genética , Sinalização do Cálcio/genética , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , NF-kappa B/genética , Neoplasias/patologia , Fosforilação , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Hippo pathway plays essential roles in organ size control and cancer prevention via restricting its downstream effector, Yes-associated protein (YAP). Previous studies have revealed an oncogenic function of YAP in reprogramming glucose metabolism, while the underlying mechanism remains to be fully clarified. Accumulating evidence suggests long noncoding RNAs (lncRNAs) as attractive therapeutic targets, given their roles in modulating various cancer-related signaling pathways. In this study, we report that lncRNA breast cancer anti-estrogen resistance 4 (BCAR4) is required for YAP-dependent glycolysis. Mechanistically, YAP promotes the expression of BCAR4, which subsequently coordinates the Hedgehog signaling to enhance the transcription of glycolysis activators HK2 and PFKFB3. Therapeutic delivery of locked nucleic acids (LNAs) targeting BCAR4 attenuated YAP-dependent glycolysis and tumor growth. The expression levels of BCAR4 and YAP are positively correlated in tissue samples from breast cancer patients, where high expression of both BCAR4 and YAP is associated with poor patient survival outcome. Taken together, our study not only reveals the mechanism by which YAP reprograms glucose metabolism, but also highlights the therapeutic potential of targeting YAP-BCAR4-glycolysis axis for breast cancer treatment.
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Glucose/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Sequência de Bases , Neoplasias da Mama/genética , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Feminino , Glicólise/genética , Células HEK293 , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Modelos Biológicos , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Transcrição Gênica , Resultado do Tratamento , Regulação para Cima/genéticaRESUMO
In the past decade, chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising immunotherapeutic approach for combating cancers, demonstrating remarkable efficacy in relapsed/refractory hematological malignancies in both pediatric and adult patients. CAR-natural killer (CAR-NK) cell complements CAR-T cell therapy by offering several distinct advantages. CAR-NK cells do not require HLA compatibility and exhibit low safety concerns. Moreover, CAR-NK cells are conducive to "off-the-shelf" therapeutics, providing significant logistic advantages over CAR-T cells. Both CAR-T and CAR-NK cells have shown consistent and promising results in hematological malignancies. However, their efficacy against solid tumors remains limited due to various obstacles including limited tumor trafficking and infiltration, as well as an immuno-suppressive tumor microenvironment. In this review, we discuss the recent advances and current challenges of CAR-T and CAR-NK cell immunotherapies, with a specific focus on the obstacles to their application in solid tumors. We also analyze in depth the advantages and drawbacks of CAR-NK cells compared to CAR-T cells and highlight CAR-NK CAR optimization. Finally, we explore future perspectives of these adoptive immunotherapies, highlighting the increasing contribution of cutting-edge biotechnological tools in shaping the next generation of cellular immunotherapy.
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Engineering cells for adoptive therapy requires overcoming limitations in cell viability and, in the efficiency of transgene delivery, the duration of transgene expression and the stability of genomic integration. Here we report a gene-delivery system consisting of a Sleeping Beauty (SB) transposase encoded into a messenger RNA delivered by an adeno-associated virus (AAV) encoding an SB transposon that includes the desired transgene, for mediating the permanent integration of the transgene. Compared with lentiviral vectors and with the electroporation of plasmids of transposon DNA or minicircle DNA, the gene-delivery system, which we named MAJESTIC (for 'mRNA AAV-SB joint engineering of stable therapeutic immune cells'), offers prolonged transgene expression, as well as higher transgene expression, therapeutic-cell yield and cell viability. MAJESTIC can deliver chimeric antigen receptors (CARs) into T cells (which we show lead to strong anti-tumour activity in vivo) and also transduce natural killer cells, myeloid cells and induced pluripotent stem cells with bi-specific CARs, kill-switch CARs and synthetic T-cell receptors.
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Dependovirus , Transposases , Transposases/genética , Transposases/metabolismo , Dependovirus/genética , Elementos de DNA Transponíveis/genética , RNA Mensageiro/genética , Técnicas de Transferência de GenesRESUMO
Natural killer (NK) cells have clinical potential against cancer; however, multiple limitations hinder the success of NK cell therapy. Here, we performed unbiased functional mapping of tumor-infiltrating NK (TINK) cells using in vivo adeno-associated virus (AAV)-SB (Sleeping Beauty)-CRISPR (clustered regularly interspaced short palindromic repeats) screens in four solid tumor mouse models. In parallel, we characterized single-cell transcriptomic landscapes of TINK cells, which identified previously unexplored subpopulations of NK cells and differentially expressed TINK genes. As a convergent hit, CALHM2-knockout (KO) NK cells showed enhanced cytotoxicity and tumor infiltration in mouse primary NK cells and human chimeric antigen receptor (CAR)-NK cells. CALHM2 mRNA reversed the CALHM2-KO phenotype. CALHM2 KO in human primary NK cells enhanced their cytotoxicity, degranulation and cytokine production. Transcriptomics profiling revealed CALHM2-KO-altered genes and pathways in both baseline and stimulated conditions. In a solid tumor model resistant to unmodified CAR-NK cells, CALHM2-KO CAR-NK cells showed potent in vivo antitumor efficacy. These data identify endogenous genetic checkpoints that naturally limit NK cell function and demonstrate the use of CALHM2 KO for engineering enhanced NK cell-based immunotherapies.
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Pooled screening creates a pool of cells with genetic variants, allowing for the simultaneous examination for changes in behavior or function. By selectively inducing mutations or perturbing expression, it enables scientists to systematically investigate the function of genes or genetic elements. Emerging gene editing tools, such as CRISPR, coupled with advances in sequencing and computational capabilities, provide growing opportunities to understand biological processes in humans, animals, and plants as well as to identify potential targets for therapeutic interventions and agricultural research. In this review, we highlight the recent advances of pooled screens using next-generation gene editing tools along with relevant challenges and describe potential future directions of this technology.
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The SARS-CoV-2 variant, Omicron (B.1.1.529), rapidly swept the world since its emergence. Compared with previous variants, Omicron has a high number of mutations, especially those in its spike glycoprotein that drastically dampen or abolish the efficacy of currently available vaccines and therapeutic antibodies. Several major sublineages of Omicron evolved, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75, which rapidly changing the global and regional landscape of the pandemic. Although vaccines are available, therapeutic antibodies remain critical for infected and especially hospitalized patients. To address this, we have designed and generated a panel of human/humanized therapeutic bispecific antibodies against Omicron and its sub-lineage variants, with activity spectrum against other lineages. Among these, the top clone CoV2-0213 has broadly potent activities against multiple SARS-CoV-2 ancestral and Omicron lineages, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75. We have solved the cryo-EM structure of the lead bi-specific antibody CoV-0213 and its major Fab arm MB.02. Three-dimensional structural analysis shows distinct epitope of antibody - spike receptor binding domain (RBD) interactions and reveals that both Fab fragments of CoV2-0213 can simultaneously target one single spike RBD or two adjacent ones in the same spike trimer, further corroborating its mechanism of action. CoV2-0213 represents a unique and potent broad-spectrum SARS-CoV-2 neutralizing bispecific antibody (nbsAb) against the currently circulating major Omicron variants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3, and BA.4/5). CoV2-0213 is primarily human and ready for translational testing as a countermeasure against the ever-evolving pathogen.
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All betacoronaviruses (ß-CoVs) encode non-structural protein 1 (Nsp1), an essential pathogenicity factor that potently restricts host gene expression. Among the ß-CoV family, MERS-CoV is the most distantly related member to SARS-CoV-2, and the mechanism for host translation inhibition by MERS-CoV Nsp1 remains controversial. Herein, we show that MERS-CoV Nsp1 directly interacts with the 40S ribosomal subunit. Using cryogenic electron microscopy (cryo-EM), we report a 2.6-Å structure of the MERS-CoV Nsp1 bound to the human 40S ribosomal subunit. The extensive interactions between C-terminal domain of MERS-CoV Nsp1 and the mRNA entry channel of the 40S ribosomal subunit are critical for its translation inhibition function. This mechanism of MERS-CoV Nsp1 is strikingly similar to SARS-CoV and SARS-CoV-2 Nsp1, despite modest sequence conservation. Our results reveal that the mechanism of host translation inhibition is conserved across ß-CoVs and highlight a potential therapeutic target for the development of antivirals that broadly restrict ß-CoVs.
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Coronavírus da Síndrome Respiratória do Oriente Médio , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , SARS-CoV-2/genética , RNA Mensageiro/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
Adoptive cell therapy has shown clinical success in patients with hematological malignancies. Immune cell engineering is critical for production, research, and development of cell therapy; however, current approaches for generation of therapeutic immune cells face various limitations. Here, we establish a composite gene delivery system for the highly efficient engineering of therapeutic immune cells. This system, termed MAJESTIC ( m RNA A AV-Sleeping-Beauty J oint E ngineering of S table T herapeutic I mmune C ells), combines the merits of mRNA, AAV vector, and transposon into one composite system. In MAJESTIC, the transient mRNA component encodes a transposase that mediates permanent genomic integration of the Sleeping Beauty (SB) transposon, which carries the gene-of-interest and is embedded within the AAV vector. This system can transduce diverse immune cell types with low cellular toxicity and achieve highly efficient and stable therapeutic cargo delivery. Compared with conventional gene delivery systems, such as lentiviral vector, DNA transposon plasmid, or minicircle electroporation, MAJESTIC shows higher cell viability, chimeric antigen receptor (CAR) transgene expression, therapeutic cell yield, as well as prolonged transgene expression. CAR-T cells generated by MAJESTIC are functional and have strong anti-tumor activity in vivo . This system also demonstrates versatility for engineering different cell therapy constructs such as canonical CAR, bi-specific CAR, kill switch CAR, and synthetic TCR; and for CAR delivery into various immune cells, including T cells, natural killer cells, myeloid cells, and induced pluripotent stem cells.
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As a clinical vaccine, lipid nanoparticle (LNP) mRNA has demonstrated potent and broad antibody responses, leading to speculation about its potential for antibody discovery. Here, we developed RAMIHM, a highly efficient strategy for developing fully human monoclonal antibodies that employs rapid mRNA immunization of humanized mice followed by single B cell sequencing (scBCR-seq). We immunized humanized transgenic mice with RAMIHM and generated 15 top-ranked clones from peripheral blood, plasma B, and memory B cell populations, demonstrating a high rate of antigen-specificity (93.3%). Two Omicron-specific neutralizing antibodies with high potency and one broad-spectrum neutralizing antibody were discovered. Furthermore, we extended the application of RAMIHM to cancer immunotherapy targets, including a single transmembrane protein CD22 and a multi-transmembrane G protein-coupled receptor target, GPRC5D, which is difficult for traditional protein immunization methods. RAMIHM-scBCR-seq is a broadly applicable platform for the rapid and efficient development of fully human monoclonal antibodies against an assortment of targets.
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Anticorpos Monoclonais , Imunização , Camundongos , Humanos , Animais , Anticorpos Monoclonais/genética , RNA Mensageiro/genética , Vacinação , Anticorpos Neutralizantes/genética , Camundongos TransgênicosRESUMO
Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential, however, multiple current limitations exist that naturally hinder the successful implementation of NK cell therapy against cancer, including their effector function, persistence, and tumor infiltration. To unbiasedly reveal the functional genetic landscape underlying critical NK cell characteristics against cancer, we perform perturbomics mapping of tumor infiltrating NK cells by joint in vivo AAV-CRISPR screens and single cell sequencing. We establish a strategy with AAV-SleepingBeauty(SB)- CRISPR screening leveraging a custom high-density sgRNA library targeting cell surface genes, and perform four independent in vivo tumor infiltration screens in mouse models of melanoma, breast cancer, pancreatic cancer, and glioblastoma. In parallel, we characterize single-cell transcriptomic landscapes of tumor-infiltrating NK cells, which identifies previously unexplored sub-populations of NK cells with distinct expression profiles, a shift from immature to mature NK (mNK) cells in the tumor microenvironment (TME), and decreased expression of mature marker genes in mNK cells. CALHM2, a calcium homeostasis modulator that emerges from both screen and single cell analyses, shows both in vitro and in vivo efficacy enhancement when perturbed in chimeric antigen receptor (CAR)-NK cells. Differential gene expression analysis reveals that CALHM2 knockout reshapes cytokine production, cell adhesion, and signaling pathways in CAR- NKs. These data directly and systematically map out endogenous factors that naturally limit NK cell function in the TME to offer a broad range of cellular genetic checkpoints as candidates for future engineering to enhance NK cell-based immunotherapies.
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Eukaryotic cells contain subcellular organelles with spatiotemporal regulation to coordinate various biochemical reactions. The various organelles perform their essential biological functions by employing specific biomolecules, including nucleic acids. Recent studies have revealed that noncoding RNAs (ncRNAs) are highly compartmentalized in cells and that their spatial distribution is intimately related to their functions. Dysregulation of subcellular ncRNAs can disrupt cellular homeostasis and cause human diseases. Mitochondria are responsible for energy generation to fuel cell growth and proliferation. Therefore, identifying mitochondria-associated ncRNAs helps to reveal new regulatory mechanisms and physiological functions of mitochondria. In this review, we summarize the latest advances in subcellular ncRNAs derived from either the nuclear or mitochondrial genome. We also discuss available biological approaches for investigating organelle-specific ncRNAs. Exploring the distribution and function of subcellular ncRNAs may facilitate the understanding of endomembrane dynamics and provide potential strategies for clinical transformation. This article is categorized under: RNA Export and Localization > RNA Localization Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Methods > RNA Analyses in Cells.
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RNA Longo não Codificante , RNA não Traduzido , Humanos , RNA não Traduzido/genética , Interferência de RNA , RNA , Núcleo Celular , MitocôndriasRESUMO
Purpose: To evaluate the impact of histological subtype on the survival of patients with renal cell carcinoma (RCC) and tumor thrombus (TT). Patients and methods: We retrospectively analyzed 350 patients with RCC and TT admitted to Chinese People's Liberation Army General Hospital between January 2006 and June 2021. The patients underwent radical nephrectomy and thrombectomy using robot-assisted laparoscopic, laparoscopic, or open surgery. The clinical and pathological parameters of the patients were taken from their medical records. Survival was calculated with the Kaplan-Meier method. Univariate and multivariate Cox regression analyses were performed to evaluate the prognostic significance of variables on overall survival (OS) and progression-free survival (PFS). Results: TT levels 0-IV were observed in 132 (37.71%), 43 (12.29%), 134 (38.29%), 20 (5.71) and 21 (6.00%) patients, respectively. Papillary (pRCC), clear cell, and other histological subtypes of RCC were detected in 28 (8.00%), 286 (81.71%), and 36 (10.29%) patients, respectively. Compared to the clear cell cohort, collecting systemic invasion (46.43 vs. 25.17%; p = 0.030) and lymph node metastasis (39.29 vs. 11.54%; p < 0.01) were more common in the pRCC cohort. Kaplan-Meier analyses showed that patients with pRCC and other subtypes had significantly worse OS and PFS compared to patients with the clear cell subtype (p < 0.05). Multivariate analyses revealed that histology was independently associated with reduced OS and PFS, including among patients without lymph node and distant metastasis (N0M0). Conclusion: Papillary or other subtypes have a considerably shorter OS and PFS compared to clear cell subtype in RCC patients with TT. Strict follow-up and surveillance should be performed for papillary or other subtypes RCC with TT.
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The Omicron variant of SARS-CoV-2 recently swept the globe and showed high level of immune evasion. Here, we generate an Omicron-specific lipid nanoparticle (LNP) mRNA vaccine candidate, and test its activity in animals, both alone and as a heterologous booster to WT mRNA vaccine. Our Omicron-specific LNP-mRNA vaccine elicits strong antibody response in vaccination-naïve mice. Mice that received two-dose WT LNP-mRNA show a > 40-fold reduction in neutralization potency against Omicron than WT two weeks post boost, which further reduce to background level after 3 months. The WT or Omicron LNP-mRNA booster increases the waning antibody response of WT LNP-mRNA vaccinated mice against Omicron by 40 fold at two weeks post injection. Interestingly, the heterologous Omicron booster elicits neutralizing titers 10-20 fold higher than the homologous WT booster against Omicron variant, with comparable titers against Delta variant. All three types of vaccination, including Omicron alone, WT booster and Omicron booster, elicit broad binding antibody responses against SARS-CoV-2 WA-1, Beta, Delta variants and SARS-CoV. These data provide direct assessments of an Omicron-specific mRNA vaccination in vivo, both alone and as a heterologous booster to WT mRNA vaccine.
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COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Lipossomos , Camundongos , Nanopartículas , RNA Mensageiro/genética , SARS-CoV-2/genética , Vacinação , Vacinas Sintéticas , Vacinas de mRNARESUMO
The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has high transmissibility and recently swept the globe. Due to the extensive number of mutations, this variant has high level of immune evasion, which drastically reduced the efficacy of existing antibodies and vaccines. Thus, it is important to test an Omicron-specific vaccine, evaluate its immune response against Omicron and other variants, and compare its immunogenicity as boosters with existing vaccine designed against the reference wildtype virus (WT). Here, we generated an Omicron-specific lipid nanoparticle (LNP) mRNA vaccine candidate, and tested its activity in animals, both alone and as a heterologous booster to existing WT mRNA vaccine. Our Omicron-specific LNP-mRNA vaccine elicited strong and specific antibody response in vaccination-naive mice. Mice that received two-dose WT LNP-mRNA, the one mimicking the commonly used Pfizer/Moderna mRNA vaccine, showed a >40-fold reduction in neutralization potency against Omicron variant than that against WT two weeks post second dose, which further reduced to background level >3 months post second dose. As a booster shot for two-dose WT mRNA vaccinated mice, a single dose of either a homologous booster with WT LNP-mRNA or a heterologous booster with Omicron LNP-mRNA restored the waning antibody response against Omicron, with over 40-fold increase at two weeks post injection as compared to right before booster. Interestingly, the heterologous Omicron LNP-mRNA booster elicited neutralizing titers 10-20 fold higher than the homologous WT booster against the Omicron variant, with comparable titers against the Delta variant. All three types of vaccination, including Omicron mRNA alone, WT mRNA homologous booster, and Omicron heterologous booster, elicited broad binding antibody responses against SARS-CoV-2 WA-1, Beta, and Delta variants, as well as other Betacoronavirus species such as SARS-CoV, but not Middle East respiratory syndrome coronavirus (MERS-CoV). These data provided direct proof-of-concept assessments of an Omicron-specific mRNA vaccination in vivo, both alone and as a heterologous booster to the existing widely-used WT mRNA vaccine form.
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Immune checkpoint blockade therapies targeting the PD-L1/PD-1 axis have demonstrated clear clinical benefits. Improved understanding of the underlying regulatory mechanisms might contribute new insights into immunotherapy. Here, we identify transmembrane and ubiquitin-like domain-containing protein 1 (TMUB1) as a modulator of PD-L1 post-translational modifications in tumor cells. Mechanistically, TMUB1 competes with HECT, UBA and WWE domain-containing protein 1 (HUWE1), a E3 ubiquitin ligase, to interact with PD-L1 and inhibit its polyubiquitination at K281 in the endoplasmic reticulum. Moreover, TMUB1 enhances PD-L1 N-glycosylation and stability by recruiting STT3A, thereby promoting PD-L1 maturation and tumor immune evasion. TMUB1 protein levels correlate with PD-L1 expression in human tumor tissue, with high expression being associated with poor patient survival rates. A synthetic peptide engineered to compete with TMUB1 significantly promotes antitumor immunity and suppresses tumor growth in mice. These findings identify TMUB1 as a promising immunotherapeutic target.
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Antígeno B7-H1 , Neoplasias , Animais , Humanos , Camundongos , Antígeno B7-H1/metabolismo , Glicosilação , Imunoterapia , Neoplasias/genética , Neoplasias/terapia , Evasão Tumoral , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
RATIONALE: The airflow limitation that defines severity of chronic obstructive pulmonary disease (COPD) is caused by a combination of small airway obstruction and emphysematous lung destruction. OBJECTIVES: To examine the hypothesis that small airway obstructive and emphysematous destructive lesions are produced by differential expression of genes associated with tissue repair. METHODS: The expression of 54 genes associated with repair of repetitively damaged tissue was measured in 136 paired samples of small bronchioles and surrounding lung tissue separated by laser capture microdissection. These samples were collected from 63 patients at different levels of disease severity who required surgery for either lung cancer or lung transplantation for very severe COPD. Gene expression was measured by quantitative polymerase chain reaction in these paired samples and compared with the FEV(1) by linear regression analysis. MEASUREMENTS AND MAIN RESULTS: After corrections for false discovery rates, only 2 of 10 genes (serpin peptidase inhibitor/plasminogen activator inhibitor member 2 and matrix metalloproteinase [MMP] 10) increased, whereas 8 (MMP2, integrin-alpha1, vascular endothelial growth factor, a disintegrin and metallopeptidase domain 33, scatter factor/hepatocyte growth factor, tissue inhibitor of matrix metalloproteinase-2, fibronectin, and collagen 3alpha1) decreased in small airways in association with FEV(1). In contrast, 8/12 genes (early growth response factor 1, MMP1, MMP9, MMP10, plasminogen activator urokinase, plasminogen activator urokinase receptor, tumor necrosis factor, and IL13) increased and 4/12 (MMP2, tissue inhibitor of matrix metalloproteinase-1, collagen 1alpha1, and transforming growth factor-beta3) decreased in the surrounding lung tissue in association with progression of COPD. CONCLUSIONS: The progression of COPD is associated with the differential expression of a cluster of genes that favor the degradation of the tissue surrounding the small conducting airways.
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Remodelação das Vias Aéreas/genética , Expressão Gênica/genética , Família Multigênica/genética , Doença Pulmonar Obstrutiva Crônica/genética , Enfisema/genética , Volume Expiratório Forçado/genética , Perfilação da Expressão Gênica/métodos , Humanos , Microdissecção/métodos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Índice de Gravidade de DoençaRESUMO
Emerging evidence has shown that open reading frames inside long noncoding RNAs (lncRNAs) could encode micropeptides. However, their roles in cellular energy metabolism and tumor progression remain largely unknown. Here, we identified a 94 amino acid-length micropeptide encoded by lncRNA LINC00467 in colorectal cancer. We also characterized its conservation across higher mammals, localization to mitochondria, and the concerted local functions. This peptide enhanced the ATP synthase construction by interacting with the subunits α and γ (ATP5A and ATP5C), increased ATP synthase activity and mitochondrial oxygen consumption rate, and thereby promoted colorectal cancer cell proliferation. Hence, this micropeptide was termed ATP synthase-associated peptide (ASAP). Furthermore, loss of ASAP suppressed patient-derived xenograft growth with attenuated ATP synthase activity and mitochondrial ATP production. Clinically, high expression of ASAP and LINC00467 predicted poor prognosis of colorectal cancer patients. Taken together, our findings revealed a colorectal cancer-associated micropeptide as a vital player in mitochondrial metabolism and provided a therapeutic target for colorectal cancer.
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Trifosfato de Adenosina/biossíntese , Neoplasias Colorretais/etiologia , Proteínas Mitocondriais/fisiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Peptídeos/farmacologia , RNA Longo não Codificante/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Progressão da Doença , Humanos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Organelles use specialized molecules to regulate their essential cellular processes. However, systematically elucidating the subcellular distribution and function of molecules such as long non-coding RNAs (lncRNAs) in cellular homeostasis and diseases has not been fully achieved. Here, we reveal the diverse and abundant subcellular distribution of organelle-associated lncRNAs from mitochondria, lysosomes and endoplasmic reticulum. Among them, we identify the mitochondrially localized lncRNA growth-arrest-specific 5 (GAS5) as a tumour suppressor in maintaining cellular energy homeostasis. Mechanistically, energy-stress-induced GAS5 modulates mitochondrial tricarboxylic acid flux by disrupting metabolic enzyme tandem association of fumarate hydratase, malate dehydrogenase and citrate synthase, the canonical members of the tricarboxylic acid cycle. GAS5 negatively correlates with levels of its associated mitochondrial metabolic enzymes in tumours and benefits overall survival in individuals with breast cancer. Together, our detailed annotation of subcellular lncRNA distribution identifies a functional role for lncRNAs in regulating cellular metabolic homeostasis, highlighting organelle-associated lncRNAs as potential clinical targets to manipulate cellular metabolism and diseases.