APC/C-regulated CPT1C promotes tumor progression by upregulating the energy supply and accelerating the G1/S transition.

BACKGROUND: In addition to functioning as a precise monitoring mechanism in cell cycle, the anaphase-promoting complex/cyclosome (APC/C) is reported to be involved in regulating multiple metabolic processes by facilitating the ubiquitin-mediated degradation of key enzymes. Fatty acid oxidation is a metabolic pathway utilized by tumor cells that is crucial for malignant progression; however, its association with APC/C remains to be explored. METHODS: Cell cycle synchronization, immunoblotting, and propidium iodide staining were performed to investigate the carnitine palmitoyltransferase 1 C (CPT1C) expression manner. Proximity ligation assay and co-immunoprecipitation were performed to detect interactions between CPT1C and APC/C. Flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt (MTS) assays, cell-scratch assays, and transwell assays and xenograft transplantation assays were performed to investigate the role of CPT1C in tumor progression in vitro and in vivo. Immunohistochemistry was performed on tumor tissue microarray to evaluate the expression levels of CPT1C and explore its potential clinical value. RESULTS: We identified CPT1C as a novel APC/C substrate. CPT1C protein levels exhibited cell cycle-dependent fluctuations, peaking at the G1/S boundary. Elevated CPT1C accelerated the G1/S transition, facilitating tumor cell proliferation in vitro and in vivo. Furthermore, CPT1C enhanced fatty acid utilization, upregulated ATP levels, and decreased reactive oxygen species levels, thereby favoring cell survival in a harsh metabolic environment. Clinically, high CPT1C expression correlated with poor survival in patients with esophageal squamous cell carcinoma. CONCLUSIONS: Overall, our results revealed a novel interplay between fatty acid utilization and cell cycle machinery in tumor cells. Additionally, CPT1C promoted tumor cell proliferation and survival by augmenting cellular ATP levels and preserving redox homeostasis, particularly under metabolic stress. Therefore, CPT1C could be an independent prognostic indicator in esophageal squamous cell carcinoma.

Spatial transcriptomics delineates molecular features and cellular plasticity in lung adenocarcinoma progression.

Indolent (lepidic) and aggressive (micropapillary, solid, and poorly differentiated acinar) histologic subtypes often coexist within a tumor tissue of lung adenocarcinoma (LUAD), but the molecular features associated with different subtypes and their transitions remain elusive. Here, we combine spatial transcriptomics and multiplex immunohistochemistry to elucidate molecular characteristics and cellular plasticity of distinct histologic subtypes of LUAD. We delineate transcriptional reprogramming and dynamic cell signaling that determine subtype progression, especially hypoxia-induced regulatory network. Different histologic subtypes exhibit heterogeneity in dedifferentiation states. Additionally, our results show that macrophages are the most abundant cell type in LUAD, and identify different tumor-associated macrophage subpopulations that are unique to each histologic subtype, which might contribute to an immunosuppressive microenvironment. Our results provide a systematic landscape of molecular profiles that drive LUAD subtype progression, and demonstrate potentially novel therapeutic strategies and targets for invasive lung adenocarcinoma.

Comparative transcriptome characterization of esophageal squamous cell carcinoma and adenocarcinoma.

Background: Esophageal cancers are primarily categorized as esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). While various (epi) genomic alterations associated with tumor development in ESCC and EAC have been documented, a comprehensive comparison of the transcriptomes in these two cancer subtypes remains lacking. Methods: We collected 551 gene expression profiles from publicly available sources, including normal, ESCC, and EAC tissues or cell lines. Subsequently, we conducted a systematic analysis to compare the transcriptomes of these samples at various levels, including gene expression, promoter activity, alternative splicing (AS), alternative polyadenylation (APA), and gene fusion. Results: Seven distinct cluster gene expression patterns were identified among the differentially expressed genes in normal, ESCC, and EAC tissues. These patterns were enriched in the PI3K-Akt signaling pathway and the activation of extracellular matrix organization and exhibited repression of epidermal development. Notably, we observed additional genes or unique expression levels enriched in these shared pathways and biological processes related to tumor development and immune activation. In addition to the differentially expressed genes, there was an enrichment of lncRNA co-expression networks and downregulation of promoter activity associated with the repression of epidermal development in both ESCC and EAC. This indicates a common feature between these two cancer subtypes. Furthermore, differential AS and APA patterns in ESCC and EAC appear to partially affect the expression of host genes associated with bacterial or viral infections in these subtypes. No gene fusions were observed between ESCC and EAC, thus highlighting the distinct molecular mechanisms underlying these two cancer subtypes. Conclusions: We conducted a comprehensive comparison of ESCC and EAC transcriptomes and uncovered shared and distinct transcriptomic signatures at multiple levels. These findings suggest that ESCC and EAC may exhibit common and unique mechanisms involved in tumorigenesis.

CDK7-YAP-LDHD axis promotes D-lactate elimination and ferroptosis defense to support cancer stem cell-like properties.

Reprogrammed cellular metabolism is essential for maintaining cancer stem cells (CSCs) state. Here, we report that mitochondrial D-lactate catabolism is a necessary initiating oncogenic event during tumorigenesis of esophageal squamous cell carcinoma (ESCC). We discover that cyclin-dependent kinase 7 (CDK7) phosphorylates nuclear Yes-associated protein 1 (YAP) at S127 and S397 sites and enhances its transcription function, which promotes D-lactate dehydrogenase (LDHD) protein expression. Moreover, LDHD is enriched significantly in ESCC-CSCs rather than differentiated tumor cells and high LDHD status is connected with poor prognosis in ESCC patients. Mechanistically, the CDK7-YAP-LDHD axis helps ESCC-CSCs escape from ferroptosis induced by D-lactate and generates pyruvate to satisfy energetic demands for their elevated self-renewal potential. Hence, we conclude that esophageal CSCs adopt a D-lactate elimination and pyruvate accumulation mode dependent on CDK7-YAP-LDHD axis, which drives stemness-associated hallmarks of ESCC-CSCs. Reasonably, targeting metabolic checkpoints may serve as an effective strategy for ESCC therapy.

Gut microbiota-mediated nucleotide synthesis attenuates the response to neoadjuvant chemoradiotherapy in rectal cancer.

Preoperative neoadjuvant chemoradiotherapy (nCRT) is a standard treatment for locally advanced rectal cancer (LARC) patients, yet little is known about the mediators underlying the heterogeneous patient response. In this longitudinal study, we performed 16S rRNA sequencing on 353 fecal specimens and find reduced microbial diversity after nCRT. Multi-omics data integration reveals that Bacteroides vulgatus-mediated nucleotide biosynthesis associates with nCRT resistance in LARC patients, and nonresponsive tumors are characterized by the upregulation of genes related to DNA repair and nucleoside transport. Nucleosides supplementation or B. vulgatus gavage protects cancer cells from the 5-fluorouracil or irradiation treatment. An analysis of 2,205 serum samples from 735 patients suggests that uric acid is a potential prognosis marker for LARC patients receiving nCRT. Our data unravel the role of intestinal microbiota-mediated nucleotide biosynthesis in the response of rectal tumors to nCRT, and highlight the importance of deciphering the cross-talk between cancer cells and gut microorganisms during cancer therapies.

The metabolic genomic atlas reveals potential drivers and clinically relevant insights into the etiology of esophageal squamous cell carcinoma.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers globally, with a poor prognosis and ambiguous therapy target. As a hallmark of cancer, metabolism reprogramming plays a critical role in the development of ESCC; however, the genomic alterations underlying this reconfiguration are still largely unknown. Methods: We have comprehensively studied the metabolic genomic variations in an integrated ESCC cohort of 490 patients and characterized the somatic alterations associated with various metabolic pathways. Results: The somatic mutations and copy number alterations (CNAs) occurred heterogeneously in all patients. Using CNA-based clustering, we stratified patients into three clusters and Cluster3 with more deletions marked for worse prognosis. Our findings revealed detailed genetic alterations in components of metabolic pathways and highlighted the role of metal ion channel transporters and non-neuronal/neuronal synapse systems in the development of ESCC. We found a subset of potential metabolic drivers and functionally validated RYR2, MGST3, and CYP8B1 involved in the ESCC-associated malignancy. Another key finding was that we identified 27 metabolic genes with genomic alterations that could serve as independent prognostic factors and figured out two genetic panels that could stratify patients into distinct prognostic groups. Conclusion: Collectively, our study provided a deep insight into the metabolic landscape in ESCC, extending our understanding of the metabolic reconfiguration underlying the genomic basis of ESCC. Furthermore, our findings revealed potential prognostic factors of ESCC, which are expected to contribute to the accurate determination of the prognosis in the clinic.

The m6A-induced lncRNA CASC8 promotes proliferation and chemoresistance via upregulation of hnRNPL in esophageal squamous cell carcinoma.

Long noncoding RNAs (lncRNAs) are dysregulated in many cancers. Here, we identified the molecular mechanisms of lncRNA Cancer Susceptibility Candidate 8 (CASC8) in promoting the malignancy of esophageal squamous cell carcinoma (ESCC). CASC8 was highly overexpressed in ESCC tissues and upregulation of CASC8 predicted poor prognosis in ESCC patients. Moreover, CASC8 decreased the cisplatin sensitivity of ESCC cells and promoted ESCC tumor growth in vivo. Mechanistically, CASC8 interacted with heterogeneous nuclear ribonucleoprotein L (hnRNPL) and inhibited its polyubiquitination and proteasomal degradation, thus stabilizing hnRNPL protein levels and activating the Bcl2/caspase3 pathway. Additionally, AlkB Homolog 5, RNA demethylase (ALKBH5)-mediated m6A demethylation stabilized the CASC8 transcript, resulting in CASC8 upregulation. Taken together, these findings identified an oncogenic function of CASC8 in the progression of ESCC, which suggest that CASC8 might become a potential prognostic biomarker in ESCC.

Nuclear genome-derived circular RNA circPUM1 localizes in mitochondria and regulates oxidative phosphorylation in esophageal squamous cell carcinoma.

Circular RNAs (circRNAs) were shown to play an important role in the occurrence and progression of tumors. However, the functions of nuclear genome-derived circRNAs localized in mitochondria of tumor cells remain largely elusive. Here, we report that circPUM1, a circular RNA derived from back-splicing of pre-mRNAs of nuclear genome PUM1, localizes in mitochondria. The expression level of circPUM1 is positively correlated with HIF1α accumulation under CoCl2-induced intracellular hypoxic-like condition in esophageal squamous cell carcinoma (ESCC) cell lines. Importantly, circPUM1 acts as a scaffold for the interaction between UQCRC1 and UQCRC2 in ESCC cell lines. Knock-down of circPUM1 would result in lower intracellular oxygen concentration, downregulated oxidative phosphorylation, decrease of mitochondrial membrane potential, increase of ROS generation and shrinking of mitochondria, respectively. CircPUM1 depletion induces dysfunction of the mitochondrial complex III and the cleavage of caspase3 spontaneously. Interestingly, disruption of circPUM1 led to pyroptosis that initiates the cell death of ESCC cell lines. Therefore, we conclude that circPUM1 plays a critical role in maintaining the stability of mitochondrial complex III to enhance oxidative phosphorylation for ATP production of ESCC cells and moreover propose that ESCC cells exploit circPUM1 during cell adaptation.

Genomic and epigenomic evolution of acquired resistance to combination therapy in esophageal squamous cell carcinoma.

BACKGROUNDTargeted arterial infusion of verapamil combined with chemotherapy (TVCC) is an effective clinical interventional therapy for esophageal squamous cell carcinoma (ESCC), but multidrug resistance (MDR) remains the major cause of relapse or poor prognosis, and the underlying molecular mechanisms of MDR, temporal intratumoral heterogeneity, and clonal evolutionary processes of resistance have not been determined.METHODSTo elucidate the roles of genetic and epigenetic alterations in the evolution of acquired resistance during therapies, we performed whole-exome sequencing on 16 serial specimens from 7 patients with ESCC at every cycle of therapeutic intervention from 3 groups, complete response, partial response, and progressive disease, and we performed whole-genome bisulfite sequencing for 3 of these 7 patients, 1 patient from each group.RESULTSPatients with progressive disease exhibited a substantially higher genomic and epigenomic temporal heterogeneity. Subclonal expansions driven by the beneficial new mutations were observed during combined therapies, which explained the emergence of MDR. Notably, SLC7A8 was identified as a potentially novel MDR gene, and functional assays demonstrated that mutant SLC7A8 promoted the resistance phenotypes of ESCC cell lines. Promoter methylation dynamics during treatments revealed 8 drug resistance protein-coding genes characterized by hypomethylation in promoter regions. Intriguingly, promoter hypomethylation of SLC8A3 and mutant SLC7A8 were enriched in an identical pathway, protein digestion and absorption, indicating a potentially novel MDR mechanism during treatments.CONCLUSIONOur integrated multiomics investigations revealed the dynamics of temporal genetic and epigenetic inter- and intratumoral heterogeneity, clonal evolutionary processes, and epigenomic changes, providing potential MDR therapeutic targets in treatment-resistant patients with ESCC during combined therapies.FUNDINGNational Natural Science Foundation of China, Science Foundation of Peking University Cancer Hospital, CAMS Innovation Fund for Medical Sciences, Major Program of Shenzhen Bay Laboratory, Guangdong Basic and Applied Basic Research Foundation, and the third round of public welfare development and reform pilot projects of Beijing Municipal Medical Research Institutes.

MAGE-C3 promotes cancer metastasis by inducing epithelial-mesenchymal transition and immunosuppression in esophageal squamous cell carcinoma.

BACKGROUND: Evading immune surveillance is necessary for tumor metastasis. Thus, there is an urgent need to better understand the interaction between metastasis and mechanisms of tumor immune evasion. In this study, we aimed to clarify a novel mechanism that link tumor metastasis and immunosuppression in the development of esophageal squamous cell carcinoma (ESCC). METHODS: The expression of melanoma-associated antigen C3 (MAGE-C3) was detected using immunohistochemistry. Transwell assays were used to evaluate the migration and invasion ability of esophageal squamous cell carcinoma (ESCC) cells. Metastasis assays in mice were used to evaluate metastatic ability in vivo. Lymphocyte-mediated cytotoxicity assays were performed to visualize the immune suppression function on tumor cells. RNA sequencing was performed to identify differentially expressed genes between MAGE-C3 overexpressing ESCC cells and control cells. Gene ontology (GO) enrichment analyses was performed to identify the most altered pathways influenced by MAGE-C3. The activation of the interferon-γ (IFN-γ) pathway was analyzed using Western blotting, GAS luciferase reporter assays, immunofluorescence, and flow cytometry. The role of MAGE-C3 in the IFN-γ pathway was determined by Western blotting and immunoprecipitation. Furthermore, immunohistochemistry and flow cytometry analysis monitored the changes of infiltrated T cell populations in murine lung metastases. RESULTS: MAGE-C3 was overexpressed in ESCC tissues. High expression of MAGE-C3 had a significant association with the risk of lymphatic metastasis and poor survival in patients with ESCC. Functional experiments revealed that MAGE-C3 promoted tumor metastasis by activating the epithelial-mesenchymal transition (EMT). MAGE-C3 repressed antitumor immunity and regulated cytokine secretion of T cells, implying an immunosuppressive function. Mechanistically, MAGE-C3 facilitated IFN-γ signaling and upregulated programmed cell death ligand 1 (PD-L1) by binding with IFN-γ receptor 1 (IFNGR1) and strengthening the interaction between IFNGR1 and signal transducer and activator of transcription 1 (STAT1). Interestingly, MAGE-C3 displayed higher tumorigenesis in immune-competent mice than in immune-deficient nude mice, confirming the immunosuppressive role of MAGE-C3. Furthermore, mice bearing MAGE-C3-overexpressing tumors showed worse survival and more lung metastases with decreased CD8+ infiltrated T cells and increased programmed cell death 1 (PD-1)+ CD8+ infiltrated T cells. CONCLUSION: MAGE-C3 enhances tumor metastasis through promoting EMT and protecting tumors from immune surveillance, and could be a potential prognostic marker and therapeutic target.

Genomic landscapes of Chinese sporadic autism spectrum disorders revealed by whole-genome sequencing.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with considerable clinical and genetic heterogeneity. In this study, we identified all classes of genomic variants from whole-genome sequencing (WGS) dataset of 32 Chinese trios with ASD, including de novo mutations, inherited variants, copy number variants (CNVs) and genomic structural variants. A higher mutation rate (Poisson test, P < 2.2 × 10-16) in exonic (1.37 × 10-8) and 3'-UTR regions (1.42 × 10-8) was revealed in comparison with that of whole genome (1.05 × 10-8). Using an integrated model, we identified 87 potentially risk genes (P < 0.01) from 4832 genes harboring various rare deleterious variants, including CHD8 and NRXN2, implying that the disorders may be in favor to multiple-hit. In particular, frequent rare inherited mutations of several microcephaly-associated genes (ASPM, WDR62, and ZNF335) were found in ASD. In chromosomal structure analyses, we found four de novo CNVs and one de novo chromosomal rearrangement event, including a de novo duplication of UBE3A-containing region at 15q11.2-q13.1, which causes Angelman syndrome and microcephaly, and a disrupted TNR due to de novo chromosomal translocation t(1; 5)(q25.1; q33.2). Taken together, our results suggest that abnormalities of centrosomal function and chromatin remodeling of the microcephaly-associated genes may be implicated in pathogenesis of ASD. Adoption of WGS as a new yet efficient technique to illustrate the full genetic spectrum in complex disorders, such as ASD, could provide novel insights into pathogenesis, diagnosis and treatment.

Novel mutations in PDE6B causing human retinitis pigmentosa.

AIM: To identify the genetic defects of a Chinese patient with sporadic retinitis pigmentosa (RP). METHODS: Ophthalmologic examinations were performed on the sporadic RP patient, 144 genes associated with retinal diseases were scanned with capture next generation sequencing (CNGS) approach. Two heterozygous mutations in PDE6B were confirmed in the pedigree by Sanger sequencing subsequently. The carrier frequency of PDE6B mutations of reported PDE6B mutations based on the available two public exome databases (1000 Genomes Project and ESP6500 Genomes Project) and one in-house exome database was investigated. RESULTS: We identified compound heterozygosity of two novel nonsense mutations c.1133G>A (p.W378X) and c.2395C>T (p.R799X) in PDE6B, one reported causative gene for RP. Neither of the two mutations in our study was presented in three exome databases. Two mutations (p.R74C and p.T604I) in PDE6B have relatively high frequencies in the ESP6500 and in-house databases, respectively, while no common dominant mutation in each of the database or across all databases. CONCLUSION: We demonstrates that compound heterozygosity of two novel nonsense mutations in PDE6B could lead to RP. These results collectively point to enormous potential of next-generation sequencing in determining the genetic etiology of RP and how various mutations in PDE6B contribute to the genetic heterogeneity of RP.

A recurrent deletion mutation in OPA1 causes autosomal dominant optic atrophy in a Chinese family.

Autosomal dominant optic atrophy (ADOA) is the most frequent form of hereditary optic neuropathy and occurs due to the degeneration of the retinal ganglion cells. To identify the genetic defect in a family with putative ADOA, we performed capture next generation sequencing (CNGS) to screen known retinal disease genes. However, six exons failed to be sequenced by CNGS in optic atrophy 1 gene (OPA1). Sequencing of those exons identified a 4 bp deletion mutation (c.2983-1_2985del) in OPA1. Furthermore, we sequenced the transcripts of OPA1 from the patient skin fibroblasts and found there is six-nucleotide deletion (c.2984-c.2989, AGAAAG). Quantitative-PCR and Western blotting showed that OPA1 mRNA and its protein expression have no obvious difference between patient skin fibroblast and control. The analysis of protein structure by molecular modeling suggests that the mutation may change the structure of OPA1 by formation of an alpha helix protruding into an existing pocket. Taken together, we identified an OPA1 mutation in a family with ADOA by filling the missing CNGS data. We also showed that this mutation affects the structural intactness of OPA1. It provides molecular insights for clinical genetic diagnosis and treatment of optic atrophy.

Molecular diagnosis of putative Stargardt disease by capture next generation sequencing.

Stargardt Disease (STGD) is the commonest genetic form of juvenile or early adult onset macular degeneration, which is a genetically heterogeneous disease. Molecular diagnosis of STGD remains a challenge in a significant proportion of cases. To address this, seven patients from five putative STGD families were recruited. We performed capture next generation sequencing (CNGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Seven disease-causing mutations in ABCA4 and two in PROM1 were identified by CNGS, which provides a confident genetic diagnosis in these five families. We also provided a genetic basis to explain the differences among putative STGD due to various mutations in different genes. Meanwhile, we show for the first time that compound heterozygous mutations in PROM1 gene could cause cone-rod dystrophy. Our findings support the enormous potential of CNGS in putative STGD molecular diagnosis.

Targeted exome sequencing identified novel USH2A mutations in Usher syndrome families.

Usher syndrome (USH) is a leading cause of deaf-blindness in autosomal recessive trait. Phenotypic and genetic heterogeneities in USH make molecular diagnosis much difficult. This is a pilot study aiming to develop an approach based on next-generation sequencing to determine the genetic defects in patients with USH or allied diseases precisely and effectively. Eight affected patients and twelve unaffected relatives from five unrelated Chinese USH families, including 2 pseudo-dominant ones, were recruited. A total of 144 known genes of inherited retinal diseases were selected for deep exome resequencing. Through systematic data analysis using established bioinformatics pipeline and segregation analysis, a number of genetic variants were released. Eleven mutations, eight of them were novel, in the USH2A gene were identified. Biparental mutations in USH2A were revealed in 2 families with pseudo-dominant inheritance. A proband was found to have triple mutations, two of them were supposed to locate in the same chromosome. In conclusion, this study revealed the genetic defects in the USH2A gene and demonstrated the robustness of targeted exome sequencing to precisely and rapidly determine genetic defects. The methodology provides a reliable strategy for routine gene diagnosis of USH.

Exome-assistant: a rapid and easy detection of disease-related genes and genetic variations from exome sequencing.

BACKGROUND: Protein-coding regions in human genes harbor 85% of the mutations that are associated with disease-related traits. Compared with whole-genome sequencing of complex samples, exome sequencing serves as an alternative option because of its dramatically reduced cost. In fact, exome sequencing has been successfully applied to identify the cause of several Mendelian disorders, such as Miller and Schinzel-Giedio syndrome. However, there remain great challenges in handling the huge data generated by exome sequencing and in identifying potential disease-related genetic variations. RESULTS: In this study, Exome-assistant (http://122.228.158.106/exomeassistant), a convenient tool for submitting and annotating single nucleotide polymorphisms (SNPs) and insertion/deletion variations (InDels), was developed to rapidly detect candidate disease-related genetic variations from exome sequencing projects. Versatile filter criteria are provided by Exome-assistant to meet different users' requirements. Exome-assistant consists of four modules: the single case module, the two cases module, the multiple cases module, and the reanalysis module. The two cases and multiple cases modules allow users to identify sample-specific and common variations. The multiple cases module also supports family-based studies and Mendelian filtering. The identified candidate disease-related genetic variations can be annotated according to their sample features. CONCLUSIONS: In summary, by exploring exome sequencing data, Exome-assistant can provide researchers with detailed biological insights into genetic variation events and permits the identification of potential genetic causes of human diseases and related traits.