NBS1 lactylation is required for efficient DNA repair and chemotherapy resistance.

The Warburg effect is a hallmark of cancer that refers to the preference of cancer cells to metabolize glucose anaerobically rather than aerobically1,2. This results in substantial accumulation of lacate, the end product of anaerobic glycolysis, in cancer cells3. However, how cancer metabolism affects chemotherapy response and DNA repair in general remains incompletely understood. Here we report that lactate-driven lactylation of NBS1 promotes homologous recombination (HR)-mediated DNA repair. Lactylation of NBS1 at lysine 388 (K388) is essential for MRE11-RAD50-NBS1 (MRN) complex formation and the accumulation of HR repair proteins at the sites of DNA double-strand breaks. Furthermore, we identify TIP60 as the NBS1 lysine lactyltransferase and the 'writer' of NBS1 K388 lactylation, and HDAC3 as the NBS1 de-lactylase. High levels of NBS1 K388 lactylation predict poor patient outcome of neoadjuvant chemotherapy, and lactate reduction using either genetic depletion of lactate dehydrogenase A (LDHA) or stiripentol, a lactate dehydrogenase A inhibitor used clinically for anti-epileptic treatment, inhibited NBS1 K388 lactylation, decreased DNA repair efficacy and overcame resistance to chemotherapy. In summary, our work identifies NBS1 lactylation as a critical mechanism for genome stability that contributes to chemotherapy resistance and identifies inhibition of lactate production as a promising therapeutic cancer strategy.

CYR61 Acts as an Intracellular Microtubule-Associated Protein and Coordinates Mitotic Progression via PLK1-FBW7 Pathway.

Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61 remains unclear. Here, we found that CYR61 is important for proper cell cycle progression. Specifically, CYR61 interacts with microtubules and promotes microtubule polymerization to ensure mitotic entry. Moreover, CYR61 interacts with PLK1 and accumulates during the mitotic process, followed by degradation as mitosis concludes. The proteolysis of CYR61 requires the PLK1 kinase activity, which directly phosphorylates two conserved motifs on CYR61, enhancing its interaction with the SCF E3 complex subunit FBW7 and mediating its degradation by the proteasome. Mutations of phosphorylation sites of Ser167 and Ser188 greatly increase CYR61's stability, while deletion of CYR61 extends prophase and metaphase and delays anaphase onset. In summary, our findings highlight the precise control of the intracellular CYR61 by the PLK1-FBW7 pathway, accentuating its significance as a microtubule-associated protein during mitotic progression.

Transcription factor EHF interacting with coactivator AJUBA aggravates malignancy and acts as a therapeutic target for gastroesophageal adenocarcinoma.

Transcriptional dysregulation of genes is a hallmark of tumors and can serve as targets for cancer drug development. However, it is extremely challenging to develop small-molecule inhibitors to target abnormally expressed transcription factors (TFs) except for the nuclear receptor family of TFs. Little is known about the interaction between TFs and transcription cofactors in gastroesophageal adenocarcinoma (GEA) or the therapeutic effects of targeting TF and transcription cofactor complexes. In this study, we found that ETS homologous factor (EHF) expression is promoted by a core transcriptional regulatory circuitry (CRC), specifically ELF3-KLF5-GATA6, and interference with its expression suppressed the malignant biological behavior of GEA cells. Importantly, we identified Ajuba LIM protein (AJUBA) as a new coactivator of EHF that cooperatively orchestrates transcriptional network activity in GEA. Furthermore, we identified KRAS signaling as a common pathway downstream of EHF and AJUBA. Applicably, dual targeting of EHF and AJUBA by lipid nanoparticles cooperatively attenuated the malignant biological behaviors of GEA in vitro and in vivo. In conclusion, EHF is upregulated by the CRC and promotes GEA malignancy by interacting with AJUBA through the KRAS pathway. Targeting of both EHF and its coactivator AJUBA through lipid nanoparticles is a novel potential therapeutic strategy.

Mitophagy is a novel protective mechanism for drug-tolerant persister (DTP) cancer cells.

Drug-tolerant persister (DTP) cancer cells drive residual tumor and relapse. However, the mechanisms underlying DTP state development are largely unexplored. In a recent study, we determined that PINK1-mediated mitophagy favors DTP generation in the context of MAPK inhibition therapy. DTP cells that persist in the presence of a MAPK inhibitor exhibit mitochondriadependent metabolism. During DTP state development, MYC depletion alleviates the transcriptional repression of PINK1, resulting in PINK1 upregulation and mitophagy activation. PINK1-mediated mitophagy is essential for mitochondrial homeostasis in DTP cells. Either knockdown of PINK1 or inhibition of mitophagy eradicates DTP cells and achieves complete responses to MAPK inhibition therapy. This study reveals a novel role of mitophagy as a protective mechanism for DTP development.

PINK1-Mediated Mitophagy Promotes Oxidative Phosphorylation and Redox Homeostasis to Induce Drug-Tolerant Persister Cancer Cells.

The drug-tolerant persister (DTP) state enables cancer cells to evade cytotoxic stress from anticancer therapy. However, the mechanisms governing DTP generation remain poorly understood. Here, we observed that lung adenocarcinoma (LUAD) cells and organoids entered a quiescent DTP state to survive MAPK inhibitor treatment. DTP cells following MAPK inhibition underwent a metabolic switch from glycolysis to oxidative phosphorylation (OXPHOS). PTEN-induced kinase 1 (PINK1), a serine/threonine kinase that initiates mitophagy, was upregulated to maintain mitochondrial homeostasis during DTP generation. PINK1-mediated mitophagy supported DTP cell survival and contributed to poor prognosis. Mechanistically, MAPK pathway inhibition resulted in MYC-dependent transcriptional upregulation of PINK1, leading to mitophagy activation. Mitophagy inhibition using either clinically applicable chloroquine or depletion of PINK1 eradicated drug tolerance and allowed complete response to MAPK inhibitors. This study uncovers PINK1-mediated mitophagy as a novel tumor protective mechanism for DTP generation, providing a therapeutic opportunity to eradicate DTP and achieve complete responses. SIGNIFICANCE: DTP cancer cells that cause relapse after anticancer therapy critically depend on PINK1-mediated mitophagy and metabolic reprogramming, providing a therapeutic opportunity to eradicate persister cells to prolong treatment efficacy.

RNA-binding protein MEX3A controls G1/S transition via regulating the RB/E2F pathway in clear cell renal cell carcinoma.

MEX3A is an RNA-binding protein that mediates mRNA decay through binding to 3' untranslated regions. However, its role and mechanism in clear cell renal cell carcinoma remain unknown. In this study, we found that MEX3A expression was transcriptionally activated by ETS1 and upregulated in clear cell renal cell carcinoma. Silencing MEX3A markedly reduced clear cell renal cell carcinoma cell proliferation in vitro and in vivo. Inhibiting MEX3A induced G1/S cell-cycle arrest. Gene set enrichment analysis revealed that E2F targets are the central downstream pathways of MEX3A. To identify MEX3A targets, systematic screening using enhanced cross-linking and immunoprecipitation sequencing, and RNA-immunoprecipitation sequencing assays were performed. A network of 4,000 genes was identified as potential targets of MEX3A. Gene ontology analysis of upregulated genes bound by MEX3A indicated that negative regulation of the cell proliferation pathway was highly enriched. Further assays indicated that MEX3A bound to the CDKN2B 3' untranslated region, promoting its mRNA degradation. This leads to decreased levels of CDKN2B and an uncontrolled cell cycle in clear cell renal cell carcinoma, which was confirmed by rescue experiments. Our findings revealed that MEX3A acts as a post-transcriptional regulator of abnormal cell-cycle progression in clear cell renal cell carcinoma.

Circular RNA circHERC4 as a novel oncogenic driver to promote tumor metastasis via the miR-556-5p/CTBP2/E-cadherin axis in colorectal cancer.

BACKGROUND: The main cause of death in colorectal cancer patients is metastasis. Accumulating evidences suggest that circRNA plays pivotal roles in cancer initiation and development. However, the underlying molecular mechanisms of circRNAs that orchestrate cancer metastasis remain vague and need further clarification. METHODS: Two paired CRC and adjacent normal tissues were used to screen the upregulated circRNAs by circRNA-seq; then, cell invasion assay was applied to confirm the functional invasion-related circRNAs. According to the above methods, circHERC4 (hsa_circ_0007113) was selected for further research. Next, we investigated the clinical significance of circHERC4 in a large cohort of patients with CRC. The oncogenic activity of circHERC4 was investigated in both CRC cell lines and animal xenograft studies. Finally, we explored the molecular mechanisms underlying circHERC4 as a malignant driver. RESULTS: We demonstrated that circHERC4 was aberrantly elevated in CRC tissues (P < 0.001), and was positively associated with lymph node metastasis and advanced tumor grade (P < 0.01). Notably, the expression of circHERC4 was associated with worse survival in patients with CRC. Silencing of circHERC4 significantly inhibited the proliferation and migration of two highly aggressive CRC cell lines and reduced liver and lung metastasis in vivo. Mechanistically, we revealed that circHERC4 inactivated the tumor suppressor, miR-556-5p, leading to the activation of CTBP2/E-cadherin pathway which promotes tumor metastasis in CRC. CONCLUSIONS: CircHERC4 exerts critical roles in promoting tumor aggressiveness through miR-556-5p/CTBP2/E-cadherin pathway and is a prognostic biomarker of the disease, suggesting that circHERC4 may serve as an exploitable therapeutic target for patients with CRC.

Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon.

BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown. RESULTS: We found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3'-end. Consequently, the secondary structures were changed, the RNA-protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3'-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs. CONCLUSIONS: Our study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study.

Activation of bivalent factor DLX5 cooperates with master regulator TP63 to promote squamous cell carcinoma.

To reconstruct systematically hyperactive transcription factor (TF)-dependent transcription networks in squamous cell carcinomas (SCCs), a computational method (ELMER) was applied to 1293 pan-SCC patient samples, and 44 hyperactive SCC TFs were identified. As a top candidate, DLX5 exhibits a notable bifurcate re-configuration of its bivalent promoter in cancer. Specifically, DLX5 maintains a bivalent state in normal tissues; its promoter is hypermethylation, leading to DLX5 transcriptional silencing in esophageal adenocarcinoma (EAC). In stark contrast, DLX5 promoter gains active histone marks and becomes transcriptionally activated in ESCC, which is directly mediated by SOX2. Functionally, silencing of DLX5 substantially inhibits SCC viability both in vitro and in vivo. Mechanistically, DLX5 cooperates with TP63 in regulating ∼2000 enhancers and promoters, which converge on activating cancer-promoting pathways. Together, our data establish a novel and strong SCC-promoting factor and elucidate a new epigenomic mechanism - bifurcate chromatin re-configuration - during cancer development.

CEBPG promotes esophageal squamous cell carcinoma progression by enhancing PI3K-AKT signaling.

CCAAT/enhancer binding proteins (CEBPs, including CEBPA, CEBPB, CEBPD, CEBPE, CEBPG, and CEBPZ) play critical roles in a variety of physiological and pathological processes. However, the molecular characteristics and biological significance of CEBPs in esophageal squamous cell carcinoma (ESCC) have rarely been reported. Here, we show that most of the CEBPs are upregulated and accompanied with copy number amplifications in ESCC. Of note, high CEBPG expression is regulated by the ESCC specific transcription factor TP63 and serves as a prognostic factor for poor survival in ESCC patients. Functionally, CEBPG significantly promotes the proliferation and migration of ESCC cells both in vitro and in vivo. Mechanistically, CEBPG activates the PI3K-AKT signaling pathway through directly binding to distal enhancers and/or promoters of genes involved in this pathway, including genes of CCND1, MYC, CDK2, etc. These findings provide new insights into CEBPs dysregulation in ESCC and elucidate a crucial role for CEBPG in the progression of ESCC, highlighting its potential therapeutic value for ESCC treatment.

A Single-Cell Transcriptomic Atlas of Human Neocortical Development during Mid-gestation.

We performed RNA sequencing on 40,000 cells to create a high-resolution single-cell gene expression atlas of developing human cortex, providing the first single-cell characterization of previously uncharacterized cell types, including human subplate neurons, comparisons with bulk tissue, and systematic analyses of technical factors. These data permit deconvolution of regulatory networks connecting regulatory elements and transcriptional drivers to single-cell gene expression programs, significantly extending our understanding of human neurogenesis, cortical evolution, and the cellular basis of neuropsychiatric disease. We tie cell-cycle progression with early cell fate decisions during neurogenesis, demonstrating that differentiation occurs on a transcriptomic continuum; rather than only expressing a few transcription factors that drive cell fates, differentiating cells express broad, mixed cell-type transcriptomes before telophase. By mapping neuropsychiatric disease genes to cell types, we implicate dysregulation of specific cell types in ASD, ID, and epilepsy. We developed CoDEx, an online portal to facilitate data access and browsing.

The Dynamic Landscape of Open Chromatin during Human Cortical Neurogenesis.

Non-coding regions comprise most of the human genome and harbor a significant fraction of risk alleles for neuropsychiatric diseases, yet their functions remain poorly defined. We created a high-resolution map of non-coding elements involved in human cortical neurogenesis by contrasting chromatin accessibility and gene expression in the germinal zone and cortical plate of the developing cerebral cortex. We link distal regulatory elements (DREs) to their cognate gene(s) together with chromatin interaction data and show that target genes of human-gained enhancers (HGEs) regulate cortical neurogenesis and are enriched in outer radial glia, a cell type linked to human cortical evolution. We experimentally validate the regulatory effects of predicted enhancers for FGFR2 and EOMES. We observe that common genetic variants associated with educational attainment, risk for neuropsychiatric disease, and intracranial volume are enriched within regulatory elements involved in cortical neurogenesis, demonstrating the importance of this early developmental process for adult human cognitive function.

Chromosome conformation elucidates regulatory relationships in developing human brain.

Three-dimensional physical interactions within chromosomes dynamically regulate gene expression in a tissue-specific manner. However, the 3D organization of chromosomes during human brain development and its role in regulating gene networks dysregulated in neurodevelopmental disorders, such as autism or schizophrenia, are unknown. Here we generate high-resolution 3D maps of chromatin contacts during human corticogenesis, permitting large-scale annotation of previously uncharacterized regulatory relationships relevant to the evolution of human cognition and disease. Our analyses identify hundreds of genes that physically interact with enhancers gained on the human lineage, many of which are under purifying selection and associated with human cognitive function. We integrate chromatin contacts with non-coding variants identified in schizophrenia genome-wide association studies (GWAS), highlighting multiple candidate schizophrenia risk genes and pathways, including transcription factors involved in neurogenesis, and cholinergic signalling molecules, several of which are supported by independent expression quantitative trait loci and gene expression analyses. Genome editing in human neural progenitors suggests that one of these distal schizophrenia GWAS loci regulates FOXG1 expression, supporting its potential role as a schizophrenia risk gene. This work provides a framework for understanding the effect of non-coding regulatory elements on human brain development and the evolution of cognition, and highlights novel mechanisms underlying neuropsychiatric disorders.

A quantitative framework to evaluate modeling of cortical development by neural stem cells.

Neural stem cells have been adopted to model a wide range of neuropsychiatric conditions in vitro. However, how well such models correspond to in vivo brain has not been evaluated in an unbiased, comprehensive manner. We used transcriptomic analyses to compare in vitro systems to developing human fetal brain and observed strong conservation of in vivo gene expression and network architecture in differentiating primary human neural progenitor cells (phNPCs). Conserved modules are enriched in genes associated with ASD, supporting the utility of phNPCs for studying neuropsychiatric disease. We also developed and validated a machine learning approach called CoNTExT that identifies the developmental maturity and regional identity of in vitro models. We observed strong differences between in vitro models, including hiPSC-derived neural progenitors from multiple laboratories. This work provides a systems biology framework for evaluating in vitro systems and supports their value in studying the molecular mechanisms of human neurodevelopmental disease.

Genome-wide analysis of a Wnt1-regulated transcriptional network implicates neurodegenerative pathways.

Wnt proteins are critical to mammalian brain development and function. The canonical Wnt signaling pathway involves the stabilization and nuclear translocation of ß-catenin; however, Wnt also signals through alternative, noncanonical pathways. To gain a systems-level, genome-wide view of Wnt signaling, we analyzed Wnt1-stimulated changes in gene expression by transcriptional microarray analysis in cultured human neural progenitor (hNP) cells at multiple time points over a 72-hour time course. We observed a widespread oscillatory-like pattern of changes in gene expression, involving components of both the canonical and the noncanonical Wnt signaling pathways. A higher-order, systems-level analysis that combined independent component analysis, waveform analysis, and mutual information-based network construction revealed effects on pathways related to cell death and neurodegenerative disease. Wnt effectors were tightly clustered with presenilin1 (PSEN1) and granulin (GRN), which cause dominantly inherited forms of Alzheimer's disease and frontotemporal dementia (FTD), respectively. We further explored a potential link between Wnt1 and GRN and found that Wnt1 decreased GRN expression by hNPs. Conversely, GRN knockdown increased WNT1 expression, demonstrating that Wnt and GRN reciprocally regulate each other. Finally, we provided in vivo validation of the in vitro findings by analyzing gene expression data from individuals with FTD. These unbiased and genome-wide analyses provide evidence for a connection between Wnt signaling and the transcriptional regulation of neurodegenerative disease genes.

Expression of connective tissue growth factor (CTGF/CCN2) in breast cancer cells is associated with increased migration and angiogenesis.

Connective tissue growth factor (CTGF/CCN2) belongs to the CCN family of matricellular proteins, comprising Cyr61, CTGF, NovH and WISP1-3. The CCN proteins contain an N-terminal signal peptide followed by four conserved domains sharing sequence similarities with the insulin-like growth factor binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a C-terminal growth factor cysteine knot domain. To investigate the role of CCN2 in breast cancer, we transfected MCF-7 cells with full-length CCN2, and with four mutant constructs in which one of the domains had been deleted. MCF-7 cells stably expressing full-length CCN2 demonstrated reduced cell proliferation, increased migration in Boyden chamber assays and promoted angiogenesis in chorioallantoic membrane assays compared to control cells. Deletion of the C-terminal cysteine knot domain, but not of any other domain-deleted mutants, abolished activities mediated by full-length CCN2. We have dissected the role of CCN2 in breast tumorigenesis on a structural basis.

Connective tissue growth factor associated with oncogenic activities and drug resistance in glioblastoma multiforme.

Connective tissue growth factor (CTGF or CCN2) is a secreted protein that belongs to the CCN [cysteine-rich CYR61/CTGF/nephroblastoma-overexpressed gene] family. These proteins have been implicated in various biological processes, including stimulation of cell proliferation, migration, angiogenesis and tumorigenesis. In a previous study, we found that CTGF mRNA was elevated in primary gliomas, and a significant correlation existed between CTGF mRNA levels versus tumor grade, histology and patient survival. In this study, the role of CTGF in glioma tumorigenesis was explored. Forced expression of CTGF in glioblastoma multiforme (GBM) cells accelerated their growth in liquid culture and soft agar, stimulated cells migration in Boyden chamber assays and significantly increased their ability to form large, vascularized tumors in nude mice. CTGF induced the expression of the antiapoptotic proteins, Bcl-xl, Survivin and Flip. Overexpression of CTGF caused the U343 GBM cells to survive for longer than 40 days in serum-free medium and resist antitumor drugs including tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand, VELCADE (bortezomib, proteasome inhibitor) and temozolomide. Our data suggest that CTGF plays an important role in glioma progression, by supporting tumor cells survival and drug resistance.

Functional genomic analysis of frataxin deficiency reveals tissue-specific alterations and identifies the PPARgamma pathway as a therapeutic target in Friedreich's ataxia.

Friedreich's ataxia (FRDA), the most common inherited ataxia, is characterized by focal neurodegeneration, diabetes mellitus and life-threatening cardiomyopathy. Frataxin, which is significantly reduced in patients with this recessive disorder, is a mitochondrial iron-binding protein, but how its deficiency leads to neurodegeneration and metabolic derangements is not known. We performed microarray analysis of heart and skeletal muscle in a mouse model of frataxin deficiency, and found molecular evidence of increased lipogenesis in skeletal muscle, and alteration of fiber-type composition in heart, consistent with insulin resistance and cardiomyopathy, respectively. Since the peroxisome proliferator-activated receptor gamma (PPARgamma) pathway is known to regulate both processes, we hypothesized that dysregulation of this pathway could play a key role in frataxin deficiency. We confirmed this by showing a coordinate dysregulation of the PPARgamma coactivator Pgc1a and transcription factor Srebp1 in cellular and animal models of frataxin deficiency, and in cells from FRDA patients, who have marked insulin resistance. Finally, we show that genetic modulation of the PPARgamma pathway affects frataxin levels in vitro, supporting PPARgamma as a novel therapeutic target in FRDA.