Enhanced ApcMin/+ adenoma formation after epithelial CUL4B deletion by recruitment of myeloid-derived suppressor cells.

Colorectal cancer (CRC) stands as a prevalent malignancy globally. A pivotal event in CRC pathogenesis involves the loss-of-function mutation in the APC gene, leading to the formation of benign polyps. Despite the well-established role of APC, the contribution of CUL4B to CRC initiation in the pre-tumorous stage remains poorly understood. In this investigation, we generated a murine model by crossing ApcMin/+ mice with Cul4bΔIEC mice to achieve specific deletion of Cul4b in the gut epithelium against an ApcMin/+ background. By employing histological methods, RNA-sequencing (RNA-seq), and flow cytometry, we assessed alterations and characterized the immune microenvironment. Our results unveiled that CUL4B deficiency in gut epithelium expedited ApcMin/+ adenoma formation. Notably, CUL4B in adenomas restrained the accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs). In vivo inhibition of MDSCs significantly delayed the growth of CUL4B deleted ApcMin/+ adenomas. Furthermore, the addition of MDSCs to in vitro cultured ApcMin/+; Cul4bΔIEC adenoma organoids mitigated their alterations. Mechanistically, CUL4B directly interacted with the promoter of Csf3, the gene encoding granulocyte-colony stimulating factor (G-CSF) by coordinating with PRC2. Inhibiting CUL4B epigenetically activated the expression of G-CSF, promoting the recruitment of MDSCs. These findings offer novel insights into the tumor suppressor-like roles of CUL4B in regulating ApcMin/+ adenomas, suggesting a potential therapeutic strategy for CRC initiation and progression in the context of activated Wnt signaling.

CUL4B mutations impair human cortical neurogenesis through PP2A-dependent inhibition of AKT and ERK.

Mutation in CUL4B gene is one of the most common causes for X-linked intellectual disability (XLID). CUL4B is the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complex. While the roles of CUL4B in cancer progression and some developmental processes like adipogenesis, osteogenesis, and spermatogenesis have been studied, the mechanisms underlying the neurological disorders in patients with CUL4B mutations are poorly understood. Here, using 2D neuronal culture and cerebral organoids generated from the patient-derived induced pluripotent stem cells and their isogenic controls, we demonstrate that CUL4B is required to prevent premature cell cycle exit and precocious neuronal differentiation of neural progenitor cells. Moreover, loss-of-function mutations of CUL4B lead to increased synapse formation and enhanced neuronal excitability. Mechanistically, CRL4B complex represses transcription of PPP2R2B and PPP2R2C genes, which encode two isoforms of the regulatory subunit of protein phosphatase 2 A (PP2A) complex, through catalyzing monoubiquitination of H2AK119 in their promoter regions. CUL4B mutations result in upregulated PP2A activity, which causes inhibition of AKT and ERK, leading to premature cell cycle exit. Activation of AKT and ERK or inhibition of PP2A activity in CUL4B mutant organoids rescues the neurogenesis defect. Our work unveils an essential role of CUL4B in human cortical development.

Death and survival from executioner caspase activation.

Executioner caspases are evolutionarily conserved regulators of cell death under apoptotic stress. Activated executioner caspases drive apoptotic cell death through cleavage of diverse protein substrates or pyroptotic cell death in the presence of gasdermin E. On the other hand, activation of executioner caspases can also trigger pro-survival and pro-proliferation signals. In recent years, a growing body of studies have demonstrated that cells can survive from executioner caspase activation in response to stress and that the survivors undergo molecular and phenotypic alterations. This review focuses on death and survival from executioner caspase activation, summarizing the role of executioner caspases in apoptotic and pyroptotic cell death and discussing the potential mechanism and consequences of survival from stress-induced executioner caspase activation.

CUL4B functions as a tumor suppressor in KRAS-driven lung tumors by inhibiting the recruitment of myeloid-derived suppressor cells.

Lung cancer is the leading cause of cancer-related death worldwide. KRAS mutations are the most common oncogenic alterations found in lung cancer. Unfortunately, treating KRAS-mutant lung adenocarcinoma (ADC) remains a major oncotherapeutic challenge. Here, we used both autochthonous and transplantable KRAS-mutant tumor models to investigate the role of tumor-derived CUL4B in KRAS-driven lung cancers. We showed that knockout or knockdown of CUL4B promotes lung ADC growth and progression in both models. Mechanistically, CUL4B directly binds to the promoter of Cxcl2 and epigenetically represses its transcription. CUL4B deletion increases the expression of CXCL2, which binds to CXCR2 on myeloid-derived suppressor cells (MDSCs) and promotes their migration to the tumor microenvironment. Targeting of MDSCs significantly delayed the growth of CUL4B knockdown KRAS-mutant tumors. Collectively, our study provides mechanistic insights into the novel tumor suppressor-like functions of CUL4B in regulating KRAS-driven lung tumor development.

CUL4B enhances the malignant phenotype of esophageal squamous cell carcinoma by suppressing TGFBR3 expression.

Cullin 4B (CUL4B), which acts as a scaffold protein in CUL4B-RING ubiquitin ligase complexes (CRL4B), is frequently overexpressed in cancer and represses tumor suppressors through epigenetic mechanisms. However, the expression and function of CUL4B in esophageal squamous cell carcinoma (ESCC) have not been well illustrated. In this study, we show that upregulation of CUL4B in ESCC cells enhances proliferation, invasion and cisplatin (CDDP)-resistance, while knockdown of CUL4B significantly represses the malignant activities. Mechanistically, we demonstrate that CUL4B promotes proliferation and migration of ESCC cells through inhibiting expression of transforming growth factor beta receptor III (TGFBR3). CRL4B complex binds to the promoter of TGFBR3, and represses its transcription by catalyzing monoubiquitination at H2AK119 and coordinating with PRC2 and HDAC complexes. Taken together, our findings establish a critical role for the CUL4B/TGFBR3 axis in the regulation of ESCC malignancy.

CUL4B orchestrates mesenchymal stem cell commitment by epigenetically repressing KLF4 and C/EBPδ.

Dysregulated lineage commitment of mesenchymal stem cells (MSCs) contributes to impaired bone formation and an imbalance between adipogenesis and osteogenesis during skeletal aging and osteoporosis. The intrinsic cellular mechanism that regulates MSC commitment remains unclear. Here, we identified Cullin 4B (CUL4B) as a critical regulator of MSC commitment. CUL4B is expressed in bone marrow MSCs (BMSCs) and downregulated with aging in mice and humans. Conditional knockout of Cul4b in MSCs resulted in impaired postnatal skeletal development with low bone mass and reduced bone formation. Moreover, depletion of CUL4B in MSCs aggravated bone loss and marrow adipose accumulation during natural aging or after ovariectomy. In addition, CUL4B deficiency in MSCs reduced bone strength. Mechanistically, CUL4B promoted osteogenesis and inhibited adipogenesis of MSCs by repressing KLF4 and C/EBPδ expression, respectively. The CUL4B complex directly bound to Klf4 and Cebpd and epigenetically repressed their transcription. Collectively, this study reveals CUL4B-mediated epigenetic regulation of the osteogenic or adipogenic commitment of MSCs, which has therapeutic implications in osteoporosis.

Reshaping immunometabolism in the tumour microenvironment to improve cancer immunotherapy.

The evolving understanding of cellular metabolism has revealed a the promise of strategies aiming to modulate anticancer immunity by targeting metabolism. The combination of metabolic inhibitors with immune checkpoint blockade (ICB), chemotherapy and radiotherapy may offer new approaches to cancer treatment. However, it remains unclear how these strategies can be better utilized despite the complex tumour microenvironment (TME). Oncogene-driven metabolic changes in tumour cells can affect the TME, limiting the immune response and creating many barriers to cancer immunotherapy. These changes also reveal opportunities to reshape the TME to restore immunity by targeting metabolic pathways. Further exploration is required to determine how to make better use of these mechanistic targets. Here, we review the mechanisms by which tumour cells reshape the TME and cause immune cells to transition into an abnormal state by secreting multiple factors, with the ultimate goal of proposing targets and optimizing the use of metabolic inhibitors. Deepening our understanding of changes in metabolism and immune function in the TME will help advance this promising field and enhance immunotherapy.

Anastasis enhances metastasis and chemoresistance of colorectal cancer cells through upregulating cIAP2/NFκB signaling.

Chemotherapy is a common strategy to treat cancer. However, acquired resistance and metastasis are the major obstacles to successful treatment. Anastasis is a process by which cells survive executioner caspase activation when facing apoptotic stress. Here we demonstrate that colorectal cancer cells can undergo anastasis after transient exposure to chemotherapeutic drugs. Using a lineage tracing system to label and isolate cells that have experienced executioner caspase activation in response to drug treatment, we show that anastasis grants colorectal cancer cells enhanced migration, metastasis, and chemoresistance. Mechanistically, treatment with chemotherapeutic drugs induces upregulated expression of cIAP2 and activation of NFκB, which are required for cells to survive executioner caspase activation. The elevated cIAP2/NFκB signaling persists in anastatic cancer cells to promote migration and chemoresistance. Our study unveils that cIAP2/NFκB-dependent anastasis promotes acquired resistance and metastasis after chemotherapy.

Chemotherapy-induced executioner caspase activation increases breast cancer malignancy through epigenetic de-repression of CDH12.

Cancer relapse and metastasis are major obstacles for effective treatment. One important mechanism to eliminate cancer cells is to induce apoptosis. Activation of executioner caspases is the key step in apoptosis and was considered "a point of no return". However, in recent years, accumulating evidence has demonstrated that cells can survive executioner caspase activation in response to apoptotic stimuli through a process named anastasis. Here we show that breast cancer cells that have survived through anastasis (anastatic cells) after exposure to chemotherapeutic drugs acquire enhanced proliferation and migration. Mechanistically, cadherin 12 (CDH12) is persistently upregulated in anastatic cells and promotes breast cancer malignancy via activation of ERK and CREB. Moreover, we demonstrate that executioner caspase activation induced by chemotherapeutic drugs results in loss of DNA methylation and repressive histone modifications in the CDH12 promoter region, leading to increased CDH12 expression. Our work unveils the mechanism underlying anastasis-induced enhancement in breast cancer malignancy, offering new therapeutic targets for preventing post-chemotherapy cancer relapse and metastasis.

Depletion of CUL4B in macrophages ameliorates diabetic kidney disease via miR-194-5p/ITGA9 axis.

Diabetic kidney disease (DKD) is the most prevalent chronic kidney disease. Macrophage infiltration in the kidney is critical for the progression of DKD. However, the underlying mechanism is far from clear. Cullin 4B (CUL4B) is the scaffold protein in CUL4B-RING E3 ligase complexes. Previous studies have shown that depletion of CUL4B in macrophages aggravates lipopolysaccharide-induced peritonitis and septic shock. In this study, using two mouse models for DKD, we demonstrate that myeloid deficiency of CUL4B alleviates diabetes-induced renal injury and fibrosis. In vivo and in vitro analyses reveal that loss of CUL4B suppresses migration, adhesion, and renal infiltration of macrophages. Mechanistically, we show that high glucose upregulates CUL4B in macrophages. CUL4B represses expression of miR-194-5p, which leads to elevated integrin α9 (ITGA9), promoting migration and adhesion. Our study suggests the CUL4B/miR-194-5p/ITGA9 axis as an important regulator for macrophage infiltration in diabetic kidneys.

CRL4B complex-mediated H2AK119 monoubiquitination restrains Th1 and Th2 cell differentiation.

CD4+ T helper (Th) cell differentiation is regulated by lineage-specific expression of transcription factors, which is tightly associated with epigenetic modifications, including histone acetylation and methylation. However, the factors regulating histone modifications involved in Th cell differentiation remain largely unknown. We herein demonstrated a critical role of Cullin 4B (CUL4B) in restricting Th1 and Th2 cell differentiation. CUL4B, which is assembled into the CUL4B-RING E3 ligase (CRL4B) complex, participates in various physiological and developmental processes through epigenetic repression of transcription. Depletion of Cul4b in CD4+ T cells enhanced Th1 and Th2 cell differentiation. In vivo, an aggravated Th2 response caused by the absence of CUL4B was observed in a murine asthma model. Mechanistically, the CRL4B complex promoted monoubiquitination at H2AK119 (H2AK119ub1) and polycomb repressive complex 2 (PRC2)-mediated trimethylation at H3K27 (H3K27me3) at Tbx21 and Maf and consequently repressed their expression during Th cell differentiation. Our study suggests that CRL4B complex-mediated H2AK119ub1 deposition functions to prevent the aberrant expression of Th1 and Th2 lineage-specific genes.

Anastasis confers ovarian cancer cells increased malignancy through elevated p38 MAPK activation.

Activation of executioner caspases was once considered as a point of no return in apoptosis. However, in recent years, accumulating evidence has demonstrated that cells can survive executioner caspase activation in response to apoptotic stimuli through a process called anastasis. In this study, we developed a reporter system, mCasExpress, to track mammalian cells that survive executioner caspase activation. We demonstrate that anastatic ovarian cancer cells acquire enhanced migration following their transient exposure to apoptotic stimulus TRAIL or Paclitaxel. Moreover, anastatic cancer cells secrete more pro-angiogenic factors that enable tumor angiogenesis, growth and metastasis. Mechanistically, we demonstrate that activation of p38 MAPK, which occurs in a caspase-dependent manner in response to apoptotic stress to promote anastasis, persists at a higher level in anastatic cancer cells even after removal of apoptotic stimuli. Importantly, p38 is essential for the elevated migratory and angiogenic capacity in the anastatic cells. Our work unveils anastasis as a potential driver of tumor angiogenesis and metastasis.

Correlation between RASSF1A Methylation in Cell-Free DNA and the Prognosis of Cancer Patients: A Systematic Review and Meta-Analysis.

Background: Although the effects of methylation of the Ras association domain-containing protein 1 isoform A (RASSF1A) gene in cell-free DNA on the outcomes of patients with different types of cancer have been reported, the results are inconsistent. Objective: : To explore the relationships between RASSF1A methylation in cell-free DNA and the outcomes of cancer patients. Methods: The PubMed, Embase, and Web of Science databases were searched for papers related to this topic on December 8, 2021. The retrieved articles were screened by two independent researchers, following which the methodological quality of the selected studies was evaluated using the Newcastle-Ottawa Scale. Additionally, hazard ratios were calculated, and publication bias of the studies was determined using Egger's test. Results: Nine relevant publications involving a combined total of 1254 patients with different types of cancer were included in this study. The combined results of the random effects models yielded a hazard ratio of 1.73 (95% confidence interval: 1.31, 2.29; P < 0.001), which suggested there was a significant association between RASSF1A methylation and overall survival, and patients with an RASSF1A methylation status had a significantly increased risk of total death. Moreover, the Egger test result suggested there was no significant publication bias among the included studies. Conclusions: The methylation of RASSF1A in cell-free DNA in cancer patients was observably associated with an increased risk of poor overall survival.

Cullin 4b-RING ubiquitin ligase targets IRGM1 to regulate Wnt signaling and intestinal homeostasis.

Hierarchical organization of intestine relies on the self-renewal and tightly regulated differentiation of intestinal stem cells (ISCs). Although signals like Wnt are known to sustain the continued intestinal renewal by maintaining ISCs activity and lineage commitment, molecular mechanisms underlying ISCs 'stemness' and supportive niche have not been well understood. Here, we found that CUL4B-RING ubiquitin ligase (CRL4B) regulates intestinal homeostasis by targeting immunity-related GTPase family M member 1 (IRGM1) for proteasomal degradation. CUL4B was mainly expressed at ISCs zone. Deletion of Cul4b led to reduced self-renewal of ISCs and a decreased lineage differentiation towards secretory progenitors through downregulated Wnt signals. Besides, Cul4b-null mice exhibited impaired Paneth cells number and structure. Mechanistically, CRL4B complex were associated with WD40 proteins and targeted IRGM1 at K270 for ubiquitination and proteosomal degradation. Impaired intestinal function caused by CUL4B deletion was rescued by down-regulation of its substrate IRGM1. Our results identified CUL4B as a novel regulator of ISCs and revealed a new 26 S proteasome degradation mechanism in intestine self-renewal and lineage commitment.

Profiling and Characterization of microRNAs Responding to Sodium Butyrate Treatment in Gastric Cancer Cells.

BACKGROUND: Short-chain fatty acids exert anti-cancer effects on tumor cells. OBJECTIVE: We aimed to reveal the signaling network altered by butyrate in Gastric Cancer (GC) using small RNA sequencing (sRNA-seq). METHODS: The effects of butyrate on the biological behavior of NCI-N87 and KATO III cells in vitro were assessed by functional assays and half-maximal inhibitory concentrations (IC50) of butyrate in KATO III cells were calculated. sRNA-seq was performed on KATO III cells. Differentially expressed miRNAs (DE-miRNAs) were identified between butyrate treatment and control groups using DESeq2, and miRNA targets were predicted. A protein-protein interaction (PPI) network of DE-miRNA targets was created using Metascape. Key MCODE complexes were identified using the MCODE algorithm and cluster Profiler. The relationship between DE-miRNA and GC overall survival (OS) was evaluated using Kaplan-Meier curves. RESULTS: Butyrate dose-dependently inhibited NCI-N87 and KATO III cell viability. KATO III cells were more sensitive to butyrate than NCI-N87 cells. Butyrate promoted apoptosis and inhibited KATO III cell migration. Total 324 DE-miRNAs were identified in KATO III cells, and 459 mRNAs were predicted as targets of 83 DE-miRNAs. Two key protein complexes were identified in a PPI network of the 459 targets. A key signaling network responding to butyrate was generated using targets in these key complexes and their miRNA regulators. The DE-miRNAs in the key signaling network were related to the OS of GC. CONCLUSION: Butyrate altered the biological behavior of GC cells, which may be achieved by regulating miRNAs and related oncogenic pathways.

miR-425 regulates lipophagy via SIRT1 to promote sorafenib resistance in liver cancer.

Liver cancer is one of the most malignant cancer, with poor outcomes and a high incidence rate, and current treatment approaches to prevent tumor progression and development remain unsatisfactory. Therefore, it is urgent to explore novel methods to inhibit tumor growth and metastasis. Autophagy is a highly conserved process associated with metastasis and drug resistance. Lipids are selectively recognized and degraded via autophagy; thus, autophagy is a crucial process to maintain tumor self-protection. MicroRNA (miR)-425 is a tumor-associated gene involved in liver cancer development that can induce cell proliferation and drug resistance. Using Cell Counting Kit-8 assays, western blot analysis and immunofluorescence assays, the present study revealed that inhibition of miR-425 promoted lipophagy by mediating the autophagy process, which in turn helps to promote sorafenib resistance. Using a bioinformatics website, it was revealed that autophagy promoted lipophagy by targeting silent information regulator 2 homolog 1 (SIRT1). The results of luciferase reporter assays supported this finding, and rescue experiments provided additional evidence. Overall, the current results suggested that inhibition of miR-425 expression increased SIRT1 expression to promote lipophagy, leading to the inhibition of liver cancer cell proliferation.

Prognostic Score Model Based on Ten Differentially Methylated Genes for Predicting Clinical Outcomes in Patients with Adenocarcinoma of the Colon.

PURPOSE: We aimed to screen novel genetic biomarkers for use in a prognostic score (PS) model for the accurate prediction of survival outcomes for patients with colon adenocarcinoma (COAD). METHODS: Gene expression and methylation data were downloaded from The Cancer Genome Atlas database, and the samples were randomly divided into training and validation sets for the screening of differentially methylated genes (DMGs) and differentially expressed genes (DEGs). Co-methylated genes were screened using weighted gene co-expression network analysis. Functional enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery. Univariate and multivariate Cox regression analyses were performed to identify prognosis-related genes and clinical factors. Receiver operating characteristic curve analysis was carried out to evaluate the predictive performance of the PS model. RESULTS: In total, 1434 DEGs and 1038 DMGs were screened in the training set, among which 284 were found to be overlapping genes. For 127 of these overlapping genes, the methylation and expression levels were significantly negatively correlated. An optimal signature from 10 DMGs was identified to construct the PS model. Patients with a high PS seemed to have worse outcomes than those with a low PS. Moreover, cancer recurrence and the PS model status were independent prognostic factors. CONCLUSION: This PS model based on an optimal 10-gene signature would help in the stratification of patients with COAD and improve the assessment of their clinical outcomes.

CUL4B Promotes Temozolomide Resistance in Gliomas by Epigenetically Repressing CDNK1A Transcription.

Resistance to temozolomide (TMZ), the first-line chemotherapeutic drug for glioblastoma (GBM) and anaplastic gliomas, is one of the most significant obstacles in clinical treatment. TMZ resistance is regulated by complex genetic and epigenetic networks. Understanding the mechanisms of TMZ resistance can help to identify novel drug targets and more effective therapies. CUL4B has been shown to be upregulated and promotes progression and chemoresistance in several cancer types. However, its regulatory effect and mechanisms on TMZ resistance have not been elucidated. The aim of this study was to decipher the role and mechanism of CUL4B in TMZ resistance. Western blot and public datasets analysis showed that CUL4B was upregulated in glioma specimens. CUL4B elevation positively correlated with advanced pathological stage, tumor recurrence, malignant molecular subtype and poor survival in glioma patients receiving TMZ treatment. CUL4B expression was correlated with TMZ resistance in GBM cell lines. Knocking down CUL4B restored TMZ sensitivity, while upregulation of CUL4B promoted TMZ resistance in GBM cells. By employing senescence ß-galactosidase staining, quantitative reverse transcription PCR and Chromatin immunoprecipitation experiments, we found that CUL4B coordinated histone deacetylase (HDAC) to co-occupy the CDKN1A promoter and epigenetically silenced CDKN1A transcription, leading to attenuation of TMZ-induced senescence and rendering the GBM cells TMZ resistance. Collectively, our findings identify a novel mechanism by which GBM cells develop resistance to TMZ and suggest that CUL4B inhibition may be beneficial for overcoming resistance.