Background:The ubiquitin domain-containing protein 1 (UBTD1) is involved in protein homeostasis and cell cycle regulation, and emerging evidence suggests its role in tumor biology. However, its function in ovarian cancer (OC) remains unclear. OC is highly lethal due to late diagnosis, metastasis, and platinum resistance. This study is aimed at investigating the role of UBTD1 in OC by integrating single-cell transcriptomics, mutation and HRD-related analyses, tumor microenvironment evaluation, and experimental validation. Methods:We examined UBTD1 expression, prognosis, somatic mutation features, and immune microenvironment characteristics utilizing public databases, such as the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Single-cell RNA-seq data were analyzed using Seurat, hdWGCNA, and CopyKAT to characterize UBTD1-associated malignant-cell states and inferred CNV burden. Based on the median UBTD1 expression, TCGA-OV samples were divided into high and low expression groups to compare mutation spectra, DNA repair pathway activity, stromal scores, and immune cell infiltration. Functional assays were performed in A2780 and SK-OV-3 cell lines following lentiviral shRNAs-mediated UBTD1 knockdown. RNA sequencing and rescue experiments were used to explore downstream pathways. Results:Integrated single-cell and TCGA-OV analyses revealed that UBTD1-low tumors were enriched for genomic instability-related features, including increased inferred CNV burden and higher tumor mutation burden. By comparison, UBTD1-high tumors showed increased stromal scores and modest enrichment of selected immune-cell populations, including macrophages and neutrophils. Experimentally, UBTD1 was upregulated in OC and associated with poor prognosis. UBTD1 knockdown inhibited malignant phenotypes, enhanced cisplatin sensitivity, promoted apoptosis, and suppressed TNF/AP-1/FOS-related signaling. FOS overexpression partially reversed the effects of UBTD1 silencing. Conclusions:UBTD1 expression defines distinct mutation and microenvironmental states in OC and functionally promotes malignant phenotypes, potentially through TNF/AP-1/FOS-related signaling.

The maintenance of excitatory synaptic activity is crucial for cognitive function and genetic mutations are responsible for the pathogenesis of related brain disorders. However, the roles of these pathogenic factors in synaptic dysregulation and cognitive malfunction are still poorly understood. In this study, a conditional knockout mouse model lacking ZC4H2-an X-linked gene implicated in ZC4H2-associated rare disorder (ZARD) -in forebrain excitatory neurons is generated and these mice exhibit cognitive malfunction, recapitulating the intellectual disability manifestation of ZARD. Mechanistically, ZC4H2 harbors a protein interaction network with key excitatory synaptic regulators and ZC4H2 interacts directly with AMPA receptors (AMPARs) and regulates their ubiquitination at the postsynaptic sites, thereby maintaining AMPARs protein stability and synaptic expression. ZC4H2 deficiency specifically and aberrantly increases AMPAR-mediated excitatory synaptic transmission and impairs synaptic plasticity of long-term potentiation. More importantly, pharmacological treatment with perampanel, an AMPAR-specific antagonist, successfully restores the excitatory synaptic activity and cognitive function of ZC4H2-deficient mice. Together, we establish that ZC4H2 is a postsynaptic regulator for AMPARs and excitatory synaptic activity and highlight that the dysregulation of these biological processes is a crucial etiology underlying ZARD-associated intellectual disability.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for approximately 85 % of cases, and is associated with high incidence and mortality rates. The presence of cancer stem cells (CSCs) contributes significantly to treatment resistance and poor patient prognosis. This study investigates the role of tubulin epsilon and delta complex 2 (TEDC2) in the progression and stemness of NSCLC. We found that TEDC2 was aberrantly overexpressed in various solid tumors, specifically in lung adenocarcinoma (LUAD), and its expression negatively correlated with patient prognosis. Functional assays demonstrated that the knockdown of TEDC2 inhibited the proliferation, migration, and maintenance of stemness in tumor cells, primarily through the inhibition of the Hedgehog (Hh) signaling pathway. Additionally, TEDC2 knockdown enhanced the sensitivity of NSCLC cells to chemotherapy drug cisplatin (DDP). In vivo animal models further demonstrated that knockdown of TEDC2 could inhibit tumor cell stemness and enhance the therapeutic efficacy of DDP. These findings highlight TEDC2 as a critical regulator promoting NSCLC progression, which may serve as a potential therapeutic target. Targeting TEDC2 could enhance the efficacy of existing treatment strategies, providing a new avenue for improving clinical outcomes of NSCLC patients.

Head and neck squamous cell carcinoma (HNSCC) is an epithelial carcinoma characterized by its distinct geographical distribution, exhibiting a higher prevalence in Southeast Asia. Despite the approval of immune checkpoint blockade (ICB) therapy for treating advanced recurrent HNSCC, the extent of patient benefit remains limited. Elucidating the molecular regulatory mechanisms of immunosuppressive tumor microenvironment in HNSCC is crucial for improving current treatment status and patient outcomes. Our findings show that knockdown of NCAPH suppresses cell proliferation, migration, and xenograft tumor growth, while enhancing radiotherapy-induced cellular apoptosis. Importantly, we found that NCAPH binds to PD-L1 and disrupts its degradation, competing with HIP1R (Huntingtin-interacting protein 1-related) and leading to the stabilization of PD-L1 protein, which contributes to the formation of immunosuppressive tumor microenvironment. To inhibit the interaction between NCAPH and PD-L1, we created a peptide known as NPIDP (NCAPH and PD-L1 Interaction Disrupting Peptide) that effectively disrupts the interaction between NCAPH and PD-L1. Furthermore, topotecan, a well-characterized topoisomerase I inhibitor, was identified to bind NCAPH and promote its proteasomal degradation. Notably, we demonstrated that NPIDP and topotecan suppress tumor immune evasion both in vitro and in vivo. In summary, our findings reveal the critical role of NCAPH in regulating tumor immune surveillance, suggesting that NCAPH could serve as a potential biomarker and therapeutic target for HNSCC in the future.

Non-small cell lung cancer (NSCLC) is a prevalent classification of human lung cancer with a variety of clinical pathological features. Several key factors and associated signaling pathways have played pivotal roles in the progression of NSCLC and serve as potential therapeutic targets. However, the therapeutic efficacy is still limited, and novel biomarkers and key regulators are inevitable. We found a human-specific long non-coding RNA (lncRNA, ENST00000504300) induced by the inflammatory pathway, termed SLC7A11AR (SLC7A11 associated lncRNA), which was highly expressed in lung adenocarcinoma (LUAD) cell lines but not in lung squamous cell carcinoma (LUSC). Our research showed that higher SLC7A11AR expression correlates with a poorer clinical prognosis. Depleting SLC7A11AR restrains tumor cell proliferation, migration, and xenograft tumor formation by promoting ferroptosis. Bioinformatic analysis and dual luciferase reporter assays revealed that SLC7A11AR binds directly to miR-150-5p, weakening the inhibition on its downstream target SLC7A11, a key ferroptosis inhibitor in NSCLC. In cancerous tissues, SLC7A11AR was upregulated, while miR-150-5p was downregulated compared to control tissues. Enforced miR-150-5p expression inhibited tumor growth. Moreover, ASOs against SLC7A11AR alone or with a ferroptosis agonist significantly suppressed tumor progression. Our results suggest that the SLC7A11AR/miR-150-5p/SLC7A11 axis plays an oncogenic role in LUAD development and has the potential to be novel therapeutic targets, presenting new opportunities for LUAD treatment in the future.

Prostate cancer (PCa) is a prevalent malignant tumor of the urinary system and remains the most common cancer among males. In this study, we showed that YTHDF1, one of the reader proteins involved in the N6-methyladenosine (m6A) modification signaling pathway, is highly expressed in PCa cancerous tissues and cells, which correlates with poor clinical outcomes. Our study revealed that YTHDF1 knockdown inhibits tumor cell proliferation, migration, and xenograft tumor formation by decreasing p27 protein stability through proteasome degradation signaling. Consistently, YTHDF1 depletion markedly reduced the clonogenic growth of Pten or/and TP53-deficient organoids. Candidate p27-targeting E3 ubiquitin ligases screening identified RNF7 as the direct downstream target for YTHDF1 in an m6A-dependent manner. The subsequent high translation of RNF7 results in the efficient degradation of the cell cycle inhibitor p27 and malignant tumor cell growth. In addition, we provided evidence showing that YTHDF1 or RNF7 depletion sensitizes tumor cells to chemotherapy drug cisplatin by increasing cellular apoptosis. Our findings revealed that the neddylation inhibitor MLN4924 effectively inhibited prostate cancer progression in vitro and in vivo. Our study highlights the YTHDF1/RNF7/p27 axis as a crucial component in PCa, suggesting its potential as a novel therapeutic target.

ObjectiveFamilial adenomatous polyposis (FAP) is a genetic syndrome characterized by multiple polyps at various evolutionary stages, which, if left untreated, inevitably progress to colorectal cancer (CRC). In this study, we present a comprehensive analysis of the evolutionary history of FAP-CRC from precancerous adenoma to carcinoma.DesignTissues were collected from gastrointestinal endoscopy or surgical resection. Exome sequencing was performed on multiple regions of adenocarcinoma (n = 8), villous adenoma (n = 10), tubular adenoma (n = 9) and blood samples were obtained from 9 patients belonging to 7 Chinese FAP families. Phylogenetic trees were reconstructed, and evolutionary analysis was conducted to reveal the temporal sequence of events leading to CRC.ResultsInherited germline mutation sites in APC gene were identified in FAP01 (p.S1281*, COSM19212), FAP03 (p.S384Tfs*19), FAP04 (p.E1538*, COSM6041693), FAP05 (p.Q1062*, COSM3696862), and FAP07-FAP09 (p.V677Sfs*3). Notably, p.V677Sfs*3 mutation was recognized as a novel germline mutation in APC, supported by evidence of genotype-phenotype correlation in pedigree analysis. Adenomas exhibited lower mutational rates than FAP-CRC and displayed recurrent alterations in well-known chromosomal instability (CIN) genes (APC, RAS, SMAD4 and TP53) and DNA damage repair genes (SUZ12, KMT2C, BCLAF1, RUNX1, and ARID1B), suggesting the presence of genomic instability. Furthermore, a progressive increase in the HRD score (a measure of “genomic scars”) was observed from tubular adenomas to villous adenomas and ultimately to carcinomas. TP53 emerged as the primary driver gene for adenoma-carcinoma transition, with driver mutations consistently appearing simultaneously rather than sequentially acquired from adenomas to carcinomas. Clonal evolution demonstrated that liver metastases can originate from the same cancer-primed cell present in a primary cancerous lesion.ConclusionWe identified a novel pathogenic variant in APC, namely, p.V677Sfs*3. The process of carcinogenesis in FAP-CRC supports the classical cancerization model, where an initial APC mutation leads to the activation of the WNT signaling pathway and CIN. Subsequently, additional mutations occur in other putative CIN genes (e.g., DNA repair, chromatin remodeling), ultimately leading to the development of microsatellite stable (MSS) tumors. Our study provides a comprehensive understanding of the genomic landscapes that underlie the transition from adenoma to carcinoma.

ObjectiveTo evaluate the association between a short-period, high-dose in utero aspirin exposure and child neurocognitive development.DesignA propensity score-matched analysis of a multicentre prospective cohort study.SettingThe US Collaborative Perinatal Project (1959-1976).PopulationA total of 50 565 singleton live births with maternal information.MethodsWe performed a propensity score matching to balance maternal characteristics between women with and without aspirin exposure. Inverse probability-weighted marginal structural models were used to estimate associations between aspirin exposure and child neurocognitive assessments.Main outcome measuresChild neurocognitive development was assessed using the Bayley Scales at 8 months, the Stanford Binet Intelligence Scale at 4 years, and the Wechsler Intelligence Scale and Wide-Range Achievement Test (WRAT) at 7 years.ResultsChildren exposed to aspirin in utero were associated with an 8%-16% reduced risk of having suspect/abnormal or below-average scores in most neurocognitive assessments. A trend of lower risks of having suspect/abnormal or below-average scores was further observed in children with in utero aspirin exposure for more than 7 days, particularly on Bayley Mental (relative risk [RR] 0.82, 95% CI 0.74-0.92), WRAT Reading (RR 0.88, 95% CI 0.78-0.98) and WRAT Arithmetic tests (RR 0.76, 95% CI 0.66-0.86). This association was mainly observed in the second trimester of pregnancy.ConclusionsIn utero aspirin exposure was associated with improved child neurocognitive development in a prospective cohort study. Further studies are warranted to evaluate the impact of long-period and low-dose in utero aspirin exposure on child short- and long-term neurodevelopment.

Abstract Gliomas, the most lethal brain tumors, often exhibit resistance to conventional chemotherapy and/or radiotherapy. This study reveals that sertindole, a potent dopamine D2 receptor antagonist primarily designed as an antipsychotic medication for schizophrenia, effectively inhibits glioma progression. Our findings demonstrate that sertindole suppresses the proliferation of U251 and U87 tumor cells, impedes cell cycle progression in vitro, and curtails xenograft tumor growth in vivo. Moreover, we present compelling evidence demonstrating the ability of sertindole to enhance the cellular response to the chemotherapeutic agent temozolomide both in vitro and in vivo. Additionally, our findings reveal that sertindole effectively suppresses the self‐renewal capacity and expression of stemness‐associated genes, such as Nanog and Sox2, in glioma tumor cells and glioma stem cells. A mechanistic investigation demonstrated that sertindole enhances the formation of autophagosome–lysosome complexes while concurrently impeding autophagic flux through the inhibition of lysosomal hydrolytic enzymes CTSD and CTSB, ultimately resulting in decreased growth of tumor cells. In conclusion, our findings suggest that sertindole has the potential to develop into a potent antiglioma therapeutic agent.

A cholic acid-conjugated oxaliplatin, LLC-202, is developed as a novel prodrug for liver cancer. The conjugate is obtained by using 3-NH2-cyclobutane-1,1-dicarboxylate as a linker between the oxaliplatin analogue and cholic acid moiety and cholic acid is strongly bonded to the linker via an amide bond. Pharmacokinetic experiment shows that LLC-202 is mainly distributed and accumulated in the liver after intravenous administration to Sprague-Dawley rats, revealing the liver-targeting profile. Compared to oxaliplatin, LLC-202 is more easily taken up by human liver cancer cells than normal human liver cells. LLC-202 exhibits higher in vitro anticancer activity and higher efficacy comparable to oxaliplatin in treating primary hepatocellular carcinoma in C57BL/6 mice. It can significantly prolong the survival time of tumor-bearing mice by inducing apoptosis and inhibiting proliferation of cancer cells. In addition, LLC-202 shows less cytotoxicity toward normal human liver cells than oxaliplatin. Its acute toxicity in healthy Kunming (KM) mice after i.v. administration is comparable to oxaliplatin. Histopathological examination reveals that the main toxicity of LLC-202 in mice is the depression of bone marrow hematopoietic cells. The results suggest that LLC-202 has great potential for further development as a new prodrug specific for liver cancer.

Gliomas are the most aggressive type of malignant brain tumors. Recent studies have demonstrated that the existence of glioma stem cells (GSCs) is critical for glioma recurrence, metastasis, and chemo- or radio-therapy resistance. Temozolomide (TMZ) has been used as an initial therapy for gliomas. However, the overall survival time is still limiting due to the lack of effective targets and treatment options. Therefore, identifying novel biomarkers for gliomas, especially for GSCs, is important to improve the clinical outcome in the future. In this study, we identify a human-specific long non-coding RNA (lncRNA, ENSG00000250377), termed GSCAR (glioma stem cell associated lncRNA), which is highly expressed in glioma cancerous tissues and cell lines. We reveal that GSCAR positively correlates with tumor grade. Glioma patients with GSCAR high expression exhibit shortened overall survival time, compared to patients with GSCAR low expression. Furthermore, we show that GSCAR knockdown by shRNAs or antisense oligonucleotide (ASO) reduces tumor cell proliferation, migration and xenograft tumor formation abilities. Mechanistic study shows that GSCAR acts as a ceRNA (competing endogenous RNA) for miR-6760-5p to promote the expression of oncogene SRSF1 (serine and arginine rich splicing factor 1). In addition, GSCAR mediates the protein complex formation between DHX9 (DExH-Box helicase 9) and IGF2BP2 (insulin-like growth factor 2 mRNA-binding protein 2), leading to the stabilization of SOX2 (sex-determining region Y-box 2) mRNA and then the transcriptional activation of GSCAR. Depleting GSCAR reduces SOX2 expression and GSC self-renewal ability, but promotes tumor cell responses to TMZ. These findings uncover that GSCAR/miR-6760-5p/SRSF1 axis and GSCAR/DHX9-IGF2BP2/SOX2 positive feedback loop are critical for glioma progression, which could be used as prognostic biomarkers and therapeutic targets in the future.

SARS-CoV-2 primary strain-based vaccination exerts a protective effect against Omicron variants-initiated infection, symptom occurrence, and disease severity in a booster-dependent manner. Yet, the underlying mechanisms remain unclear. During the 2022 Omicron outbreak in Shanghai, we enrolled 122 infected adults and 50 uninfected controls who had been unvaccinated or vaccinated with two or three doses of COVID-19 inactive vaccines and performed integrative analysis of 41-plex CyTOF, RNA-seq, and Olink on their peripheral blood samples. The frequencies of HLA-DRhi classical monocytes, non-classical monocytes, and Th1-like Tem tended to increase, whereas the frequency of Treg was reduced by booster vaccine, and they influenced symptom occurrence in a vaccine dose-dependent manner. Intercorrelation and mechanistic analysis suggested that the booster vaccination induced monocytic training, which would prime monocytic activation and maturation rather than differentiating into myeloid-derived suppressive cells upon Omicron infections. Overall, our study provides insights into how booster vaccination elaborates protective immunity across SARS-CoV-2 variants.

Objectives Despite of strenuous research in the past decades, the etiology of schizophrenia (SCZ) still remains incredibly controversial. Previous genetic analysis has uncovered a close association of Unc-51 like kinase 4 (ULK4), a family member of Unc-51-like serine/threonine kinase, with SCZ. However, animal behavior data which may connect Ulk4 deficiency with psychiatric disorders, particularly SCZ are still missing. Methods We generated Emx1-Cre:Ulk4flox/flox conditional knockout (CKO) mice, in which Ulk4 was deleted in the excitatory neurons of cerebral cortex and hippocampus. Results The cerebral cellular architecture was maintained but the spine density of pyramidal neurons was reduced in Ulk4 CKO mice. CKO mice showed deficits in the spatial and working memories and sensorimotor gating. Levels of p-Akt and p-GSK-3α/β were markedly reduced in the CKO mice indicating an elevation of GSK-3 signaling. Mechanistically, Ulk4 may regulate the GSK-3 signaling via putative protein complex comprising of two phosphatases, protein phosphatase 2A (PP2A) and 1α (PP1α). Indeed, the reduction of p-Akt and p-GSK-3α/β was rescued by administration of inhibitor acting on PP2A and PP1α in CKO mice. Conclusions Our data identified potential downstream signaling pathway of Ulk4, which plays important roles in the cognitive functions and when defective, may promote SCZ-like pathogenesis and behavioral phenotypes in mice.

Gliomas are the most aggressive primary brain tumors. However, no significant improvement in survival has been achieved with the addition of temozolomide (TMZ) or radiation as initial therapy, although many clinical efforts have been carried out to target various signaling pathways or putative driver mutations. Here, we report that glycosyltransferase 8 domain containing 1 (GLT8D1), induced by HIF-1α under a hypoxic niche, significantly correlates with a higher grade of glioma, and a worse clinical outcome. Depletion of GLT8D1 inhibits self-renewal of glioma stem cell (GSC) in vitro and represses tumor growth in glioma mouse models. GLT8D1 knockdown promotes cell cycle arrest at G2/M phase and cellular apoptosis with or without TMZ treatment. We reveal that GLT8D1 impedes CD133 degradation through the endosomal-lysosomal pathway by N-linked glycosylation and protein-protein interaction. Directly blocking the GLT8D1/CD133 complex formation by CD133N1~108 (referred to as FECD133), or inhibiting GLT8D1 expression by lercanidipine, suppresses Wnt/β-catenin signaling dependent tumorigenesis both in vitro and in patient-derived xenografts mouse model. Collectively, these findings offer mechanistic insights into how hypoxia promotes GLT8D1/CD133/Wnt/β-catenin signaling during glioma progression, and identify GLT8D1 as a potential therapeutic target in the future.

Objective:To analyze the autosomal dominant inheritance of five families of adenomatous polyposis (FAP).Methods:Five FAP families were investigated and examined. Peripheral blood and feces were taken for whole-exon sequencing to screen colorectal cancergenes. Colonoscopy was performed and biopsy tissues were taken for pathological analysis.Results:In the five families, FAP occurred in successive generations. When the father or mother had FAP, the probability of FAP occurred in the offspring was equal (50%) without gender difference. All the patients had adenomatous polyposis coli gene (APC) mutation. Some patients had more than two types of tumors.Conclusions:In families with a history of FAP, FAP generally does not develop in juveniles, and APC gene testing and colonoscopy are needed in adults. Once adenoma is found, endoscopic treatment should be performed as soon as possible to avoid the occurrence of colorectal cancer.

The incidence of cutaneous melanoma (CM) has been increasing annually worldwide. In this study, we identify that MrgprF, a MAS related GPR family member, is decreased in cutaneous melanoma tissues and cell lines due to hypermethylation of its promoter region, and show that patients with CM expressing high levels of MrgprF exhibit an improved clinical outcome. We demonstrate that MrgprF forced expression inhibits tumor cell proliferation, migration, xenograft tumor growth, and metastasis. On the contrary, MrgprF knockdown promotes tumor cell proliferation and transformation of immortalized human keratinocyte-HaCaT cells, supporting the inhibitory role of MrgprF during tumor progression. Mechanistic studies reveal that MrgprF reduces the phosphoinositol‑3‑kinase (PI3K) complex formation between p101 and p110γ subunits, the critical step for phosphatidylinositol-(3, 4)-P2 (PIP2) conversion to phosphatidylinositol-(3, 4, 5)-P3 (PIP3), and then reduces the activation of PI3K/Akt signaling. This effect can be reversed by Akt specific agonist SC79. In addition, AMG 706, a previously documented inhibitor for endothelial cell proliferation, is identified as a potential agonist for MrgprF, and can impede tumor growth both in vitro and in vivo. Taken together, our findings suggest that MrgprF, a novel tumor suppressor in cutaneous melanoma, may be useful as a therapeutic target in the future.

Exposure to radiation causes DNA damage; hence, continuous surveillance and timely DNA repair are important for genome stability. Epigenetic modifications alter the chromatin architecture, thereby affecting the efficiency of DNA repair. However, how epigenetic modifiers coordinate with the DNA repair machinery to modulate cellular radiosensitivity is relatively unknown. Here, we report that loss of the demethylase ribosomal oxygenase 1 (RIOX1) restores cell proliferation and reduces cell death after exposure to ionizing radiation. Furthermore, RIOX1 depletion enhances homologous recombination (HR) repair but not nonhomologous end-joining (NHEJ) repair in irradiated bone marrow cells and oral mucosal epithelial cells. Mechanistic study demonstrates that RIOX1 removes monomethylation at K491 of cyclic GMP-AMP synthase (cGAS) to release cGAS from its interaction with the methyl-lysine reader protein SAGA complex-associated factor 29 (SGF29), which subsequently enables cGAS to interact with poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase 1 (PARP1) at DNA break sites, thereby blocking PARP1-mediated recruitment of Timeless. High expression of RIOX1 maintains cGAS K491me at a low level, which impedes HR repair and reduces cellular tolerance to ionizing radiation. This study highlights a novel RIOX1-dependent mechanism involved in the non-immune function of cGAS that is essential for the regulation of ionizing radiation-elicited HR repair.

Lung cancer is the leading cause of cancer death worldwide, and around 85% of patients are grouped into non-small cell lung cancer (NSCLC) based on the histological characteristics.1 The survival of motor neurons (SMN) complex has been demonstrated to play critical roles in the biogenesis of ribonucleoprotein complexes (RNPs), and the reduced expression of the causative gene SMN leads to spinal muscular atrophy (SMA).2, 3 The SMN complex mainly consists of SMN, Gemin2, 3, 4, 5, 6 and 7. SMN interacts with Gemin2, 3, 5 and 7 directly, whereas Gemin4 and 6 binding to SMN relies on Gemin3 and 7, respectively. In this study, we revealed the oncogenic role of Gemin6/AURKB/c-Myc axis in lung adenocarcinoma, and the promising anticancer potential of sertindole treatment with probable clinical application in the future. As the functional roles of SMN complex in NSCLC remain elusive, we examined the expressions of individual components of SMN complex, and found that Gemin6 is unanimously upregulated in multiple types of human cancer, including NSCLC (Figure 1A,B; Figure S1A–C). Gemin6 high expression was identified to correlate with worse clinical outcome (Figure 1C–H; Figure S1D,E; Table S1). Furthermore, the ROC curve analysis of the SMN complex factors showed that Gemin6 exhibits the highest AUC value of 0.936 (Figure 1I). As expected, real-time RT-PCR and immunoblot assays validated that Gemin6 was highly expressed in NSCLC tissues and cell lines (Figure 1J–M; Figure S1F,G). We also identified the mutation pattern of Gemin6 in pan-cancers including NSCLC (Figure S1H; Table S2). Hypomethylation in Gemin6 promoter region was uncovered to be positively associated with its transcript expression level in multiple tumours (Figure 1N; Figure S1I,J). In addition, the decreased expression of Gemin6 in normal control cell line BEAS-2B and tumour cells could be reversed by DNA methylases inhibitor 5-azacytidine (5-Aza) treatment (Figure 1O). Gemin6 transcript was then inhibited with two lenti-viral shRNAs in A549 and H1975 (Figure 2A; Figure S2A). We found that Gemin6 knockdown repressed tumour cell proliferation examined by growth curve, BrdU incorporation and colony formation assays (Figure 2B–G; Figure S2B–D). Furthermore, we uncovered that the cell cycle was arrested at G0/G1 phase after Gemin6 knockdown, and the key regulators for G0/G1 cell cycle transition, including CDK2, CDK4 and CDK6 were also markedly reduced upon Gemin6 knockdown (Figure 2H–J; Figure S2E–G). The cell migration ability was also reduced upon Gemin6 knockdown both in vitro and in vivo (Figure 2K–M; Figure S2H–P). In line with the findings in vitro, the xenograft tumour masses, tumour weights and volumes in Gemin6 inhibition groups were markedly impeded compared to the control group (Figure 2N–Q; Figure S2N). To decipher the underlying mechanism by which Gemin6 regulates NSCLC progression, we found that Gemin6 was involved in c-Myc, but not E2F, related signaling pathway (Figure 3A,B; Figure S3A). However, the reduced c-Myc proteins, but not the transcripts, in Gemin6 knockdown groups were detected compared to control (Figure 3C–F; Figure S3B–E). As expected, Gemin6 knockdown-reduced cell proliferation ability could be overcome by c-Myc overexpression (Figure 3G). These findings prompted us to hypothesise that Gemin6 regulates the post-translational modification of c-Myc proteins. As documented by other studies, c-Myc proteins could be stabilised by deubiquitinases USP36/USP28,4, 5 or degraded by E3 ligase FBXW7/SKP2.6, 7 To our surprise, the above c-Myc regulators' mRNA expressions were not markedly deregulated upon Gemin6 inhibition (Figure 3H; Figure S3F,G). However, recent findings identified that phosphorylation of c-Myc Serine 67 site mediated by AURKB, prevents its degradation in a proteasome signaling pathway-dependent manner,8 which was uncovered to be significantly reduced upon Gemin6 knockdown (Figure 3I; Figure S3H). Furthermore, we found that Gemin6 expression positively correlates with AURKB, and the mRNA and protein expressions of AURKB were both decreased in Gemin6 knockdown cells (Figure 3J,K; Figure S3I). In addition, we showed that AURKB overexpression reversed Gemin6 knockdown-reduced cell proliferation and migration abilities (Figure 3L–Q). In line with the findings that SMN complex regulates the biogenesis of RNPs, we revealed that the SMN complex formation, the maturation process, but not the stability of AURKB mRNA, were decreased upon Gemin6 knockdown (Figure 3R–T; Figure S3J–L). As expected, we showed that AURKB serves as a prognostic biomarker and is highly expressed in NSCLC, which correlates with worse overall survival (OS) and disease-specific survival (DSS) rates in LUAD (Figure 3U,V; Figure S3M–P). To further explore the clinical value of Gemin6, we then examined the potential drug repurposing activities of 10 drugs, selectively targeting dopamine or serotonin receptors from FDA-Approved Drug Library Mini, in NSCLC by blocking Gemin6 expression (Table S3).9 Compound sertindole was identified as the only one with the activity decreasing Gemin6 proteins to less than 70% of control level (Figure 4A; Figure S4A). Sertindole has been previously identified as an antagonist targeting dopamine D2 receptors, serotonin 5HT2A receptors and α1-adrenoceptors, which is mainly produced in the central nervous system and gastrointestinal tract.10 The cell viability after sertindole treatment was examined, and more dramatic inhibitory effect on tumour cell (A549 and H1975) survival was detected (Figure 4B). Furthermore, we found that sertindole treatment decreased the mRNA expressions of Gemin6 and AURKB, but not c-Myc (Figure 4C,D; Figure S4B). The cell proliferation and cell migration abilities were also repressed after sertindole treatment, which were markedly reversed by Gemin6 or c-Myc forced expression, respectively (Figure 4E–I; Figure S4C–I). As expected, the xenograft tumour masses, tumour weights and volumes in sertindole treatment group were markedly inhibited compared to control group, evidenced by deceased Ki67, Gemin6 and c-Myc IHC-positive signals (Figure 4J–O). We observed that AURKB mRNA was reduced upon Gemin6 inhibition. However, how Gemin6 acts alone or cooperates with other SMN complex components to regulate AURKB mRNA splicing or transcription is still unclear. Therefore, it will be necessary to examine the integrity of RNPs or SMN complex upon Gemin6 knockdown in the future. We showed that sertindole, previous identified antagonist targeting dopamine D2 receptors, serotonin 5HT2A receptors and α1-adrenoceptors, inhibited NSCLC progression by inhibiting c-Myc, who lies at the crossroads of these signaling pathways, and reveals the promising anticancer potentials of sertindole against NSCLC even with lung-to-brain metastases in the future (Figure 4P). This study was supported by National Key Research and Development Program of China (2021YFF1000602), National Nature Science Foundation of China (U1902216, U2102206, 82173110, 82002439, 82060515, 81960423, 82160453, 82160593) and Yunnan Applied Basic Research Projects (2019FJ009, 202001AS070037, 2019HB076, 2019FE001-042, 202001AT070027, 202001AC070712). The specific phosphorylation antibody against Serine 67 site on c-Myc was kindly provided by Dr. Hudan Liu at Wuhan University, China. The NSCLC tissue microarray was provided by Dr. Songqing Fan at the Second Xiangya Hospital, Central South University, Changsha, Hunan, China. The authors declare that there is no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Abstract The interaction between intestinal epithelial cells (IECs) and lymphocytes is known to regulate mucosal barrier function, immune responses, and by extension, to play an etiologic role in the development of chronic inflammation in Ulcerative colitis (UC). However, the biologic function of lymphocytes and their relationship with IECs is insufficiently understood. In this study, we report, for the first time, the establishment of 3-dimensional (3D) co-culture systems of lymphocytes and mouse primary colonic organoids.C57BL/6 mice were treated with dextran sulfate sodium to induce acute colonic inflammation. Primary colonic organoids were established from cells isolated from mouse colons. Lymphocytes isolated from spleens by Ficoll density gradient assays. Colonoids and lymphocytes were co-cultured. The effects of the tested drugs on the co-culture system were assayed by Enzyme-linked Immune Sorbent Assay (ELISA) for inflammatory molecules. Inflammatory stimulation resulted in a significant increase in inflammatory cytokines in the supernatant of the colonoid-lymphocyte organoid system. Treatment with drugs known to be effective in patients with UC demonstrated a reduction in inflammatory cytokines. These findings argue that the colonoid-lymphocyte system can be utilized to model inflammation in vitro as well as for drug discovery. To validate these findings, we tested three other drugs. We produced experimental evidence that, indeed, these three drugs also induced a decrease in inflammatory cytokines of varying degrees. We report, for the first time, the successful establishment of a 3D colonoid-lymphocyte system and we validate its ability to predict the anti-inflammatory properties of several small molecules.