p27 specifically decreases in squamous carcinoma, and mediates NNK-induced transformation of human bronchial epithelial cells.

Lung cancer remains the leading cause of cancer-related deaths, with cigarette smoking being the most critical factor, linked to nearly 90% of lung cancer cases. NNK, a highly carcinogenic nitrosamine found in tobacco, is implicated in the lung cancer-causing effects of cigarette smoke. Although NNK is known to mutate or activate certain oncogenes, its potential interaction with p27 in modulating these carcinogenic effects is currently unexplored. Recent studies have identified specific downregulation of p27 in human squamous cell carcinoma, in contrast to adenocarcinoma. Additionally, exposure to NNK significantly suppresses p27 expression in human bronchial epithelial cells. Subsequent studies indicates that the downregulation of p27 is pivotal in NNK-induced cell transformation. Mechanistic investigations have shown that reduced p27 expression leads to increased level of ITCH, which facilitates the degradation of Jun B protein. This degradation in turn, augments miR-494 expression and its direct regulation of JAK1 mRNA stability and protein expression, ultimately activating STAT3 and driving cell transformation. In summary, our findings reveal that: (1) the downregulation of p27 increases Jun B expression by upregulating Jun B E3 ligase ITCH, which then boosts miR-494 transcription; (2) Elevated miR-494 directly binds to 3'-UTR of JAK1 mRNA, enhancing its stability and protein expression; and (3) The JAK1/STAT3 pathway is a downstream effector of p27, mediating the oncogenic effect of NNK in lung cancer. These findings provide significant insight into understanding the participation of mechanisms underlying p27 inhibition of NNK induced lung squamous cell carcinogenic effect.

Extrachromosomal circular DNA promotes prostate cancer progression through the FAM84B/CDKN1B/MYC/WWP1 axis.

BACKGROUND: Extrachromosomal circular DNA (eccDNA), a kind of circular DNA that originates from chromosomes, carries complete gene information, particularly the oncogenic genes. This study aimed to examine the contributions of FAM84B induced by eccDNA to prostate cancer (PCa) development and the biomolecules involved. METHODS: The presence of eccDNA in PCa cells and the FAM84B transcripts that eccDNA carries were verified by outward and inward PCR. The effect of inhibition of eccDNA synthesis on FAM84B expression in PCa cells was analyzed by knocking down Lig3. The impact of FAM84B on the growth and metastases of PCa cells was verified by Cell Counting Kit-8 (CCK8), EdU, transwell assays, and a xenograft mouse model. Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) and dual-luciferase reporter assays were carried out to examine the effect of FAM84B/MYC on WWP1 transcription, and a co-immunoprecipitation (Co-IP) assay was conducted to verify the modification of CDKN1B by WWP1. The function of this molecular axis in PCa was explored by rescue assays. RESULTS: The inhibited eccDNA synthesis significantly downregulated FAM84B in PCa cells, thereby attenuating the growth and metastasis of PCa. FAM84B promoted the transcription of WWP1 by MYC by activating the expression of MYC coterminous with the 8q24.21 gene desert in a beta catenin-dependent approach. WWP1 transcription promoted by MYC facilitated the ubiquitination and degradation of CDKN1B protein and inversely attenuated the repressive effect of CDKN1B on MYC expression. Exogenous overexpression of CDKN1B blocked FAM84B-activated MYC/WWP1 expression, thereby inhibiting PCa progression. CONCLUSIONS: FAM84B promoted by eccDNA mediates degradation of CDKN1B via MYC/WWP1, thereby accelerating PCa progression.

Fatty Acid Synthase Promotes Hepatocellular Carcinoma Growth via S-Phase Kinase-Associated Protein 2/p27KIP1 Regulation.

Background and Objectives: Aberrant upregulation of fatty acid synthase (FASN), catalyzing de novo synthesis of fatty acids, occurs in various tumor types, including human hepatocellular carcinoma (HCC). Although FASN oncogenic activity seems to reside in its pro-lipogenic function, cumulating evidence suggests that FASN's tumor-supporting role might also be metabolic-independent. Materials and Methods: In the present study, we show that FASN inactivation by specific small interfering RNA (siRNA) promoted the downregulation of the S-phase kinase associated-protein kinase 2 (SKP2) and the consequent induction of p27KIP1 in HCC cell lines. Results: Expression levels of FASN and SKP2 directly correlated in human HCC specimens and predicted a dismal outcome. In addition, forced overexpression of SKP2 rendered HCC cells resistant to the treatment with the FASN inhibitor C75. Furthermore, FASN deletion was paralleled by SKP2 downregulation and p27KIP1 induction in the AKT-driven HCC preclinical mouse model. Moreover, forced overexpression of an SKP2 dominant negative form or a p27KIP1 non-phosphorylatable (p27KIP1-T187A) construct completely abolished AKT-dependent hepatocarcinogenesis in vitro and in vivo. Conclusions: In conclusion, the present data indicate that SKP2 is a critical downstream effector of FASN and AKT-dependent hepatocarcinogenesis in liver cancer, envisaging the possibility of effectively targeting FASN-positive liver tumors with SKP2 inhibitors or p27KIP1 activators.

HES1 is required for mouse fetal hematopoiesis.

BACKGROUND: Hematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling. METHODS: In this study, we investigated the role of HES1, a member of HES family, in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry, histopathology analysis, and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay. RESULTS: We found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers, decreases hematopoietic stem progenitor cell (HSPC) pool, results in defective multi-lineage differentiation. Functionally, fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1fl/flFlt3Cre embryos are resulted from decreased proliferation and elevated apoptosis, associated with de-repressed HES1 targets, p27 and PTEN in Hes1-KO fetal HSPCs. Finally, pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo. CONCLUSION: Together, our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis, and provide new insight into the differences between fetal and adult HSC maintenance.

C-terminally phosphorylated p27 activates self-renewal driver genes to program cancer stem cell expansion, mammary hyperplasia and cancer.

In many cancers, a stem-like cell subpopulation mediates tumor initiation, dissemination and drug resistance. Here, we report that cancer stem cell (CSC) abundance is transcriptionally regulated by C-terminally phosphorylated p27 (p27pT157pT198). Mechanistically, this arises through p27 co-recruitment with STAT3/CBP to gene regulators of CSC self-renewal including MYC, the Notch ligand JAG1, and ANGPTL4. p27pTpT/STAT3 also recruits a SIN3A/HDAC1 complex to co-repress the Pyk2 inhibitor, PTPN12. Pyk2, in turn, activates STAT3, creating a feed-forward loop increasing stem-like properties in vitro and tumor-initiating stem cells in vivo. The p27-activated gene profile is over-represented in STAT3 activated human breast cancers. Furthermore, mammary transgenic expression of phosphomimetic, cyclin-CDK-binding defective p27 (p27CK-DD) increases mammary duct branching morphogenesis, yielding hyperplasia and microinvasive cancers that can metastasize to liver, further supporting a role for p27pTpT in CSC expansion. Thus, p27pTpT interacts with STAT3, driving transcriptional programs governing stem cell expansion or maintenance in normal and cancer tissues.

Enzyme-independent role of EZH2 in regulating cell cycle progression via the SKP2-KIP/CIP pathway.

While EZH2 enzymatic activity is well-known, emerging evidence suggests that EZH2 can exert functions in a methyltransferase-independent manner. In this study, we have uncovered a novel mechanism by which EZH2 positively regulates the expression of SKP2, a critical protein involved in cell cycle progression. We demonstrate that depletion of EZH2 significantly reduces SKP2 protein levels in several cell types, while treatment with EPZ-6438, an EZH2 enzymatic inhibitor, has no effect on SKP2 protein levels. Consistently, EZH2 depletion leads to cell cycle arrest, accompanied by elevated expression of CIP/KIP family proteins, including p21, p27, and p57, whereas EPZ-6438 treatment does not modulate their levels. We also provide evidence that EZH2 knockdown, but not enzymatic inhibition, suppresses SKP2 mRNA expression, underscoring the transcriptional regulation of SKP2 by EZH2 in a methyltransferase-independent manner. Supporting this, analysis of the Cancer Genome Atlas database reveals a close association between EZH2 and SKP2 expression in human malignancies. Moreover, EZH2 depletion but not enzymatic inhibition positively regulates the expression of major epithelial-mesenchymal transition (EMT) regulators, such as ZEB1 and SNAIL1, in transformed cells. Our findings shed light on a novel mechanism by which EZH2 exerts regulatory effects on cell proliferation and differentiation through its methyltransferase-independent function, specifically by modulating SKP2 expression.

UHRF2 accumulates in early G1-phase after serum stimulation or mitotic exit to extend G1 and total cell cycle length.

Ubiquitin like with PHD and ring finger domains 2 (UHRF2) regulates the cell cycle and epigenetics as a multi-domain protein sharing homology with UHRF1. UHRF1 functions with DNMT1 to coordinate daughter strand methylation during DNA replication, but UHRF2 can't perform this function, and its roles during cell cycle progression are not well defined. UHRF2 role as an oncogene vs. tumor suppressor differs in distinct cell types. UHRF2 interacts with E2F1 to control Cyclin E1 (CCNE1) transcription. UHRF2 also functions in a reciprocal loop with Cyclin E/CDK2 during G1, first as a direct target of CDK2 phosphorylation, but also as an E3-ligase with direct activity toward both Cyclin E and Cyclin D. In this study, we demonstrate that UHRF2 is expressed in early G1 following either serum stimulation out of quiescence or in cells transiting directly out of M-phase, where UHRF2 protein is lost. Further, UHRF2 depletion in G2/M is reversed with a CDK1 specific inhibitor. UHRF2 controls expression levels of cyclins and CDK inhibitors and controls its own transcription in a negative-feedback loop. Deletion of UHRF2 using CRISPR/Cas9 caused a delay in passage through each cell cycle phase. UHRF2 loss culminated in elevated levels of cyclins but also the CDK inhibitor p27KIP1, which regulates G1 passage, to reduce retinoblastoma phosphorylation and increase the amount of time required to reach G1/S passage. Our data indicate that UHRF2 is a central regulator of cell-cycle pacing through its complex regulation of cell cycle gene expression and protein stability.

Targeting RCC1 to block the human soft-tissue sarcoma by disrupting nucleo-cytoplasmic trafficking of Skp2.

Soft-tissue sarcomas (STS) emerges as formidable challenges in clinics due to the complex genetic heterogeneity, high rates of local recurrence and metastasis. Exploring specific targets and biomarkers would benefit the prognosis and treatment of STS. Here, we identified RCC1, a guanine-nucleotide exchange factor for Ran, as an oncogene and a potential intervention target in STS. Bioinformatics analysis indicated that RCC1 is highly expressed and correlated with poor prognosis in STS. Functional studies showed that RCC1 knockdown significantly inhibited the cell cycle transition, proliferation and migration of STS cells in vitro, and the growth of STS xenografts in mice. Mechanistically, we identified Skp2 as a downstream target of RCC1 in STS. Loss of RCC1 substantially diminished Skp2 abundance by compromising its protein stability, resulting in the upregulation of p27Kip1 and G1/S transition arrest. Specifically, RCC1 might facilitate the nucleo-cytoplasmic trafficking of Skp2 via direct interaction. As a result, the cytoplasmic retention of Skp2 would further protect it from ubiquitination and degradation. Notably, recovery of Skp2 expression largely reversed the phenotypes induced by RCC1 knockdown in STS cells. Collectively, this study unveils a novel RCC1-Skp2-p27Kip1 axis in STS oncogenesis, which holds promise for improving prognosis and treatment of this formidable malignancy.

XPO1 blockade with KPT-330 promotes apoptosis in cutaneous T-cell lymphoma by activating the p53-p21 and p27 pathways.

Dysregulated nuclear-cytoplasmic trafficking has been shown to play a role in oncogenesis in several types of solid tumors and hematological malignancies. Exportin 1 (XPO1) is responsible for the nuclear export of several proteins and RNA species, mainly tumor suppressors. KPT-330, a small molecule inhibitor of XPO1, is approved for treating relapsed multiple myeloma and diffuse large B-cell lymphoma. Cutaneous T-cell lymphoma (CTCL) is an extranodal non-Hodgkin lymphoma with an adverse prognosis and limited treatment options in advanced stages. The effect of therapeutically targeting XPO1 with KPT-330 in CTCL has not been established. We report that XPO1 expression is upregulated in CTCL cells. KPT-330 reduces cell proliferation, induces G1 cell cycle arrest and apoptosis. RNA-sequencing was used to explore the underlying mechanisms. Genes associated with the cell cycle and the p53 pathway were significantly enriched with KPT-330 treatment. KPT-330 suppressed XPO1 expression, upregulated p53, p21WAF1/Cip1, and p27Kip1 and their nuclear localization, and downregulated anti-apoptotic protein (Survivin). The in vivo efficacy of KPT-330 was investigated using a bioluminescent xenograft mouse model of CTCL. KPT-330 blocked tumor growth and prolonged survival (p < 0.0002) compared to controls. These findings support investigating the use of KPT-330 and next-generation XPO1 inhibitors in CTCL.

Endothelial Cell Flow-Mediated Quiescence Is Temporally Regulated and Utilizes the Cell Cycle Inhibitor p27.

BACKGROUND: Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. METHODS: Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time-to-cell cycle reentry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA-seq (single-cell RNA sequencing) analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. RESULTS: Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous flow-exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. CONCLUSIONS: Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence misregulation that leads to vascular dysfunction and disease.

Non-canonical deubiquitination of OTUB1 induces IFNγ-mediated cell cycle arrest via regulation of p27 stability.

The deubiquitinase OTUB1, implicated as a potential oncogene in various tumors, lacks clarity in its regulatory mechanism in tumor progression. Our study investigated the effects and underlying mechanisms of OTUB1 on the breast cancer cell cycle and proliferation in IFNγ stimulation. Loss of OTUB1 abrogated IFNγ-induced cell cycle arrest by regulating p27 protein expression, whereas OTUB1 overexpression significantly enhanced p27 expression even without IFNγ treatment. Tyr26 phosphorylation residue of OTUB1 directly bound to p27, modulating its post-translational expression. Furthermore, we identified crucial lysine residues (K134, K153, and K163) for p27 ubiquitination. Src downregulation reduced OTUB1 and p27 expression, suggesting that IFNγ-induced cell cycle arrest is mediated by the Src-OTUB1-p27 signaling pathway. Our findings highlight the pivotal role of OTUB1 in IFNγ-induced p27 expression and cell cycle arrest, offering therapeutic implications.

Germ cell-specific gene 2 accelerates cell cycle in epithelial ovarian cancer by inhibiting GSK3α-p27 cascade.

Epithelial ovarian cancer (EOC) is one of the most common malignant gynecological tumors with rapid growth potential and poor prognosis, however, the molecular mechanism underlying its outgrowth remained elusive. Germ cell-specific gene 2 (GSG2) was previously reported to be highly expressed in ovarian cancer and was essential for the growth of EOC. In this study, GSG2-knockdown cells and GSG2-overexpress cells were established through lentivirus-mediated transfection with Human ovarian cancer cells HO8910 and SKOV3. Knockdown of GSG2 inhibited cell proliferation and induced G2/M phase arrest in EOC. Interestingly, the expression of p27, a well-known regulator of the cell cycle showed a most significant increase after GSG2 knockdown. Further phosphorylation-protein array demonstrated the phosphorylation of GSK3αSer21 decreased in GSG2-knockdown cells to the most extent. Notably, inhibiting GSK3α activity effectively rescued GSG2 knockdown's suppression on cell cycle as well as p27 expression in EOC. Our study substantiates that GSG2 is able to phosphorylate GSK3α at Ser21 and then leads to the reduction of p27 expression, resulting in cell cycle acceleration and cell proliferation promotion. Thus, GSG2 may have the potential to become a promising target in EOC.

Whole-transcriptome profiling reveals potential biomarkers for the reversal of thymic epithelial cell senescence by umbilical cord mesenchymal stem cells.

BACKGROUND: Reduced numbers and dysfunction of thymic epithelial cells (TECs) are important factors of thymic degeneration. Previous studies have found that umbilical cord mesenchymal stem cells (UCMSCs) reverse the structure and function of the senescent thymus in vivo. However, the transcriptomic regulation mechanism is unclear. METHODS: TECs were cultured with H2O2 for 72 hours to induce senescence. UCMSCs were cocultured with senescent TECs for 48 hours to detect SA-ß-gal, P16 and Ki67. The cocultured TECs were collected for lncRNA, mRNA and miRNA sequencing to establish a competitive endogenous regulatory network (ceRNA). And RT-qPCR, immunofluorescence staining, and western blot were used to identified key genes. RESULTS: Our results showed that H2O2 induced TEC aging and that UCMSCs reversed these changes. Compared with those in aged TECs, 2260 DE mRNAs, 1033 DE lncRNAs and 67 DE miRNAs were differentially expressed, and these changes were reversed by coculturing the cells with UCMSCs. Differential mRNA enrichment analysis of ceRNA regulation revealed that the PI3K-AKT pathway was a significant signaling pathway. UCMSC coculture upregulated VEGFA, which is the upstream factor of the PI3K-AKT signaling pathway, and the expression of the key proteins PI3K and AKT. Thus, the expression of the cell cycle suppressor P27, which is downstream of the PI3K-AKT signaling pathway, was downregulated, while the expression of the cell cycle regulators CDK2 and CCNE was upregulated. CONCLUSION: UCMSC coculture upregulated the expression of VEGFA, activated the PI3K-AKT signaling pathway, increased the expression of CDK2 and CCNE, decreased the expression of P27, and promoted the proliferation of TECs.

RB1 loss induces quiescent state through downregulation of RAS signaling in mammary epithelial cells.

While loss of function (LOF) of retinoblastoma 1 (RB1) tumor suppressor is known to drive initiation of small-cell lung cancer and retinoblastoma, RB1 mutation is rarely observed in breast cancers at their initiation. In this study, we investigated the impact on untransformed mammary epithelial cells given by RB1 LOF. Depletion of RB1 in anon-tumorigenic MCF10A cells induced reversible growth arrest (quiescence) featured by downregulation of multiple cyclins and MYC, upregulation of p27KIP1, and lack of expression of markers which indicate cellular senescence or epithelial-mesenchymal transition (EMT). We observed a similar phenomenon in human mammary epithelial cells (HMEC) as well. Additionally, we found that RB1 depletion attenuated the activity of RAS and the downstream MAPK pathway in an RBL2/p130-dependent manner. The expression of farnesyltransferase ß, which is essential for RAS maturation, was found to be downregulated following RB1 depletion also in an RBL2/p130-dependent manner. These findings unveiled an unexpected mechanism whereby normal mammary epithelial cells resist to tumor initiation upon RB1 LOF.

Targeted inhibition of SCFSKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma.

Osteosarcoma(OS) is a highly aggressive bone cancer for which treatment has remained essentially unchanged for decades. Although OS is characterized by extensive genomic heterogeneity and instability, RB1 and TP53 have been shown to be the most commonly inactivated tumor suppressors in OS. We previously generated a mouse model with a double knockout (DKO) of Rb1 and Trp53 within cells of the osteoblastic lineage, which largely recapitulates human OS with nearly complete penetrance. SKP2 is a repression target of pRb and serves as a substrate recruiting subunit of the SCFSKP2 complex. In addition, SKP2 plays a central role in regulating the cell cycle by ubiquitinating and promoting the degradation of p27. We previously reported the DKOAA transgenic model, which harbored a knock-in mutation in p27 that impaired its binding to SKP2. Here, we generated a novel p53-Rb1-SKP2 triple-knockout model (TKO) to examine SKP2 function and its potential as a therapeutic target in OS. First, we observed that OS tumorigenesis was significantly delayed in TKO mice and their overall survival was markedly improved. In addition, the loss of SKP2 also promoted an apoptotic microenvironment and reduced the stemness of DKO tumors. Furthermore, we found that small-molecule inhibitors of SKP2 exhibited anti-tumor activities in vivo and in OS organoids as well as synergistic effects when combined with a standard chemotherapeutic agent. Taken together, our results suggest that SKP2 inhibitors may reduce the stemness plasticity of OS and should be leveraged as next-generation adjuvants in this cancer.

Syndromic MEN1 parathyroid adenomas consist of both subclonal nodules and clonally independent tumors.

Primary hyperparathyroidism with parathyroid tumors is a typical manifestation of Multiple Endocrine Neoplasia Type 1 (MEN1) and is historically termed "primary hyperplasia". Whether these tumors represent a multi-glandular clonal disease or hyperplasia has not been robustly proven so far. Loss of Menin protein expression is associated with inactivation of both alleles and a good surrogate for a MEN1 gene mutation. The cyclin-dependent kinase inhibitor 1B (CDKN1B) gene is mutated in MEN4 and encodes for protein p27 whose expression is poorly studied in the syndromic MEN1 setting.Here, we analyzed histomorphology and protein expression of Menin and p27 in parathyroid adenomas of 25 patients of two independent, well-characterized MEN1 cohorts. The pattern of loss of heterozygosity (LOH) was assessed by fluorescence in situ hybridization (FISH) in one MEN1-associated parathyroid adenoma. Further, next-generation sequencing (NGS) was performed on eleven nodules of four MEN1 patients.Morphologically, the majority of MEN1 adenomas consisted of multiple distinct nodules, in which Menin expression was mostly lost and p27 protein expression reduced. FISH analysis revealed that most nodules exhibited MEN1 loss, with or without the loss of centromere 11. NGS demonstrated both subclonal evolution and the existence of clonally unrelated tumors.Syndromic MEN1 parathyroid adenomas therefore consist of multiple clones with subclones, which supports the current concept of the novel WHO classification of parathyroid tumors (2022). p27 expression was lost in a large fraction of MEN1 parathyroids and must therefore be used with caution in suggesting MEN4.

Beyond MEN1, When to Think About MEN4? Retrospective Study on 5600 Patients in the French Population and Literature Review.

CONTEXT: Germline CDKN1B variants predispose patients to multiple endocrine neoplasia type 4 (MEN4), a rare MEN1-like syndrome, with <100 reported cases since its discovery in 2006. Although CDKN1B mutations are frequently suggested to explain cases of genetically negative MEN1, the prevalence and phenotype of MEN4 patients is poorly known, and genetic counseling is unclear. OBJECTIVE: To evaluate the prevalence of MEN4 in MEN1-suspected patients and characterize the phenotype of MEN4 patients. DESIGN: Retrospective observational nationwide study. Narrative review of literature and variant class reassessment. PATIENTS: We included all adult patients with class 3/4/5 CDKN1B variants identified by the laboratories from the French Oncogenetic Network on Neuroendocrine Tumors network between 2015 and 2022 through germline genetic testing for MEN1 suspicion. After class reassessment, we compared the phenotype of symptomatic patients with class 4/5 CDKN1B variants (ie, with genetically confirmed MEN4 diagnosis) in our series and in literature with 66 matched MEN1 patients from the UMD-MEN1 database. RESULTS: From 5600 MEN1-suspected patients analyzed, 4 with class 4/5 CDKN1B variant were found (0.07%). They presented with multiple duodenal NET, primary hyperparathyroidism (PHPT) and adrenal nodule, isolated PHPT, PHPT, and pancreatic neuroendocrine tumor. We listed 29 patients with CDKN1B class 4/5 variants from the literature. Compared with matched MEN1 patients, MEN4 patients presented lower NET incidence and older age at PHPT diagnosis. CONCLUSION: The prevalence of MEN4 is low. PHPT and pituitary adenoma represent the main associated lesions, NETs are rare. Our results suggest a milder and later phenotype than in MEN1. Our observations will help to improve genetic counseling and management of MEN4 families.

UBE4B regulates p27 expression in A549 NSCLC cells through regulating the interaction of HuR and the p27 5' UTR.

Ubiquitination factor E4B (UBE4B) has a tumor-promoting effect, demonstrated by its aberrant expression in various types of cancers, and in vitro studies have shown that the retardation of cancer cell proliferation can be induced by targeting UBE4B. However, the molecular pathways through which UBE4B exerts its oncogenic activities have not yet been clearly identified and existing knowledge is limited to p53 and its subsequent downstream targets. In this study, we demonstrated that UBE4B regulates p27 expression in A549 cells via the cap-independent translation pathway following treatment with rapamycin and cycloheximide (CHX). Subsequently, we identified that UBE4B regulates p27 translation by regulating the interaction between human antigen R (HuR) and the p27 internal ribosomal entry site (IRES). First, UBE4B interacts with HuR, which inhibits p27 translation through the IRES. Secondly, the interaction between HuR and the p27 IRES was diminished by UBE4B depletion and enhanced by UBE4B overexpression. Finally, HuR depletion-induced growth retardation, accompanied by p27 accumulation, was restored by UBE4B overexpression. Collectively, these results suggest that the oncogenic properties of UBE4B in A549 cells are mediated by HuR, suggesting the potential of targeting the UBE4B-HuR-p27 axis as a therapeutic strategy for lung cancer.

SUZ12 inhibition attenuates cell proliferation of glioblastoma via post-translational regulation of CDKN1B.

BACKGROUND: Human gliomas are aggressive brain tumors characterized by uncontrolled cell proliferation. Differential expression of Polycomb repressive complex 2 (PRC2) has been reported in various subtypes of glioma. However, the role of PRC2 in uncontrolled growth in glioma and its underlying molecular mechanisms remain to be elucidated. OBJECTIVE: We aimed to investigate the functional role of PRC2 in human glioblastoma cell growth by silencing SUZ12, the non-catalytic core component of PRC2. METHODS: Knockdown of SUZ12 was achieved by infecting T98G cells with lentivirus carrying sequences specifically targeting SUZ12 (shSUZ12). Gene expression was examined by quantitative PCR and western analysis. The impact of shSUZ12 on cell growth was assessed using a cell proliferation assay. Cell cycle distribution was analyzed by flow cytometry, and protein stability was evaluated in cycloheximide-treated cells. Subcellular localization was examined through immunofluorescence staining and biochemical cytoplasmic-nuclear fractionation. Gene expression analysis was also performed on human specimens from normal brain and glioblastoma patients. RESULTS: SUZ12 knockdown (SUZ12 KD) led to widespread decrease in the PRC2-specific histone mark, accompanied by a slowdown of cell proliferation through G1 arrest. In SUZ12 KD cells, the degradation of CDKN1B protein was reduced, resulting from alterations in the MYC-SKP2-CDKN1B axis. Furthermore, nuclear localization of CDKN1B was enhanced in SUZ12 KD cells. Analysis of human glioblastoma samples yielded increased expression of EZH2 and MYC along with reduced CDKN1B compared to normal human brain tissue. CONCLUSION: Our findings suggest a novel role for SUZ12 in cell proliferation through post-translational regulation of CDKN1B in glioblastoma.