[Effects of 4-hydroxy-2(3H)-benzoxazolone on proliferation and apoptosis of pancreatic cancer L3.6 cells].

This study explored the effects of 4-hydroxy-2(3H)-benzoxazolone(HBOA) on the proliferation and apoptosis of pancreatic cancer cells and its molecular mechanism. The L3.6 cells cultured in vitro were treated with HBOA of 0-1.0 mmol·L~(-1). The cell viability was detected by the cell counting kit-8(CCK-8) method, and the half inhibitory concentration(IC_(50)) was analyzed to determine the drug concentration and time. The cell morphology was observed under an inverted microscope and by acridine orange(AO) staining. The ability of proliferation and self-renewal were evaluated through live cell counting and colony formation experiments. The cell cycle progression and cell apoptosis rate were detected by flow cytometry. The morphology of cell apoptosis was observed by scanning electron microscopy. The mRNA expression of proliferating cell nuclear antigen(PCNA), cyclinA1, cyclinA2, cyclin dependent kinase 2(CDK2), and cyclin dependent kinase inhibitor 1A(P21) were determined by qPCR. The level of reactive oxygen species(ROS), lipid peroxide, and mitochondrial membrane potential were measured by flow cytometry. The activity of protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway was detected by Western blot. Compared with the control group, the cells treated with HBOA exhibited a significant decrease in viability. Then the optimal concentration and intervention time of HBOA were determined to be 0.4 mmol·L~(-1), 0.6 mmol·L~(-1), and 48 h. Compared with the control group, groups with HBOA of 0.4 mmol·L~(-1 )and 0.6 mmol·L~(-1) showed a significant suppression in cell proliferation and colony formation ability, down-regulated mRNA of PCNA, cyclinA1, cyclinA2, and CDK2, up-regulated P21 mRNA, S-phase cell cycle arrest, and increased cell apoptosis rate. There was an appearance of apoptotic bodies, increased ROS and lipid peroxide, decreased mitochondrial membrane potential(with a significant decrease in 0.6 mmol·L~(-1) group), and down-regulated p-Akt and p-mTOR proteins. The results show that HBOA inhibits the proliferation of pancreatic cancer L3.6 cells and induces cell apoptosis, which may be related to the increase in reactive oxygen species and the inhibition of the Akt/mTOR pathway.

Clinicopathological Significance of Cyclin-Dependent Kinase 2 (CDK2) in Ductal Carcinoma In Situ and Early-Stage Invasive Breast Cancers.

Cyclin-dependent kinase 2 (CDK2) is a key cell cycle regulator, with essential roles during G1/S transition. The clinicopathological significance of CDK2 in ductal carcinomas in situ (DCIS) and early-stage invasive breast cancers (BCs) remains largely unknown. Here, we evaluated CDK2's protein expression in 479 BC samples and 216 DCIS specimens. Analysis of CDK2 transcripts was completed in the METABRIC cohort (n = 1980) and TCGA cohort (n = 1090), respectively. A high nuclear CDK2 protein expression was significantly associated with aggressive phenotypes, including a high tumour grade, lymph vascular invasion, a poor Nottingham prognostic index (all p-values < 0.0001), and shorter survival (p = 0.006), especially in luminal BC (p = 0.009). In p53-mutant BC, high nuclear CDK2 remained linked with worse survival (p = 0.01). In DCIS, high nuclear/low cytoplasmic co-expression showed significant association with a high tumour grade (p = 0.043), triple-negative and HER2-enriched molecular subtypes (p = 0.01), Comedo necrosis (p = 0.024), negative ER status (p = 0.004), negative PR status (p < 0.0001), and a high proliferation index (p < 0.0001). Tumours with high CDK2 transcripts were more likely to have higher expressions of genes involved in the cell cycle, homologous recombination, and p53 signaling. We provide compelling evidence that high CDK2 is a feature of aggressive breast cancers. The clinical evaluation of CDK2 inhibitors in early-stage BC patients will have a clinical impact.

Molecular docking approach for the design and synthesis of new pyrazolopyrimidine analogs of roscovitine as potential CDK2 inhibitors endowed with pronounced anticancer activity.

Cyclin-dependent kinase 2 (CDK2) is a vital protein for controlling cell cycle progression that is critically associated with various malignancies and its inhibition could offer a convenient therapeutic approach in designing anticancer remedies. Consequently, this study aimed to design and synthesize new CDK2 inhibitors featuring roscovitine as a template model. The purine ring of roscovitine was bioisosterically replaced with the pyrazolo[3,4-d]pyrimidine scaffold, in addition to some modifications in the side chains. A preliminary molecular docking study for the target chemotypes in the CDK2 binding domain revealed their ability to accomplish similar binding patterns and interactions to that of the lead compound roscovitine. Afterwards, synthesis of the new derivatives was accomplished. Then, the initial anticancer screening at a single dose by the NCI revealed that compounds 7a, 9c, 11c, 17a and 17b achieved the highest GI% values reaching up to 150 % indicating their remarkable activity. These derivatives were subsequently selected to undertake five-dose testing, where compounds 7a, 9c, 11c and 17a unveiled the most pronounced activity against almost the full panel with GI50 ranges; 1.41-28.2, 0.116-2.39, 0.578-60.6 and 1.75-42.4 µM, respectively and full panel GI50 (MG-MID); 8.24, 0.6, 2.46 and 6.84 µM, respectively. CDK2 inhibition assay presented compounds 7a and 9c as the most potent inhibitors with IC50 values of 0.262 and 0.281 µM, respectively which are nearly 2.4 folds higher than the reference ligand roscovitine (IC50 = 0.641 µM). Besides, flow cytometric analysis on the most susceptible and safe cell lines depicted that 7a caused cell cycle arrest at G1/S phase in renal cancer cell line (RXF393) while 9c led to cell growth arrest at S phase in breast cancer cell line (T-47D) along with pronounced apoptotic induction in the mentioned cell lines. These findings afforded new anticancer pyrazolo[3,4-d]pyrimidine, roscovitine analogs, acting via CDK2 inhibition.

Smoking-induced CCNA2 expression promotes lung adenocarcinoma tumorigenesis by boosting AT2/AT2-like cell differentiation.

Lung adenocarcinoma (LUAD), a type of non-small cell lung cancer (NSCLC), originates from not only bronchial epithelial cells but also alveolar type 2 (AT2) cells, which could differentiate into AT2-like cells. AT2-like cells function as cancer stem cells (CSCs) of LUAD tumorigenesis to give rise to adenocarcinoma. However, the mechanism underlying AT2 cell differentiation into AT2-like cells in LUAD remains unknown. We analyze genes differentially expressed and genes with significantly different survival curves in LUAD, and the combination of these two analyses yields 147 differential genes, in which 14 differentially expressed genes were enriched in cell cycle pathway. We next analyze the protein levels of these genes in LUAD and find that Cyclin-A2 (CCNA2) is closely associated with LUAD tumorigenesis. Unexpectedly, high CCNA2 expression in LUAD is restrictedly associated with smoking and independent of other driver mutations. Single-cell sequencing analyses reveal that CCNA2 is predominantly involved in AT2-like cell differentiation, while inhibition of CCNA2 significantly reverses smoking-induced AT2-like cell differentiation. Mechanistically, CCNA2 binding to CDK2 phosphorylates the AXIN1 complex, which in turn induces ubiquitination-dependent degradation of ß-catenin and inhibits the WNT signaling pathway, thereby failing AT2 cell maintenance. These results uncover smoking-induced CCNA2 overexpression and subsequent WNT/ß-catenin signaling inactivation as a hitherto uncharacterized mechanism controlling AT2 cell differentiation and LUAD tumorigenesis.

Exploring the mechanism of action of spirooxindoles as a class of CDK2 inhibitors: a structure-based computational approach.

Cyclin-dependent kinase 2 (CDK2) regulates cell cycle checkpoints in the synthesis and mitosis phases and plays a pivotal role in cancerous cell proliferation. The activation of CDK2, influenced by various protein signaling pathways, initiates the phosphorylation process. Due to its crucial role in carcinogenesis, CDK2 is a druggable hotspot target to suppress cancer cell proliferation. In this context, several studies have identified spirooxindoles as an effective class of CDK2 inhibitors. In the present study, three spirooxindoles (SOI1, SOI2, and SOI3) were studied to understand their inhibitory mechanism against CDK2 through a structure-based approach. Molecular docking and molecular dynamics (MD) simulations were performed to explore their interactions with CDK2 at the molecular level. The calculated binding free energy for the spirooxindole-based CDK2 inhibitors aligned well with experimental results regarding CDK2 inhibition. Energy decomposition (ED) analysis identified key binding residues, including I10, G11, T14, R36, F82, K89, L134, P155, T158, Y159, and T160, in the CDK2 active site and T-loop phosphorylation. Molecular mechanics (MM) energy was identified as the primary contributor to stabilizing inhibitor binding in the CDK2 protein structure. Furthermore, the analysis of binding affinity revealed that the inhibitor SOI1 binds more strongly to CDK2 compared to the other inhibitors under investigation. It demonstrated a robust interaction with the crucial residue T160 in the T-loop phosphorylation site, responsible for kinase activation. These insights into the inhibitory mechanism are anticipated to contribute to the development of potential CDK2 inhibitors using the spirooxindole scaffold.

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.

Hsa_circ_0005320 affects cell proliferation and the cell cycle via the IGF2BP3/CDK2 axis in bladder cancer.

BACKGROUND: Circular RNAs (circRNAs), which are covalently closed non-coding RNAs, are frequently dysregulated in cancer. However, their precise role in bladder cancer (BCa) remains largely unknown. METHODS: Expression of hsa_circ_0005320 in tissues and cell lines was detected using quantitative real-time PCR. Proliferation and colony forming capacity of BCa cells were assessed using Cell Counting Kit-8, ethynyl-labeled deoxyuridine, and colony formation assays. The cell cycle was analyzed using flow cytometry. Protein expression of insulin-like growth factor II mRNA-binding protein 3 (IGF2BP3) and cyclin dependent kinase 2 (CDK2) was examined using western blots. The binding of RNA and protein was validated using RNA immunoprecipitation. Additionally, xenograft tumor models were established to validate the function of hsa_circ_0005320 in vivo. RESULTS: We screened hsa_circ_0005320 from previous high-throughput sequencing and found that it was highly expressed in BCa tissues and associated with tumor differentiation and depth of invasion in BCa patients. Through functional experiments, we demonstrated that hsa_circ_0005320 promoted cell proliferation and regulated the cell cycle. Mechanistically, hsa_circ_0005320 interacted with and upregulated the expression of IGF2BP3, which binds to and enhances the stability of CDK2 mRNA. Furthermore, knockdown of hsa_circ_0005320 resulted in a reduction in tumor burden in vivo. CONCLUSIONS: Collectively, these findings highlight the pro-oncogenic role of hsa_circ_0005320 in BCa through the IGF2BP3/CDK2 axis, providing valuable insights into the mechanism of circRNAs in tumor progression.

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.

Bruceantinol works as a CDK2/4/6 inhibitor to inhibit the growth of breast cancer cells.

Bruceantinol (BOL), isolated from the dried fruit of the Brucea javanica (L.) Merr., exhibits cytotoxic effects on breast cancer cells. However, the underlying mechanism remains to be fully addressed. In this paper, the MCF-7 and MDA-MB-231 human breast cancer cell lines were used as experimental models to uncover how BOL inhibits breast cancer cell growth. The effects of BOL on cell growth, proliferation, the cell cycle, and apoptosis were investigated using the MTT assays, EdU incorporation assays, and flow cytometry, respectively. Bioinformatics techniques were applied to predict the key targets of BOL in breast cancer. Subsequent validation of these targets and the anti-breast cancer mechanism of BOL was conducted through Western blotting, RT-PCR, siRNA transfection, and molecular docking analysis. The results demonstrated that BOL dose- and time-dependently reduced the growth of both cell lines, impeded cell proliferation, disrupted the cell cycle, and induced necrosis in MCF-7 cells and apoptosis in MDA-MB-231 cells. Furthermore, CDK2/4/6 were identified as BOL targets, and their knockdown reduced cell sensitivity to BOL. BOL was found to potentially bind with CDK2/4/6 to facilitate protein degradation through the proteasome pathway. Additionally, BOL activated ERK in MDA-MB-231 cells, and this activation was required for BOL's functions in these cells. Collectively, BOL may act as an inhibitor of CDK2/4/6 to exert anti-breast cancer effects. Its effects on cell growth and CDK2/4/6 expression may also depend on ERK activation in HRs-HER2- breast cancer cells. These results suggest the potential of using BOL for treating breast cancer.

Effects of silencing NLRP3 gene on proliferation of psoriasis-like HaCaT cells and expressions of cytokines.

We aimed to explore the effects of silencing NOD-like receptor protein 3 (NLRP3) on proliferation of psoriasis-like HaCaT cells and expressions of cytokines. HaCaT cells were treated with human keratinocyte growth factor (KGF) and were divided into KGF group, negative control group, NLRP3-RNAi group and control group. Cells proliferation was detected by CCK8, cell clone formation rate was detected by clone formation assay, distribution of cells cycle was detected by flow cytometry, expressions of cyclin B1 (Cyclin B1), cyclin-dependent kinase 2 (CDK2), Ki67 and proliferating cell nuclear antigen (PCNA) proteins were detected by Western blot, and levels of interleukin (IL)-17, IL-23, IL-6 and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were increased in KGF group, percentage of cells in G0/G1 phase was decreased, percentage of cells in S phase was increased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were increased, and levels of IL-17, IL-23, IL-6 and TNF-α were increased. Compared with negative control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were decreased in NLRP3-RNAi group, percentage of cells in G0/G1 phase was increased, percentage of cells in S phase was decreased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were decreased, and levels of IL-17, IL-23, IL-6 and TNF-α were decreased. Silencing NLRP3 gene can inhibit the proliferation of psoriasis-like HaCaT cells, arrest cell cycle, inhibit the expressions of cell proliferation-related proteins and reduce levels of pro-inflammatory factors.

A naturally derived small molecule compound suppresses tumor growth and metastasis in mice by relieving p53-dependent repression of CDK2/Rb signaling and the Snail-driven EMT.

The tumor suppressor protein p53 is central to cancer biology, with its pathway reactivation emerging as a promising therapeutic strategy in oncology. This study introduced LZ22, a novel compound that selectively inhibits the growth, migration, and metastasis of tumor cells expressing wild-type p53, demonstrating ineffectiveness in cells devoid of p53 or those expressing mutant p53. LZ22's mechanism of action involves a high-affinity interaction with the histidine-96 pocket of the MDM2 protein. This interaction disrupted the MDM2-p53 binding, consequently stabilizing p53 by shielding it from proteasomal degradation. LZ22 impeded cell cycle progression and diminished cell proliferation by reinstating the p53-dependent suppression of the CDK2/Rb signaling pathway. Moreover, LZ22 alleviated the p53-dependent repression of Snail transcription factor expression and its consequent EMT, effectively reducing tumor cell migration and distal metastasis. Importantly, LZ22 administration in tumor-bearing mice did not manifest notable side effects. The findings position LZ22 as a structurally unique reactivator of p53, offering therapeutic promise for the management of human cancers with wild-type TP53.

Exploring new quinazolin-4(3H)-one derivatives as CDK2 inhibitors: Design, synthesis, and anticancer evaluation.

In the present work, five series of new 2,3-disubstituted quinazolin-4(3H)-ones 4a-c, 5a-d, 6a-g, 7a,b, and 9a-c were designed, synthesized, and screened in vitro for their cytotoxic activity against 60 cancer cell lines by the National Cancer Institute, USA. Five candidates 4c, 6a, 6b, 6d, and 6g revealed promising cytotoxicity with significant percentage growth inhibition in the range of 81.98%-96.45% against the central nervous system (CNS) (SNB-19), melanoma (MDA-MB-435), and non-small cell lung cancer (HOP-62) cell lines. The in vitro cytotoxic half maximal inhibitory concentration (IC50 ) values for the most active compounds 4c, 6a, 6b, 6d, and 6g against the most sensitive cell lines were evaluated. Additionally, screening their cyclin-dependent kinase 2 (CDK2) inhibitory activity was performed. Ortho-chloro-benzylideneamino derivative 6b emerged as the most potent compound with IC50 = 0.67 µM compared to Roscovitine (IC50 = 0.64 µM). The most active candidates arrested the cell cycle at G1, S phases, or both, leading to cell death and inducing apoptosis against CNS (SNB-19), melanoma (MDA-MB-435), and non-small cell lung cancer (HOP-62) cell lines. The molecular docking study verified the resulting outcomes for the most active candidates in the CDK2-binding pocket. Finally, physicochemical, and pharmacokinetic properties deduced that compounds 4c, 6a, 6b, 6d, and 6g displayed significant drug-likeness properties. According to the obtained results, the newly targeted compounds are regarded as promising scaffolds for the continued development of novel CDK2 inhibitors.

Synthesis, docking, MD simulation, ADMET, drug likeness, and DFT studies of novel furo[2,3-b]indol-3a-ol as promising Cyclin-dependent kinase 2 inhibitors.

A new series of furo[2,3-b]indol-3a-ol derivatives was synthesized to investigate their potential as inhibitors of the Cyclin-dependent kinase 2 (CDK2) enzyme. CDK2 is a serine/threonine protein kinase belonging to a family of kinases involved in the control of the cell cycle. Based on results from clinical studies, it has been shown that overexpression of CDK2 may play a role in the development of cancer. In order to discover highly effective derivatives, a process of in silico screening was carried out. The obtained results revealed that compound 3f. had excellent binding energies. In this study, in silico screening was used to investigate protein-ligand interactions and assess the stability of the most favorable conformation. The methods utilized included molecular docking, density functional theory (DFT) calculations using the B3LYP/6-31++G(d,p) basis set in the gas phase, molecular dynamic (MD) simulation, as well as the evaluation of drug-likeness scores. The pharmacokinetic and drug-likeness properties of the novel furo[2,3-b]indol-3a-ol derivatives suggest that these compounds have the potential to be considered viable candidates for future development as anticancer drugs.

New pyrrolidine-carboxamide derivatives as dual antiproliferative EGFR/CDK2 inhibitors.

Cancer is one of the leading causes of mortality worldwide, making it a public health concern. A novel series of pyrrolidine-carboxamide derivatives 7a-q were developed and examined in a cell viability assay utilizing a human mammary gland epithelial cell line (MCF-10A), where all the compounds exhibited no cytotoxic effects and more than 85% cell viability at a concentration of 50 µM. Antiproliferative activity was evaluated in vitro against four panels of cancer cell lines A-549, MCF-7, Panc-1, and HT-29. Compounds 7e, 7g, 7k, 7n, and 7o were the most active as antiproliferative agents capable of triggering apoptosis. Compound 7g was the most potent of all the derivatives, with a mean IC50 of 0.90 µM compared to IC50 of 1.10 µM for doxorubicin. Compound 7g inhibited A-549 (epithelial cancer cell line), MCF-7 (breast cancer cell line), and HT-29 (colon cancer cell line) more efficiently than doxorubicin. EGFR inhibitory assay results of 7e, 7g, 7k, 7n, and 7o demonstrated that the tested compounds inhibited EGFR with IC50 values ranging from 87 to 107 nM in comparison with the reference drug erlotinib (IC50 = 80 nM). 7e, 7g, 7k, 7n, and 7o inhibited CDK2 efficiently in comparison to the reference dinaciclib (IC50 = 20 nM), with IC50 values ranging from 15 to 31 nM. The results of inhibitory activity assay against different CDK isoforms revealed that the tested compounds had preferential inhibitory activity against the CDK2 isoform.

Lactylation-driven FTO targets CDK2 to aggravate microvascular anomalies in diabetic retinopathy.

Diabetic retinopathy (DR) is a leading cause of irreversible vision loss in working-age populations. Fat mass and obesity-associated protein (FTO) is an N6-methyladenosine (m6A) demethylase that demethylates RNAs involved in energy homeostasis, though its influence on DR is not well studied. Herein, we detected elevated FTO expression in vitreous fibrovascular membranes of patients with proliferative DR. FTO promoted cell cycle progression and tip cell formation of endothelial cells (ECs) to facilitate angiogenesis in vitro, in mice, and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetic microvascular leakage, and mediated EC-microglia interactions to induce retinal inflammation and neurodegeneration in vivo and in vitro. Mechanistically, FTO affected EC features via modulating CDK2 mRNA stability in an m6A-YTHDF2-dependent manner. FTO up-regulation under diabetic conditions was driven by lactate-mediated histone lactylation. FB23-2, an inhibitor to FTO's m6A demethylase activity, suppressed angiogenic phenotypes in vitro. To allow for systemic administration, we developed a nanoplatform encapsulating FB23-2 and confirmed its targeting and therapeutic efficiency in mice. Collectively, our study demonstrates that FTO is important for EC function and retinal homeostasis in DR, and warrants further investigation as a therapeutic target for DR patients.

Cells use multiple mechanisms for cell-cycle arrest upon withdrawal of individual amino acids.

Amino acids are required for cell growth and proliferation, but it remains unclear when and how amino acid availability impinges on the proliferation-quiescence decision. Here, we used time-lapse microscopy and single-cell tracking of cyclin-dependent kinase 2 (CDK2) activity to assess the response of individual cells to withdrawal of single amino acids and found strikingly different cell-cycle effects depending on the amino acid. For example, upon leucine withdrawal, MCF10A cells complete two cell cycles and then enter a CDK2-low quiescence, whereas lysine withdrawal causes immediate cell-cycle stalling. Methionine withdrawal triggers a restriction point phenotype similar to serum starvation or Mek inhibition: upon methionine withdrawal, cells complete their current cell cycle and enter a CDK2-low quiescence after mitosis. Modulation of restriction point regulators p21/p27 or cyclin D1 enables short-term rescue of proliferation under methionine and leucine withdrawal, and to a lesser extent lysine withdrawal, revealing a checkpoint connecting nutrient signaling to cell-cycle entry.

EMP3 sustains oncogenic EGFR/CDK2 signaling by restricting receptor degradation in glioblastoma.

Epithelial membrane protein 3 (EMP3) is an N-glycosylated tetraspanin with a putative trafficking function. It is highly expressed in isocitrate dehydrogenase-wild-type glioblastoma (IDH-wt GBM), and its high expression correlates with poor survival. However, the exact trafficking role of EMP3 and how it promotes oncogenic signaling in GBM remain unclear. Here, we show that EMP3 promotes EGFR/CDK2 signaling by regulating the trafficking and enhancing the stability of EGFR. BioID2-based proximity labeling revealed that EMP3 interacts with endocytic proteins involved in the vesicular transport of EGFR. EMP3 knockout (KO) enhances epidermal growth factor (EGF)-induced shuttling of EGFR into RAB7 + late endosomes, thereby promoting EGFR degradation. Increased EGFR degradation is rescued by the RAB7 negative regulator and novel EMP3 interactor TBC1D5. Phosphoproteomic and transcriptomic analyses further showed that EMP3 KO converges into the inhibition of the cyclin-dependent kinase CDK2 and the repression of EGFR-dependent and cell cycle transcriptional programs. Phenotypically, EMP3 KO cells exhibit reduced proliferation rates, blunted mitogenic response to EGF, and increased sensitivity to the pan-kinase inhibitor staurosporine and the EGFR inhibitor osimertinib. Furthermore, EGFR-dependent patient-derived glioblastoma stem cells display a transcriptomic signature consistent with reduced CDK2 activity, as well as increased susceptibility to CDK2 inhibition upon EMP3 knockdown. Lastly, using TCGA data, we showed that GBM tumors with high EMP3 expression have increased total and phosphorylated EGFR levels. Collectively, our findings demonstrate a novel EMP3-dependent mechanism by which EGFR/CDK2 activity is sustained in GBM. Consequently, EMP3's stabilizing effect provides an additional layer of tumor cell resistance against targeted kinase inhibition.

CDK2 inhibition disorders centrosome stoichiometry and alters cellular outcomes in aneuploid cancer cells.

Cyclin-dependent Kinase 2 (CDK2) inhibition prevents supernumerary centrosome clustering. This causes multipolarity, anaphase catastrophe and apoptotic death of aneuploid cancers. This study elucidated how CDK2 antagonism affected centrosome stoichiometry. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent imaging were used. Studies interrogated multipolar mitosis after pharmacologic or genetic repression of CDK2. CDK2/9 antagonism with CYC065 (Fadraciclib)-treatment disordered centrosome stoichiometry in aneuploid cancer cells, preventing centrosome clustering. This caused ring-like chromosomes or multipolar cancer cells to form before onset of cell death. Intriguingly, CDK2 inhibition caused a statistically significant increase in single centrioles rather than intact centrosomes with two centrioles in cancer cells having chromosome rings or multipolarity. Statistically significant alterations in centrosome stoichiometry were undetected in other mitotic cancer cells. To confirm this pharmacodynamic effect, CDK2 but not CDK9 siRNA-mediated knockdown augmented cancer cells with chromosome ring or multipolarity formation. Notably, engineered gain of CDK2, but not CDK9 expression, reversed emergence of cancer cells with chromosome rings or multipolarity, despite CYC065-treatment. In marked contrast, CDK2 inhibition of primary human alveolar epithelial cells did not confer statistically significant increases of cells with ring-like chromosomes or multipolarity. Hence, CDK2 antagonism caused differential effects in malignant versus normal alveolar epithelial cells. Translational relevance was confirmed by CYC065-treatment of syngeneic lung cancers in mice. Mitotic figures in tumors exhibited chromosome rings or multipolarity. Thus, CDK2 inhibition preferentially disorders centrosome stoichiometry in cancer cells. Engaging this disruption is a strategy to explore against aneuploid cancers in future clinical trials.

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation.

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

Cyclin E/CDK2 and feedback from soluble histone protein regulate the S phase burst of histone biosynthesis.

Faithful DNA replication requires that cells fine-tune their histone pool in coordination with cell-cycle progression. Replication-dependent histone biosynthesis is initiated at a low level upon cell-cycle commitment, followed by a burst at the G1/S transition, but it remains unclear how exactly the cell regulates this burst in histone biosynthesis as DNA replication begins. Here, we use single-cell time-lapse imaging to elucidate the mechanisms by which cells modulate histone production during different phases of the cell cycle. We find that CDK2-mediated phosphorylation of NPAT at the restriction point triggers histone transcription, which results in a burst of histone mRNA precisely at the G1/S phase boundary. Excess soluble histone protein further modulates histone abundance by promoting the degradation of histone mRNA for the duration of S phase. Thus, cells regulate their histone production in strict coordination with cell-cycle progression by two distinct mechanisms acting in concert.