Expression Of Cyclin-dependent Kinase Inhibitor 1A Research Articles

Decreased mitochondrial transcription factor A and mitochondrial DNA copy number promote cyclin-dependent kinase inhibitor 1A expression and reduce tumorigenic properties of colorectal cancer cells

PurposeColorectal cancer is one of the most common and deadliest cancer types worldwide. In the last years, changes in the mitochondrial DNA (mtDNA) copy number have been described to correlate with the prognostic outcome for colorectal cancer patients by impacting different tumorigenic properties. One key regulator of mtDNA is the mitochondrial transcription factor A (TFAM) that acts as a limiting factor of mtDNA copy number. Here, we investigated the effect of TFAM deficiency on mtDNA and tumorigenic properties in the human colorectal cancer cell line SW480.MethodsTFAM expression was stably downregulated in the colorectal cancer cell line SW480 using the CRISPR-Cas9 approach. To dissect the molecular alterations induced by deletion of TFAM, RNA sequencing and gene set enrichment analysis was performed on TFAM-wild-type and TFAM-deficient SW480 cells. Functional consequences of TFAM downregulation were assessed in cellular assays.ResultsWe showed that TFAM deficiency leads to decreased mtDNA copy number and reduced expression of mtDNA-encoded genes. TFAM-deficient cells also revealed higher activity of senescence-associated β-galactosidase and decreased cell growth parameters. Moreover, RNA sequencing showed that the expression of cyclin dependent kinase inhibitor 1A (CDKN1A/p21) is significantly increased in TFAM-deficient cells.ConclusionOur results suggest that TFAM-induced changes of the mitochondrial genome lead to upregulated CDKN1A/p21 expression in colorectal cancer cells identifying p21 as a new possible linker between mitochondria and nucleus.

Exploring the Role of CDKN2A in Human Cancers Using an Integrative Pan-Cancer Approach

This study aims to investigate the expression variation, biological significance, and prognostic value of cyclin-dependent kinase inhibitor 2A (CDKN2A) as a common biomarker across 33 malignancies. Various bioinformatics tools, including UALCAN, GEPIA2, OncoDB, cBioPortal, TIMER2, STRING, DAVID, and the GSCA database, were employed for this pan-cancer analysis. The results revealed significant up-regulation of CDKN2A in 24 major human cancer subtypes (P < 0.05). This up-regulation was strongly associated with poor overall survival and tumor dissemination, particularly in uterine corpus endometrial carcinoma (UCEC), colon adenocarcinoma (COAD), and liver hepatocellular carcinoma (LIHC), highlighting its potential as a prognostic biomarker. Moreover, CDKN2A overexpression was linked to diverse clinicopathological characteristics of patients. Genetic alterations recorded via cBioPortal indicated minimal mutation rates in COAD, LIHC, and UCEC. Additionally, promoter methylation, drug sensitivity, and enrichment analyses were performed to explore associations with CDKN2A expression. Overall, the findings emphasize the potential of CDKN2A as a shared diagnostic and prognostic biomarker, as well as a therapeutic target in COAD, LIHC, and UCEC, particularly in patients with varied clinicopathological traits.

Impact of CDKN2A gene expression on colon adenocarcinoma via biosignature analysis.

Colorectal adenocarcinoma (COAD) has a poor prognosis. Cyclin-dependent kinase inhibitor 2A (CDKN2A) significantly affects the development and progression of various human tumors. However, the significance and pathological mechanisms of CDKN2A in COAD remain to be elucidated. We assessed expression levels, clinical significance, biological function, co-expressed genes, and enrichment of related pathways of CDKN2A in COAD using various databases, including The University of Alabama at Birmingham Cancer Data Analysis Portal, Gene Expression Profiling Interactive Analysis, Tumor Immune Estimation Resource, Human Protein Atlas, STRING, GeneMANIA, cBioPortal, and Linked Omics. Our investigation showed that CDKN2A was highly expressed in colon adenocarcinomas (P < .001). It is weakly expressed or not expressed in normal tissues. The survival time of patients with colon adenocarcinoma with high CDKN2A expression is significantly shorter than that of patients with low expression levels (P = .011). There was a significant positive correlation between the expression level of CDKN2A in colon adenocarcinoma tissues and the infiltration of CD4+ T cells, macrophages, and neutrophils. Moreover, there was a significant negative association between the expression level of CDKN2A in colon adenocarcinoma tissues and B cell infiltration. The ten hub genes included tumor protein 53, V-myc Avian Myelocytomatosis Viral Oncogene Homolog, AKT serine/threonine kinase 1, cyclin-dependent kinase 2, phosphatase and tensin homolog deleted on chromosome ten, cyclin D1, cyclin dependent kinase 4, cyclin dependent kinase inhibitor 1A, catenin beta 1, and B-Raf proto-oncogene, serine/threonine kinase. Mutations in the CDKN2A genome in colon adenocarcinoma reduce survival. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that the differentially expressed genes were enriched in apoptotic signaling pathways and multiple pathways related to metabolic progression. Our results indicate that CDKN2A can be used as a marker of poor prognosis in patients with colon adenocarcinoma. CDKN2A may regulate the occurrence and development of colon adenocarcinomas by influencing immune cell infiltration and metabolic pathways.

The crosstalk role of CDKN2A between tumor progression and cuproptosis resistance in colorectal cancer.

Cuproptosis is a type of cell death characterized by excessive copper-lipid reactions in the tricarboxylic acid cycle, resulting in protein toxicity stress and cell death. Although known as a cuproptosis inhibitor through CRISPR-Cas9 screening, the role of cyclin-dependent kinase inhibitor 2A (CDKN2A) in cuproptosis resistance and its connection to tumor development remains unclear. In this study, we combined single-cell sequencing, spatial transcriptomics, pathological image analysis, TCGA multi-omics analysis and in vitro experimental validation to comprehensively investigate CDKN2A distribution, expression, epigenetic modification, regulation and genomic features in colorectal cancer cells. We further explored the associations between CDKN2A and cellular pathway, immune infiltration and spatial signal communication. Our findings showed an increasing trend in cuproptosis in the trajectory of tumor progression, accompanied by an upward trend of CDKN2A. CDKN2A underwent transcriptional activation by MEF2D and via the SNHG7/miR-133b axis, upregulating glycolysis, copper metabolism and copper ion efflux. CDKN2A likely drives epithelial-mesenchymal transition (EMT) and progression by activating Wnt signaling. CDKN2A is associated with high genomic instability and sensitivity to radiation and chemotherapy. Tumor regions expressing CDKN2A exhibit distinctive SPP1+ tumor-associated macrophage (TAM) infiltration and MMP7 enrichment, along with unique signaling crosstalk with adjacent areas. CDKN2A mediates cuproptosis resistance through regulating glycolysis and copper homeostasis, accompanied by a malignant phenotype and pro-tumor niche. Radiation and chemotherapy are expected to potentially serve as therapeutic approaches for cuproptosis-resistant colorectal cancer with high CDKN2A expression.

CDKN2A inhibited ferroptosis through activating JAK2/STAT3 pathway to modulate cisplatin resistance in cervical squamous cell carcinoma.

Cervical squamous cell carcinoma (CESC) is a significant threat to women's health. Resistance to cisplatin (DDP), a common treatment, hinders the therapeutic efficacy. Understanding the molecular basis of DDP resistance in CESC is imperative. Cyclin-dependent kinase inhibitor 2A (CDKN2A) expression was evaluated through quantitative real-time-PCR and western blot in clinical samples from 30 CESC patients and human cervical epithelial cells and CESC cell lines (SiHa, C33A, and Caski). It was also evaluated through bioinformatics analysis in Timer, Ualcan, and GEPIA database. Cell viability was detected by CCK-8. Apoptosis was detected by Calcein AM/PI assay. Lipid reactive oxygen species (ROS), malondialdehyde, glutathione, Fe 2+ , and iron level were detected by kits. Protein level of JAK2, STAT3, p-JAK2, p-STAT3, ACSL4, GPX4, SLC7A11, and FTL were detected by western blot. In CESC, elevated CDKN2A expression was observed. Cisplatin exhibited a dual effect, inhibiting cell proliferation and inducing ferroptosis in CESC. CDKN2A knockdown in a cisplatin-resistant cell line suppressed proliferation and induced ferroptosis. Moreover, CDKN2A was identified as an inhibitor of erastin-induced ferroptosis. Additionally, targeting the JAK2/STAT3 pathway enhanced ferroptosis in cisplatin-resistant cells. CDKN2A could inhibit ferroptosis in CESC through activating JAK2/STAT3 pathway to modulate cisplatin resistance.

Clinical significance and prospective mechanism of increased CDKN2A expression in small cell lung cancer

BackgroundAlthough it has been shown that cyclin dependent kinase inhibitor 2A (CDKN2A) plays a significant role in a number of malignancies, its clinicopathological value and function in small cell lung cancer (SCLC) is unclear and warrants additional research.MethodsThe clinical significance of CDKN2A expression in SCLC was examined by multiple methods, including comprehensive integration of mRNA level by high throughput data, Kaplan–Meier survival analysis for prognostic value, and validation of its protein expression using in-house immunohistochemistry.ResultsThe expression of CDKN2A mRNA in 357 cases of SCLC was evidently higher than that in the control group (n = 525) combing the data from 20 research centers worldwide. The standardized mean difference (SMD) was 3.07, and the area under the curve (AUC) of summary receiver operating characteristic curve (sROC) was 0.97 for the overexpression of CDKN2A. ACC, COAD, KICH, KIRC, PCPG, PRAD, UCEC, UVM patients with higher CDKN2A expression had considerably worse overall survival rates than those with lower CDKN2A expression with the hazard ratio (HR) > 1.ConclusionCDKN2A upregulation extensively enhances the carcinogenesis and progression of SCLC.

Cuproptosis-related gene CDKN2A as a molecular target for IPF diagnosis and therapeutics.

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic interstitial lung disease with limited therapeutic options. Cuproptosis is a recently proposed novel form of programmed cell death, which has been strongly implicated in the development ofvarioushumandiseases. However, the prognostic and therapeutic value of cuproptosis-related genes (CRGs) in IPF remains to be elucidated. In the present study, weighted gene co-expression network analysis (WGCNA) was employed to identify the key CRGs associated with the development of IPF. The subsequent GSEA, immune cell correlation analysis, and single-cell RNA-Seq analysis were conducted to explore the potential role of the identified CRGs in IPF. In addition, ROC curves and survival analysis were used to assess the prognostic value of the key CRGs in IPF. Moreover, we explored the molecular mechanisms of participation of identified key CRGsinthedevelopmentofpulmonary fibrogenesis throughinvivo and invitroexperiments. The expression level of cyclin-dependent kinase inhibitor 2A (CDKN2A) is upregulated in the lung tissues of IPF patients and associated with disease severity. Notably, CDKN2A was constitutively expressed by fibrosis-promoting M2 macrophages. Decreased CDKN2A expression sensitizes M2 macrophages to elesclomol-induced cuproptosis in vitro. Inhibition of CDKN2A decreases the number of viable macrophages and attenuates bleomycin-induced pulmonary fibrosis in mice. These findings indicate that CDKN2A mediates the resistance of fibrosis-promoting M2 macrophages to cuproptosis and promotes pulmonary fibrosis in mice. Our work provides fresh insights into CRGs in IPF with potential value for research in the pathogenesis, diagnosis,andanewtherapystrategyforIPF.

The cell senescence regulator p16 is a promising cancer prognostic and immune check-point inhibitor (ICI) therapy biomarker.

Cyclin-dependent kinase inhibitor 2A (CDKN2A) encodes the cell senescence regulator protein p16. The expression of p16 raises in cell senescence and has a nuclear regulation in cell aging. Meanwhile, it's also reported to inhibit the aggression of several cancers. But its clinical application and role in cancer immunotherapy needs further investigation. We collected the transcriptional data of pan-cancer and normal human tissues from The Cancer Genome Atlas and the Genotype-Tissue Expression databases. CBioPortal webtool was employed to mine the genomic alteration status of CDKN2A across cancers. Kaplan-Meier method and univariate Cox regression were performed for prognostic assessments across cancers, respectively. Gene Set Enrichment Analysis is the main method used to search the associated cancer hallmarks associated with CDKN2A. TIMER2.0 was used to analyze the immune cell infiltration relevance with CDKN2A in pan-cancer. The associations between CDKN2A and immunotherapy biomarkers or regulators were performed by spearman correlation analysis. We found CDKN2A is overexpressed in most cancers and exhibits prognosis predictive ability in various cancers. In addition, it is significantly correlated with immune-activated hallmarks, cancer immune cell infiltrations and immunoregulators. The most interesting finding is that CDKN2A can significantly predict anti-PDL1 therapy response. Finally, specific inhibitors which correlated with CDKN2A expression in different cancer types were also screened by using Connectivity Map (CMap) tool. The results revealed that CDKN2A acts as a robust cancer prognostic and immunotherapy biomarker. Its function in the regulation of cancer cell senescence might shape the tumor microenvironment and contribute to its predictive ability of immunotherapy.

Knocking Down CDKN2A in 3D hiPSC-Derived Brown Adipose Progenitors Potentiates Differentiation, Oxidative Metabolism and Browning Process.

Human induced pluripotent stem cells (hiPSCs) have the potential to be differentiated into any cell type, making them a relevant tool for therapeutic purposes such as cell-based therapies. In particular, they show great promise for obesity treatment as they represent an unlimited source of brown/beige adipose progenitors (hiPSC-BAPs). However, the low brown/beige adipocyte differentiation potential in 2D cultures represents a strong limitation for clinical use. In adipose tissue, besides its cell cycle regulator functions, the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus modulates the commitment of stem cells to the brown-like type fate, mature adipocyte energy metabolism and the browning of adipose tissue. Here, using a new method of hiPSC-BAPs 3D culture, via the formation of an organoid-like structure, we silenced CDKN2A expression during hiPSC-BAP adipogenic differentiation and observed that knocking down CDKN2A potentiates adipogenesis, oxidative metabolism and the browning process, resulting in brown-like adipocytes by promoting UCP1 expression and beiging markers. Our results suggest that modulating CDKN2A levels could be relevant for hiPSC-BAPs cell-based therapies.

MiR-484 promotes the malignant progression of glioma by inhibiting CDKN2A expression.

Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) is involved in glioma progression, but the specific molecular mechanism of CDKN2A in glioma cell migration and invasion needs to be further explored. Data related to CDKN2A expression and glioma overall survival were obtained from The Cancer Genome Atlas (TCGA) database. Then, CDKN2A expression in glioma tissues/cells or paracancer tissues/astrocytes was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot. Afterwards, Wound healing, Transwell and tube formation assay were performed to identify the invasion, migration and angiogenesis of glioma cells, respectively. TargetScan database predicted the targeted binding between miR-484 and CDKN2A, which was verified by dual luciferase reporter gene assay. Western blot and qRT-PCR were performed to detect the expression of VEGF, E-cadherin, N-cadherin and Vimentin in glioma cells. CDKN2A was low-expressed in glioma tissue/cells as compared to paracancer tissue/astrocytes, and was strongly associated to the poor prognosis of glioma. Further studies found that down-regulation of CDKN2A could promote migration, invasion and angiogenesis of glioma cells. Besides, miR-484 was high-expressed in glioma cells compared to astrocytes. Up-regulation of miR-484 could enhance migration, invasion and angiogenesis of glioma cells. In addition, up-regulated miR-484 suppressed the expression of E-cadherin, and promoted the expression of N-cadherin, Vimentin and VEGF. However, there was negative regulation of miR-484 and CDKN2A, and CDKN2A could partially offset the effect of miR-484. MiR-484 promoted cell migration, invasion and angiogenesis by inhibiting CDKN2A expression.

Accumulation of senescent cells in the stroma of aged mouse ovary.

Senescent cells play a detrimental role in age-associated pathogenesis by producing factors involved in senescence-associated secretory phenotype (SASP). The present study was conducted to examine the possibility that senescent cells are present in aged ovaries and, if so, to determine the tissue region where senescent cells accumulate using a mouse model. Female mice at 2-4 and 8-10 months were used as reproductively young and aged models, respectively; the latter included mice with and without reproductive experience. Cells positive for senescence-associated β-galactosidase (SA-β-Gal) staining, one of the markers of cellular senescence, were detected in the stromal region of aged, but not young, ovaries regardless of reproductive experience. Likewise, the localization of cells expressing CDKN2A (cyclin dependent kinase inhibitor 2A), another senescence marker, in the stromal region of aged ovaries was detected with immunohistochemistry. CDKN2A expression detected by western blotting was significantly higher in the ovaries of aged mice with reproductive experience than in those without the experience. Moreover, cells positive for both γH2AX (a senescence marker) and fluorescent SA-β-Gal staining were present in those isolated from aged ovaries. In addition, the transcript levels of several SASP factors were significantly increased in aged ovaries. These results suggest that senescent cells accumulate in the ovarian stroma and may affect ovarian function in aged mice. Additionally, reproductive experience may promote accumulation.

LncRNA LENGA acts as a tumor suppressor in gastric cancer through BRD7/TP53 signaling.

It has been established that long noncoding RNAs (lncRNAs) play a crucial role in various cancer types, and there are vast numbers of long noncoding RNA transcripts that have been identified by high-throughput methods. However, the biological function of many novel aberrantly expressed lncRNAs remains poorly elucidated, especially in gastric cancer (GC). Here, we first identified a novel lncRNA termed LENGA (Low Expression Noncoding RNA in Gastric Adenocarcinoma), which was significantly downregulated in GC tissues compared to adjacent normal tissues. Next, we found that reduced expression of LENGA in GC was also associated with a shorter life expectancy. The proliferation, migration, and invasion of GC cells were increased after LENGA knockdown but restrained after LENGA overexpression in vitro and in vivo. It was further demonstrated that LENGA physically binds to BRD7 (bromodomain-containing 7) in the bromodomain domain and acts as a scaffold that enhances the interaction between BRD7 and TP53 (tumor protein p53), regulating the expression of a subset of genes in the p53 pathway, including CDKN1A (cyclin-dependent kinase inhibitor 1A) and PCDH7 (protocadherin 7), at the transcriptional level. Consistently, the expression of CDKN1A has a positive correlation with LENGA in GC patients. Taken together, this study uncovers a novel tumor suppressor lncRNA, LENGA, and describes its biological function, molecular mechanism, and clinical significance. This highlights the potential importance of targeting the LENGA/BRD7/TP53 axis in GC treatment.

Genome-Wide Association Study Identifies CDKN1A as a Novel Locus Associated with Muscle Fiber Composition.

Muscle fiber composition is associated with physical performance, with endurance athletes having a high proportion of slow-twitch muscle fibers compared to power athletes. Approximately 45% of muscle fiber composition is heritable, however, single nucleotide polymorphisms (SNP) underlying inter-individual differences in muscle fiber types remain largely unknown. Based on three whole genome SNP datasets, we have shown that the rs236448 A allele located near the cyclin-dependent kinase inhibitor 1A (CDKN1A) gene was associated with an increased proportion of slow-twitch muscle fibers in Russian (n = 151; p = 0.039), Finnish (n = 287; p = 0.03), and Japanese (n = 207; p = 0.008) cohorts (meta-analysis: p = 7.9 × 10−5. Furthermore, the frequency of the rs236448 A allele was significantly higher in Russian (p = 0.045) and Japanese (p = 0.038) elite endurance athletes compared to ethnically matched power athletes. On the contrary, the C allele was associated with a greater proportion of fast-twitch muscle fibers and a predisposition to power sports. CDKN1A participates in cell cycle regulation and is suppressed by the miR-208b, which has a prominent role in the activation of the slow myofiber gene program. Bioinformatic analysis revealed that the rs236448 C allele was associated with increased CDKN1A expression in whole blood (p = 8.5 × 10−15) and with greater appendicular lean mass (p = 1.2 × 10−5), whereas the A allele was associated with longer durations of exercise (p = 0.044) reported amongst the UK Biobank cohort. Furthermore, the expression of CDKN1A increased in response to strength (p < 0.0001) or sprint (p = 0.00035) training. Accordingly, we found that CDKN1A expression is significantly (p = 0.002) higher in the m. vastus lateralis of strength athletes compared to endurance athletes and is positively correlated with the percentage of fast-twitch muscle fibers (p = 0.018). In conclusion, our data suggest that the CDKN1A rs236448 SNP may be implicated in the determination of muscle fiber composition and may affect athletic performance.

Targeting cell cycle and apoptosis to overcome chemotherapy resistance in acute myeloid leukemia.

Chemotherapy-resistant acute myeloid leukemia (AML), frequently driven by clonal evolution, has a dismal prognosis. A genome-wide CRISPR knockout screen investigating resistance to doxorubicin and cytarabine (Dox/AraC) in human AML cell lines identified gene knockouts involving AraC metabolism and genes that regulate cell cycle arrest (cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53) as contributing to resistance. In human AML cohorts, reduced expression of CDKN2A conferred inferior overall survival and CDKN2A downregulation occurred at relapse in paired diagnosis-relapse samples, validating its clinical relevance. Therapeutically targeting the G1S cell cycle restriction point (with CDK4/6 inhibitor, palbociclib and KAT6A inhibitor, WM-1119, to upregulate CDKN2A) synergized with chemotherapy. Additionally, direct promotion of apoptosis with venetoclax, showed substantial synergy with chemotherapy, overcoming resistance mediated by impaired cell cycle arrest. Altogether, we identify defective cell cycle arrest as a clinically relevant contributor to chemoresistance and identify rationally designed therapeutic combinations that enhance response in AML, potentially circumventing chemoresistance.

E3 ubiquitin ligase FBXW7 enhances radiosensitivity of non-small cell lung cancer cells by inhibiting SOX9 regulation of CDKN1A through ubiquitination

AbstractNon-small cell lung cancer (NSCLC) has high rates of morbidity and mortality. E3 ubiquitin ligase usually has antitumor effects. This study evaluated the mechanism of E3 ligase FBXW7 (F-box and WD repeat domain containing 7) in the radiosensitivity of NSCLC. NCI-H1299 and NCI-H1299R cells were irradiated by 0, 2, 4, and 6 Gy doses of X-ray, respectively. In addition to the measurement of cell proliferation, apoptosis, and γ-H2AX, FBXW7 expression was measured and the interaction between FBXW7 and SOX9 (SRY-box transcription factor 9) was evaluated. Ubiquitination level and protein stability of SOX9 were examined after FBXW7 overexpression. The binding relationship between SOX9 and CDKN1A (cyclin-dependent kinase inhibitor 1A) was verified. Xenograft tumor model was established to evaluate the effect of FBXW7 on radiosensitivity in vivo. FBXW7 was under-expressed in radioresistant cells. Overexpression of FBXW7 repressed NCI-H1299 and NCI-H1299R cell proliferation and colony formation and increased γ-H2AX-positive foci. Overexpression of FBXW7 increased the ubiquitination level and reduced the protein stability of SOX9. SOX9 bound to the CDKN1A promoter to inhibit CDKN1A expression. FBXW7 inhibited tumorigenesis and apoptosis and enhanced radiosensitivity of NSCLC cells in vivo via the SOX9/CDKN1A axis. Overall, FBXW7 inhibited SOX9 expression by promoting SOX9 ubiquitination and proteasome degradation, suppressing the binding of SOX9 to CDKN1A, and upregulating CDKN1A, thereby improving the radiosensitivity of NSCLC cells.

Comprehensive proteomic signature and identification of CDKN2A as a promising prognostic biomarker and therapeutic target of colorectal cancer.

BACKGROUNDThe carcinogenesis of colorectal cancer (CRC) involves many different molecules and multiple pathways, and the specific mechanism has not been elucidated until now. Existing studies on the proteomic signature profiles of CRC are relatively limited. Therefore, we herein aimed to provide a more comprehensive proteomic signature profile and discover new prognostic markers and therapeutic targets by performing proteomic analysis of CRC and paired normal tissues.AIMTo investigate the proteomic signature and identify novel protein prognostic biomarkers of CRC.METHODSCancer tissues and paired normal tissues were collected from 48 patients who underwent surgical removal at the China-Japan Friendship Hospital from January 2020 to June 2021. Data independent acquisition (DIA) quantitative proteomic analysis was performed using high-performance liquid chromatography–mass spectrometry/mass spectrometry (nano-UHPLC–MS/MS) to identify differentially expressed proteins, among which those with a P adj value (t test, BH correction) < 0.05 and an absolute fold change (|log2FC|) > 2 were identified as potential markers. Differentially expressed proteins were selected by bioinformatics analysis and validated by immunohistochemical tissue microarrays, and their association with prognosis was further analyzed with the Gene Expression Profiling Interactive Analysis database to identify prognostic protein biomarkers of CRC.RESULTSSignificantly differential protein expression was observed between cancer tissues and normal tissues. Compared with normal tissues, 1115 proteins were upregulated and 705 proteins were downregulated in CRC based on P adj < 0.05 and |log2FC| > 2, and bioinformatics analysis revealed that the differentially expressed proteins were involved in multiple biological processes associated with tumorigenesis, including ribosome biogenesis in eukaryotes, focal adhesion, extracellular matrix-receptor interactions and other tumor metabolism processes. Moreover, cyclin-dependent kinase inhibitor 2A (CDKN2A) expression was markedly upregulated in CRC, as validated by immunohistochemistry (0.228 vs 0.364, P = 0.0044), and was significantly enriched in tumor proliferation and signal transduction pathways such as the cell cycle and p53 signaling pathways. High CDKN2A expression was significantly correlated with poor prognosis (P = 0.021). These results demonstrated that CDKN2A functions as a driver of CRC.CONCLUSIONOur study provides a comprehensive proteomic signature of CRC and highlights CDKN2A as a potential powerful prognostic marker and precision therapeutic target.

Inter-individual Differences in Radiosensitivity based on CDKN1A, GADD45A, DDB2, BCL2 and TRX Gene Expression in Human Lymphocytes

Transcriptomic signatures of radioresponsive genes are recently explored as a powerful tool for biodosimetry. As cytogenetics remains the gold standard and a robust approach for wide-scale testing during radiologic emergencies, the utility of molecular signatures remains to be adequately validated. The present study analyzed the expression profiles of five highly responsive genes to radiation: B-cell lymphoma 2 (BCL2), DNA damage-binding protein 2 (DDB2), cyclin-dependent kinase inhibitor 1A (CDKN1A), growth arrest and DNA-damage-inducible alpha (GADD45A) and thioredoxin (TRX). Ex vivo exposure of peripheral blood lymphocytes from 17 healthy subjects, aged 21 to 53 years, at 2 Gy radiation appeared to mobilize the index genes involved in cell cycle arrest (GADD45A and CDKN1A) and the binding to DNA lesion to facilitate excision repair (DDB2). However, the transcription of pro-survival protein BCL2 and redox repair antioxidant protein TRX were not as reliable as molecular biodosimeters considering the variability among individual responses. Nevertheless, the significant correlations observed among the genes emphasize their synchronized roles during DNA damage and redox response. Gender-based differences in gene expression were not detected. These findings indicated the diverse transcriptional regulation of p53-dependent pathways in radiation-exposed lymphocytes. Further validation in more patients during healthy and diseased states could contribute to the clinical application of gene-based radiation biodosimetry.

Targeting Control of Cell Cycle Enhances the Activity of Conventional Chemotherapy in Chemotherapy-Resistant Acute Myeloid Leukemia

Chemotherapy-resistant acute myeloid leukemia (AML) manifesting as primary refractory or relapsed disease carries a dismal prognosis and is often driven by clonal evolution. We have performed a genome-wide CRISPR knockout screen investigating resistance to conventional AML therapy, cytarabine and doxorubicin (AraC/Dox), in 2 independent human AML cell lines. Chemoresistant populations were enriched with gRNAs disrupting cell cycle arrest genes, including cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53. Here, we validate the direct contribution of these genes to chemoresistance and demonstrate that rationally designed therapeutic regimens targeting cell cycle enhances chemotherapy response in AML.Using CRISPR-mediated gene editing, we individually inactivated CDKN2A and CHEK2 in Cas9-expressing OCI-AML3 and MV4-11 cells. CDKN2A- and CHEK2-deficient cells demonstrated proliferative advantage in the presence of AraC/Dox in co-culture competition assays, confirming direct contribution of these gene knockouts to chemoresistance. Nil to modest reductions in apoptosis were seen in CDKN2A- and CHEK2-deficient cells treated with AraC/Dox for 72 hours compared to unedited controls. However, failure of accumulation of cells in the non-cycling G 0/G 1 proportion was seen in these edited cells after chemotherapy, corresponding with maintained DNA synthesis, as measured by BrdU incorporation, and a failure to downregulate phospho-Rb protein expression, indicating ongoing active cell cycling in spite of chemotherapy. These results confirm failure of cell cycle arrest as the major mechanism of resistance with inactivation of CDKN2A or CHEK2.To assess relevance of these genes in chemotherapy response in human AML, we analysed the effect of CDKN2A expression on prognosis. Reduced expression of CDKN2A conferred inferior overall survival in 3 independent clinical cohorts. Additionally, downregulation of CDKN2A and an increase in downstream cell cycling effector, cyclin-dependent kinase 6 (CDK6) was seen at relapse in paired diagnosis-relapse AML samples (Li et al. 2016, Nature Medicine). Further, CHEK2 mutations in clonal hematopoiesis are enriched in patients with solid organ cancers following chemo- or radiotherapy, functionally demonstrating chemoresistance over non-mutated cells. These data suggest CDKN2A and CHEK2 loss-of-function is relevant in promoting chemoresistance in human hematopoiesis.We therefore investigated whether therapeutically targeting cell cycle control pathways that converge on the G 1/S restriction point could synergise with cytotoxic chemotherapy, potentially circumventing chemoresistance. The addition of MDM2 inhibitor, nutlin-3a with AraC/Dox achieved striking synergism in TP53-competent cell lines in reducing viability and promoting apoptosis. Inhibition of CDK4/6 with palbociclib alone induced cell cycle arrest without apoptosis, however combination therapy with AraC/Dox significantly potentiated apoptosis. To functionally validate the role of CDKN2A itself, we examined the inhibitor of histone acetyltransferase KAT6A, WM-1119 (Baell et al. 2018, Nature), which transcriptionally upregulates CDKN2A. WM-1119 had anti-leukemic activity across multiple cell lines, corresponding with down regulation of cell cycling and upregulation of senescence signatures. The anti-leukemic activity was prevented by CDKN2A inactivation, indicating that CDKN2A upregulation is essential for WM-1119's anti-leukemic effect. WM-1119 enhanced activity of AraC/Dox in multiple cell contexts, mediated by cooperative induction of CDKN2A. Its efficacy and synergy with AraC/Dox was further demonstrated in MLL-AF9 primary murine AML ex vivo, demonstrating broader applicability of the approach.Our findings reveal defects in cell cycle arrest pathways as clinically relevant contributors to chemoresistance in AML. Combining agents to target cell cycle components improved in vitro anti-leukemic activity of chemotherapy and warrants further exploration of their translational potential. DisclosuresBullinger: Amgen: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Menarini: Consultancy; Hexal: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Consultancy, Honoraria; Astellas: Honoraria; Gilead: Consultancy; Daiichi Sankyo: Consultancy, Honoraria; Seattle Genetics: Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Honoraria; Abbvie: Consultancy, Honoraria. Lane: BMS: Consultancy, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Novartis: Consultancy; Geron: Consultancy.

Triptolide is a Promising Therapeutic Approach in Treating Thyroid Cancer Based on in silico and in vitro Experiment.

IntroductionThyroid cancer is a familiar kind of cancer. Natural products are promising therapeutic approaches in treating thyroid cancer. Triptolide is a diterpenoid epoxide extracted from Tripterygium wilfordii. The mechanism of triptolide in the treatment of thyroid cancer has not been investigated clearly.MethodsWe evaluated triptolide targets and thyroid cancer targets with related databases. The protein–protein interaction (PPI) networks of the triptolide targets and thyroid cancer targets were constructed with Cytoscape software. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the core PPI network were obtained. Molecular docking analysis was used to evaluated the binding of triptolide with core targets. Furthermore, apoptosis assays, real-time polymerase chain reaction (RT-PCR) and Western blotting were used to evaluate the anticancer functions of triptolide.ResultsTriptolide had 34 targets, and thyroid cancer had 210 targets. The core PPI network of merged PPI networks had 164 nodes and 4513 edges. GO and KEGG enrichment analyses showed that triptolide were related to the cell cycle, apoptosis, and inflammatory signaling pathways. Molecular docking analysis showed that triptolide directly reacted with four core targets: cyclin-dependent kinase inhibitor 1A (CDKN1A), c-JUN, RELA, and tumor protein p53 (TP53). CB-Dock analysis indicated that triptolide could stably bind to core targets. Triptolide inhibited the growth but induced apoptosis of thyroid cancer cells. Triptolide increased the mRNA expression of CDKN1A and TP53 but reduced the mRNA expression of c-JUN and RELA, as shown by RT-PCR. Triptolide increased the protein levels of CDKN1A and phospho-p53 but reduced those of phospho-c-JUN and phospho-NF-κB p65, as shown by Western blotting.DiscussionWe considered that triptolide could treat thyroid cancer by inhibiting cell proliferation, inducing apoptosis and inhibiting inflammatory pathways such as the NF-κB and MAPK signaling pathways. CDKN1A, c-JUN, RELA, and TP53 were involved in the antithyroid cancer mechanism of triptolide.

The Prognostic Value of FGFR3 Expression in Patients with T1 Non-Muscle Invasive Bladder Cancer.

PurposeFibroblast growth factor receptor 3 (FGFR3) alterations are frequent in non-muscle-invasive bladder cancer (NMIBC), although current data regarding the prognostic and therapeutic relevance are inconsistent. We analyzed the prognostic role of FGFR3 mRNA expression in stage T1 NMIBC.Patients and MethodsThe mRNA expression of FGFR3 and cyclin-dependent kinase inhibitor 2A (CDKN2A) was measured by RT-qPCR in 80 patients with stage T1 NMIBC treated with transurethral resection of the bladder and correlated with clinical data and KRT5 and KRT20 expression, used as surrogate markers for basal and luminal subtypes, respectively.ResultsFGFR3 and CDKN2A transcript levels were not correlated. FGFR3 expression was associated with the expression of KRT5 (p=0.002) and KRT20 (p < 0.001). CDKN2A expression was negatively correlated with KRT5 (p=0.030). In Kaplan–Meier analysis and univariable Cox regression analysis, high FGFR3 expression was associated with significantly reduced recurrence-free survival (RFS) (HR=3.78; p < 0.001) and improved overall survival (OS) (HR=0.50; p=0.043), while high CDKN2A expression was associated with reduced OS (HR=2.34; p=0.034). Patient age was the only clinicopathological parameter associated with reduced OS (HR=2.29; p=0.022). No parameter was an independent prognostic factor in multivariable analysis. Next, we stratified the patients depending on their lineage differentiation. In univariable analysis, the prognostic effect of FGFR3 and CDKN2A was observed primarily in patients demonstrating high expression of KRT5 or KRT20, whereas high FGFR3 expression was associated with significantly reduced RFS, irrespective of instillation therapy.ConclusionStage T1 NMIBC patients with high FGFR3 expression show shorter RFS but better OS than patients with low FGFR3 expression.