CBX8 promotes lung adenocarcinoma growth and metastasis through transcriptional repression of CDKN2C and SCEL.

Dysregulation of polycomb group (PcG) proteins that mediate epigenetic gene silencing contributes to tumorigenesis. As core components of the polycomb repressive complex 1 (PRC1), chromobox (CBX) proteins recognize H3K27me3 to recruit PRC1 to maintain a repressive transcriptional state. However, the individual biological functions of these CBX proteins in tumorigenesis warrant in-depth investigation. In this study, we analyzed the mRNA expression of CBX family genes across multiple cancers using The Cancer Genome Atlas data and found different expression patterns of the five CBX genes in different types of cancer. This analyses together with the result of immunohistochemistry indicated that CBX8 expression was significantly higher in lung adenocarcinoma (LUAD) tissues compared to adjacent nontumor tissues. Overexpression approaches demonstrated that CBX8 facilitated LUAD cell proliferation and migration in vitro. Consistently, CBX8 knockdown reduced LUAD cell proliferation and migration in both cell culture and mouse models. RNA sequencing combined with real-time RT-PCR assays revealed CDKN2C and SCEL as target genes of CBX8. Furthermore, chromatin immunoprecipitation assays indicated that CBX8 directly bound to the promoters of CDKN2C and SCEL to establish H2AK119ub. CBX8 depletion reduced the enrichment of H2AK119ub on CDKN2C and SCEL promoters. Moreover, depletion of CDKN2C and SCEL restored the repressed growth and invasion ability of LUAD cells caused by CBX8 knockdown. These findings demonstrate that CBX8 promotes LUAD growth and metastasis through the transcriptional repression of CDKN2C and SCEL. Our study uncovers the oncogenic role of CBX8 in LUAD progression and provides a new target for the diagnosis and therapy of LUAD.

The Oncogenic Role of Cyclin-Dependent Kinase Inhibitor 2C in Lower-Grade Glioma.

Lower-grade gliomas (LGGs) are slow-growing, indolent tumors that usually affect younger patients and present a therapeutic challenge due to the heterogeneity of their clinical presentation. Dysregulation of cell cycle regulatory factors is implicated in the progression of many tumors, and drugs that target cell cycle machinery have shown efficacy as promising therapeutic approaches. To date, however, no comprehensive study has examined how cell cycle-related genes affect LGG outcomes. The cancer genome atlas (TCGA) data were used as the training set for differential analysis of gene expression and patient outcomes; the Chinese glioma genome atlas (CGGA) was used for validation. Levels of one candidate protein, cyclin-dependent kinase inhibitor 2C (CDKN2C), and its relationship to clinical prognosis were determined using a tissue microarray containing 34 LGG tumors. A nomogram was constructed to model the putative role of candidate factors in LGG. Cell type proportion analysis was performed to evaluate immune cell infiltration in LGG. Various genes encoding cell cycle regulatory factors showed increased expression in LGG and were significantly related to isocitrate dehydrogenase and chromosome arms 1p and 19q mutation status. CDKN2C expression independently predicted the outcome of LGG patients. High M2 macrophage values along with elevated CDKN2C expression were associated with poorer prognosis in LGG patients. CDKN2C plays an oncogenic role in LGG, which is associated with M2 macrophages.

Novel germline variants of CDKN1B and CDKN2C identified during screening for familial primary hyperparathyroidism.

PURPOSE: CDKN1B mutations were established as a cause of multiple endocrine neoplasia 4 (MEN4) syndrome in patients with MEN1 phenotype without a mutation in the MEN1 gene. In addition, variants in other cyclin-dependent kinase inhibitors (CDKIs) were found in some MEN1-like cases without the MEN1 mutation. We aimed to describe novel germline mutations of these genes in patients with primary hyperparathyroidism (PHPT). METHODS: During genetic screening for familial hyperparathyroidism, three novel CDKIs germline mutations in three unrelated cases between January 2019 and November 2021 were identified. In this report, we describe clinical features, DNA sequence analysis, and familial segregation studies based on these patients and their relatives. Genome-wide DNA study of loss of heterozygosity (LOH), copy number variation (CNV), and p27/kip immunohistochemistry was performed on tumour samples. RESULTS: DNA screening was performed for atypical parathyroid adenomas in cases 1 and 2 and for cystic parathyroid adenoma and young age at diagnosis of PHPT in case 3. Genetic analysis identified likely pathogenic variants of CDKN1B in cases 1 and 2 and a variant of the uncertain significance of CDKN2C, with uniparental disomy in the tumour sample, in case 3. Neoplasm screening of probands showed other non-endocrine tumours in case 1 (colon adenoma with dysplasia and atypical lipomas) and case 2 (aberrant T-cell population) and a non-functional pituitary adenoma in case 3. CONCLUSION: Germline mutations in CDKIs should be included in gene panels for genetic testing of primary hyperparathyroidism. New germline variants here described can be added to the current knowledge.

Clinical significance of cyclin-dependent kinase inhibitor 2C expression in cancers: from small cell lung carcinoma to pan-cancers.

BACKGROUND: Cyclin-dependent kinase inhibitor 2C (CDKN2C) was identified to participate in the occurrence and development of multiple cancers; however, its roles in small cell lung carcinoma (SCLC) remain unclear. METHODS: Differential expression analysis of CDKN2C between SCLC and non-SCLC were performed based on 937 samples from multiple centers. The prognosis effects of CDKN2C in patients with SCLC were detected using both Kaplan-Meier curves and log-rank tests. Using receiver-operating characteristic curves, whether CDKN2C expression made it feasible to distinguish SCLC was determined. The potential mechanisms of CDKN2C in SCLC were investigated by gene ontology terms and signaling pathways (Kyoto Encyclopedia of Genes and Genomes). Based on 10,080 samples, a pan-cancer analysis was also performed to determine the roles of CDKN2C in multiple cancers. RESULTS: For the first time, upregulated CDKN2C expression was detected in SCLC samples at both the mRNA and protein levels (p of Wilcoxon rank-sum test < 0.05; standardized mean difference = 2.86 [95% CI 2.20-3.52]). Transcription factor FOXA1 expression may positively regulate CDKN2C expression levels in SCLC. High CDKN2C expression levels were related to the poor prognosis of patients with SCLC (hazard ratio > 1, p < 0.05) and showed pronounced effects for distinguishing SCLC from non-SCLC (sensitivity, specificity, and area under the curve ≥ 0.95). CDKN2C expression may play a role in the development of SCLC by affecting the cell cycle. Furthermore, the first pan-cancer analysis revealed the differential expression of CDKN2C in 16 cancers (breast invasive carcinoma, etc.) and its independent prognostic significance in nine cancers (e.g., adrenocortical carcinoma). CDKN2C expression was related to the immune microenvironment, suggesting its potential usefulness as a prognostic marker in immunotherapy. CONCLUSIONS: This study identified upregulated CDKN2C expression and its clinical significance in SCLC and other multiple cancers, suggesting its potential usefulness as a biomarker in treating and differentiating cancers.

The distinct effects of P18 overexpression on different stages of hematopoiesis involve TGF-ß and NF-κB signaling.

Deficiency of P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment. However, the effects of P18 overexpression, which is involved in the inhibitory effects of RUNX1b at the early stage of hematopoiesis, have not been examined in detail. In this study, we established inducible P18/hESC lines and monitored the effects of P18 overexpression on hematopoietic differentiation. Induction of P18 from day 0 (D0) dramatically decreased production of CD34highCD43- cells and derivative populations, but not that of CD34lowCD43- cells, changed the cell cycle status and apoptosis of KDR+ cells and downregulated the key hematopoietic genes at D4, which might cause the severe blockage of hematopoietic differentiation at the early stage. By contrast, induction of P18 from D10 dramatically increased production of classic hematopoietic populations and changed the cell cycle status and apoptosis of CD45+ cells at D14. These effects can be counteracted by inhibition of TGF-ß or NF-κB signaling respectively. This is the first evidence that P18 promotes hematopoiesis, a rare property among cyclin-dependent kinase inhibitors (CKIs).

Enhanced self-renewal of human long-term hematopoietic stem cells by a sulfamoyl benzoate derivative targeting p18INK4C.

The use of umbilical cord blood transplant has been substantially limited by the finite number of hematopoietic stem and progenitor cells in a single umbilical cord blood unit. Small molecules that not only quantitatively but also qualitatively stimulate enhancement of hematopoietic stem cell (HSC) self-renewal ex vivo should facilitate the clinical use of HSC transplantation and gene therapy. Recent evidence has suggested that the cyclin-dependent kinase inhibitor, p18INK4C (p18), is a critical regulator of mice HSC self-renewal. The role of p18 in human HSCs and the effect of p18 inhibitor on human HSC expansion ex vivo need further studies. Here we report that knockdown of p18 allowed for an increase in long-term colony-forming cells in vitro. We then identified an optimized small molecule inhibitor of p18, 005A, to induce ex vivo expansion of HSCs that was capable of reconstituting human hematopoiesis for at least 4 months in immunocompromised mice, and hence, similarly reconstituted secondary recipients for at least 4 more months, indicating that cells exposed to 005A were still competent in secondary recipients. Mechanistic studies showed that 005A might delay cell division and activate both the Notch signaling pathway and expression of transcription factor HoxB4, leading to enhancement of the self-renewal of long-term engrafting HSCs and the pool of progenitor cells. Taken together, these observations support a role for p18 in human HSC maintenance and that the p18 inhibitor 005A can enhance the self-renewal of long-term HSCs.

Downregulated miRNA-22-3p promotes the progression and leads to poor prognosis of hepatocellular carcinoma through targeting CDKN2C.

PURPOSE: CDKN2C exerts critical functions during the progression of hepatocellular carcinoma (HCC). Its dysfunction is closely linked to poor prognosis of HCC. This study aimed to uncover the underlying mechanism of CDKN2C in affecting the prognosis of HCC. METHODS: Potential miRNAs that could regulate CDKN2c were predicted by bioinformatics, and their differential levels in HCC and normal liver tissues were detected. CDKN2C level in Huh7 and Hep3B cells influenced by the two candidate microRNAs, miRNA-22-3p and miRNA-182-5p, were examined. Correlation between miRNA-22-3p and CDKN2C in HCC was analyzed on LinkedOmics, and further confirmed by Pearson correlation test and dual-luciferase reporter gene assay. Thereafter, the prognostic potential of miRNA-22-3p in HCC was evaluated by Kaplan-Meier method. Furthermore, the regulatory effects of miRNA-22-3p/CDKN2C axis on proliferative ability and cell cycle progression of HCC were assessed. RESULTS: There were five miRNAs predicted to bind to CDKN2C and among them, miRNA-22-3p and miRNA-182-5p were markedly downregulated in LIHC tissues. In Huh7 and Hep3B cells, miRNA-22-3p negatively regulated CDKN2C level, while transfection of miRNA-182-5p mimic or inhibitor did not influence CDKN2C expression. MiRNA-22-3p was closely linked to poor prognosis of HCC patients. Subsequently, dual-luciferase reporter gene assay verified the binding between miRNA-22-3p and CDKN2C. CONCLUSIONS: Knockdown of miRNA-22-3p suppressed proliferative ability and arrested cell cycle progression, which were reversed by overexpression of CDKN2C. MiRNA-22-3p suppresses proliferative ability and arrests cell cycle progression in HCC through targeting CDKN2C.

PDGFRß is an essential therapeutic target for BRCA1-deficient mammary tumors.

BACKGROUND: Basal-like breast cancers (BLBCs) are a leading cause of cancer death due to their capacity to metastasize and lack of effective therapies. More than half of BLBCs have a dysfunctional BRCA1. Although most BRCA1-deficient cancers respond to DNA-damaging agents, resistance and tumor recurrence remain a challenge to survival outcomes for BLBC patients. Additional therapies targeting the pathways aberrantly activated by BRCA1 deficiency are urgently needed. METHODS: Most BRCA1-deficient BLBCs carry a dysfunctional INK4-RB pathway. Thus, we created genetically engineered mice with Brca1 loss and deletion of p16INK4A, or separately p18INK4C, to model the deficient INK4-RB signaling in human BLBC. By using these mutant mice and human BRCA1-deficient and proficient breast cancer tissues and cells, we tested if there exists a druggable target in BRCA1-deficient breast cancers. RESULTS: Heterozygous germline or epithelium-specific deletion of Brca1 in p18INK4C- or p16INK4A-deficient mice activated Pdgfrß signaling, induced epithelial-to-mesenchymal transition, and led to BLBCs. Confirming this role, targeted deletion of Pdgfrß in Brca1-deficient tumor cells promoted cell death, induced mesenchymal-to-epithelial transition, and suppressed tumorigenesis. Importantly, we also found that pharmaceutical inhibition of Pdgfrß and its downstream target Pkcα suppressed Brca1-deficient tumor initiation and progression and effectively killed BRCA1-deficient cancer cells. CONCLUSIONS: Our work offers the first genetic and biochemical evidence that PDGFRß-PKCα signaling is repressed by BRCA1, which establishes PDGFRß-PKCα signaling as a therapeutic target for BRCA1-deficient breast cancers.

Assessing the prognostic value of stemness-related genes in breast cancer patients.

Breast cancer (BC) is currently one of the deadliest tumors worldwide. Cancer stem cells (CSCs) are a small group of tumor cells with self-renewal and differentiation abilities and high treatment resistance. One of the reasons for treatment failures is the inability to completely eliminate tumor stem cells. By using the edgeR package, we identified stemness-related differentially expressed genes in GSE69280. Via Lasso-penalized Cox regression analysis and univariate Cox regression analysis, survival genes were screened out to construct a prognostic model. Via nomograms and ROC curves, we verified the accuracy of the prognostic model. We selected 4 genes (PSMB9, CXCL13, NPR3, and CDKN2C) to establish a prognostic model from TCGA data and a validation model from GSE24450 data. We found that the low-risk score group had better OS than the high-risk score group, whether using TCGA or GSE24450 data. A prognostic model including four stemness-related genes was constructed in our study to determine targets of breast cancer stem cells (BCSCs) and improve the treatment effect.

Axolotl Ambystoma mexicanum extract induces cell cycle arrest and differentiation in human acute myeloid leukemia HL-60 cells.

Acute myeloid leukemia is the most common form of acute leukemia in adults, constituting about 80% of cases. Although remarkable progress has been made in the therapeutic scenario for patients with acute myeloid leukemia, research and development of new and effective anticancer agents to improve patient outcome and minimize toxicity is needed. In this study, the antitumor activity of axolotl (AXO) Ambystoma mexicanum crude extract was assessed in vitro on the human acute myeloid leukemia HL-60 cell line. The anticancer activity was evaluated in terms of ability to influence proliferative activity, cell viability, cell cycle arrest, and differentiation. Moreover, gene expression analysis was performed to evaluate the genes involved in the regulation of these processes. The AXO crude extract exhibited antiproliferative but not cytotoxic activities on HL-60 cells, with cell cycle arrest in the G0/G1 phase. Furthermore, the AXO-treated HL-60 cells showed an increase in both the percentage of nitroblue tetrazolium positive cells and the expression of CD11b, whereas the proportion of CD14-positive cells did not change, suggesting that extract is able to induce differentiation toward the granulocytic lineage. Finally, the treatment with AXO extract caused upregulation of CEBPA, CEBPB, CEBPE, SPI1, CDKN1A, and CDKN2C, and downregulation of c-MYC. Our data clearly show the potential anticancer activity of Ambystoma mexicanum on HL-60 cells and suggest that it could help develop promising therapeutic agents for the treatment of acute myeloid leukemia.

PRMT6 serves an oncogenic role in lung adenocarcinoma via regulating p18.

Lung adenocarcinoma (LUAD), a major subtype of lung cancer, is the leading cause of cancer­related mortality worldwide. Previous studies have determined the role of the protein arginine methyltransferases (PRMTs) in the physiology and pathology of LUAD. However, to the best of our knowledge, no empirical studies have been performed determining the association between protein arginine methyltransferase 6 (PRMT6) and LUAD. The present study aimed to determine the expression levels of PRMT6 in LUAD and its association with the clinicopathological characteristics. The effect of PRMT6 knockdown on cell growth was analyzed and chromatin immunoprecipitation (ChIP) assay was used to investigate the regulatory mechanisms of PRMT6 on downstream gene expression. In addition, a xenograft model was used to determine whether the PRMT6­regulated expression levels of p18 in vitro could be validated in vivo. PRMT6 overexpression in LUAD is associated with high clinical stage, lymph node metastasis and poor clinical outcomes. Furthermore, the silencing of PRMT6 significantly reduced the enrichment of Histone H3 asymmetric demethylation at arginine 2 in the promoter region of the p18 gene, thereby activating the expression of the gene. This, in turn, induced G1/S phase cell cycle arrest, resulting in the inhibition of cell proliferation. The xenograft model also suggested that PRMT6 suppressed LUAD development by activating p18 expression in vivo. In conclusion, the findings of the present study suggested that PRMT6 may serve as an oncogene in the progression of LUAD through epigenetically suppressing p18 expression. Thus, PRMT6 may represent a novel potential therapeutic target for LUAD.

A genome-wide gain-of-function screen identifies CDKN2C as a HBV host factor.

Chronic HBV infection is a major cause of liver disease and cancer worldwide. Approaches for cure are lacking, and the knowledge of virus-host interactions is still limited. Here, we perform a genome-wide gain-of-function screen using a poorly permissive hepatoma cell line to uncover host factors enhancing HBV infection. Validation studies in primary human hepatocytes identified CDKN2C as an important host factor for HBV replication. CDKN2C is overexpressed in highly permissive cells and HBV-infected patients. Mechanistic studies show a role for CDKN2C in inducing cell cycle G1 arrest through inhibition of CDK4/6 associated with the upregulation of HBV transcription enhancers. A correlation between CDKN2C expression and disease progression in HBV-infected patients suggests a role in HBV-induced liver disease. Taken together, we identify a previously undiscovered clinically relevant HBV host factor, allowing the development of improved infectious model systems for drug discovery and the study of the HBV life cycle.

Therapy after cyclin-dependent kinase inhibition in metastatic hormone receptor-positive breast cancer: Resistance mechanisms and novel treatment strategies.

Endocrine therapy has been the standard of care for patients with metastatic hormone receptor (HR)-positive, HER2-negative breast cancer since the 1970s, improving survival while avoiding the toxicities associated with cytotoxic chemotherapy. However, all HR-positive tumors ultimately develop resistance to endocrine therapy. Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have more recently become an important component of the management of this breast cancer subtype, significantly delaying time to the disease progression and improving survival when combined with endocrine therapy. However, as with endocrine therapy alone, treatment resistance remains a universal phenomenon. As more women receive CDK4/6 inhibitors as part of their treatment, the management of de novo and acquired resistance to combined CDK4/CDK6 inhibitor plus endocrine therapy regimens has emerged as an important clinical challenge. Several resistance mechanisms have been described, including alterations in the CDK4/6/cyclin D complex or its major effector retinoblastoma protein (pRb), bypass signaling through other cyclin/CDK complexes and activation of upstream signaling pathways, in particular the PI3K/mTOR pathway, but robust biomarkers to predict resistance remain elusive, and the role for continuing CDK4/6 inhibitors after progression remains under investigation. Novel strategies being evaluated in clinical trials include the continuation of CDK4/6 inhibitors through progression, as well as triplet therapy combinations with PI3K inhibitors or immune checkpoint inhibitors.

miR-21-5p promotes cell proliferation and G1/S transition in melanoma by targeting CDKN2C.

Human melanoma is a highly malignant tumor originating from cutaneous melanocytes. The noncoding RNA microRNA (miR)-21-5p has been reported to be expressed at high levels in malignant melanocytic skin tissues, but its potential functional role in melanoma remains poorly understood. Here, we explored the cellular effects of miR-21-5p on melanoma in vitro and the underlying mechanisms. Quantitative real-time PCR was used to show that miR-21-5p is significantly up-regulated in clinical samples from patients with melanoma as compared with adjacent noncancerous tissues. Overexpression of miR-21-5p significantly enhanced, whereas knockdown attenuated, cell proliferation and G1/S transition in melanoma cell lines (A375 and M14). Luciferase reporter assays were used to show that the cyclin-dependent kinase inhibitor 2C (CDKN2C) is a downstream target of miR-21-5p. Furthermore, miR-21-5p mimics resulted in a decrease in CDKN2C expression, and CDKN2C expression was observed to be inversely correlated with miR-21-5p expression in melanoma tissues. Rescue experiments were performed to show that overexpression of CDKN2C partially reversed the effects of miR-21-5p up-regulation on A375 cells. Consistently, knockdown of CDKN2C abolished the effects of miR-21-5p down-regulation on A375 cells. Overall, our studies demonstrate that miR-21-5p can promote the growth of melanoma cells by targeting CDKN2C, which may induce G0/G1 phase arrest of melanoma cells.

Novel use of a Clinical Laboratory Improvements Amendments (CLIA)-certified Cyclin-Dependent Kinase N2C (CDKN2C) loss assay in sporadic medullary thyroid carcinoma.

BACKGROUND: The cyclin-dependent-kinase inhibitor/retinoblastoma pathway has been implicated in sporadic medullary thyroid carcinoma tumorigenesis. Somatic CDKN2C loss has been associated with decreased overall survival in medullary thyroid carcinoma patients. We evaluated CDKN2C loss in a prospective clinical environment using a novel Clinical Laboratory Improvement Amendments-certified assay to confirm its association with aggressive disease and to interrogate response to targeted therapy. METHODS: Patients with advanced sporadic medullary thyroid carcinoma underwent tumor genotyping for the purpose of management of targeted therapy and prognostication. RESULTS: Of patients with informative CDKN2C assay results, 30 (51.8%) were haploinsufficient/1N and 28 (48.3%) were 2N. Forty patients (69.0%) had a somatic RET mutation, and 36.9% had alterations of both genes. Thirty patients (51.7%) were treated with systemic therapy. Presence of genetic alterations in CDKN2C or RET did not predict treatment response. Patients with 1N CDKN2C loss had significantly shorter time-to-distant-metastasis than patients with normal copy number (P = .03). CONCLUSION: This is the first evaluation in the clinical setting of CDKN2C haploinsufficiency in sporadic medullary thyroid carcinoma. Although a larger cohort and longer follow-up will be required, loss seems to be associated with more aggressive disease and may indicate patients that might receive benefit from treatment with a CDK inhibitor.

Absence of cyclin-dependent kinase inhibitor p27 or p18 increases efficiency of iPSC generation without induction of iPSC genomic instability.

Mechanisms underlying the generation of induced pluripotent stem cells (iPSC) and keeping iPSC stability remain to be further defined. Accumulated evidences showed that iPSC reprogramming may be controlled by the cell-division-rate-dependent model. Here we reported effects of absence of mouse p27 or p18 on iPSC generation efficiency and genomic stability. Expression levels of cyclin-dependent kinases inhibitors (CDKIs), p21, p27, and p18 decreased during iPSC reprogramming. Like p21 loss, p27 or p18 deficiency significantly promoted efficiency of iPSC generation, whereas ectopic expression of p27, p18, or treatment with CDK2 or CDK4 inhibitors repressed the reprogramming rate, suggesting that CDKIs-regulated iPSC reprogramming is directly related with their functions as CDK inhibitors. However, unlike p21 deletion, absence of p27 or p18 did not increase DNA damage or chromosomal aberrations during iPSC reprogramming and at iPSC stage. Our data not only support that cell cycle regulation is critical for iPSC reprogramming, but also reveal the distinction of CDKIs in somatic cell reprogramming.

Interference of the long noncoding RNA CDKN2B-AS1 upregulates miR-181a-5p/TGFßI axis to restrain the metastasis and promote apoptosis and senescence of cervical cancer cells.

Long noncoding RNA (lncRNA) CDKN2B-AS1 has been shown to play a crucial role in the development as well as in the prognosis of various human cancers, including cervical cancer. However, the underlying mechanisms need to be further explored between CDKN2B-AS1 and cervical cancer. In the present study, RT-PCR showed that the mRNA level of CDKN2B-AS1 was significantly upregulated while the miR-181a-5p was downregulated in cervical cancer cell lines. In addition, the interference of CDKN2B-AS1 by shRNA resulted in the suppression of cell proliferation, invasion, migration and promotion of apoptosis and senescence, and either CDKN2B-AS1 overexpression or miR-181a-5p showed reversed results. Further studies demonstrated that CDKN2B-AS1 could directly interact with miR-181a-5p, and that there was an inverse correlation between miR-181a-5p and CDKN2B-AS1. In addition, we found that TGFßI was a target of miR-181a-5p and could be downregulated by CDKN2B-AS1 knockdown. Moreover, the in vivo experiments further demonstrated the contribution of CDKN2B-AS1 in cervical cancer including tumor growth, apoptosis inhibition and senescence inhibition, and CDKN2B-AS1 knockdown could inhibit the aforementioned activities. In summary, our study demonstrated that the CDKN2B-AS1/miR-181a-5p/TGFßI axis might play a vital role in cervical cancer development.

KLF1 mutation E325K induces cell cycle arrest in erythroid cells differentiated from congenital dyserythropoietic anemia patient-specific induced pluripotent stem cells.

Krüppel-like factor 1 (KLF1), a transcription factor controlling definitive erythropoiesis, is involved in sequential control of terminal cell division and enucleation via fine regulation of key cell cycle regulator gene expression in erythroid lineage cells. Type IV congenital dyserythropoietic anemia (CDA) is caused by a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A, p.E325K). We recently diagnosed a female patient with type IV CDA with the identical missense mutation. To understand the mechanism underlying the dyserythropoiesis caused by the mutation, we generated induced pluripotent stem cells (iPSCs) from the CDA patient (CDA-iPSCs). The erythroid cells that differentiated from CDA-iPSCs (CDA-erythroid cells) displayed multinucleated morphology, absence of CD44, and dysregulation of the KLF1 target gene expression. In addition, uptake of bromodeoxyuridine by CDA-erythroid cells was significantly decreased at the CD235a+/CD71+ stage, and microarray analysis revealed that cell cycle regulator genes were dysregulated, with increased expression of negative regulators such as CDKN2C and CDKN2A. Furthermore, inducible expression of the KLF1 E325K, but not the wild-type KLF1, caused a cell cycle arrest at the G1 phase in CDA-erythroid cells. Microarray analysis of CDA-erythroid cells and real-time polymerase chain reaction analysis of the KLF1 E325K inducible expression system also revealed altered expression of several KLF1 target genes including erythrocyte membrane protein band 4.1 (EPB41), EPB42, glutathione disulfide reductase (GSR), glucose phosphate isomerase (GPI), and ATPase phospholipid transporting 8A1 (ATP8A1). Our data indicate that the E325K mutation in KLF1 is associated with disruption of transcriptional control of cell cycle regulators in association with erythroid membrane or enzyme abnormalities, leading to dyserythropoiesis.

Regulation of Tumor Suppressor Gene CDKN2A and Encoded p16-INK4a Protein by Covalent Modifications.

CDKN2A is one of the most studied tumor suppressor genes. It encodes the p16-INK4a protein that plays a critical role in the cell cycle progression, differentiation, senescence, and apoptosis. Mutations in CDKN2A or dysregulation of its functional activity are frequently associated with various types of human cancer. As a cyclin-dependent kinase inhibitor, p16-INK4a forms a complex with cyclin-dependent kinases 4/6 (CDK4/6) thereby competing with cyclin D. It is believed that the helix-turn-helix structures in the content of tandem ankyrin repeats in p16-INK4a are required for the protein interaction with CDK4. Until recently, the mechanisms considered to be involved in the regulation of p16-INK4a functions and cancer development have been mutations in DNA, homozygous or heterozygous gene loss, and methylation of CDKN2A promoter region. In this review, we discuss recent findings on the regulation of p16-INK4a by covalent modifications at both transcriptional and post-translational levels.

Genomic Deletion of BAP1 and CDKN2A Are Useful Markers for Quality Control of Malignant Pleural Mesothelioma (MPM) Primary Cultures.

Malignant pleural mesothelioma (MPM) is a deadly cancer that is caused by asbestos exposure and that has limited treatment options. The current standard of MPM diagnosis requires the testing of multiple immunohistochemical (IHC) markers on formalin-fixed paraffin-embedded tissue to differentiate MPM from other lung malignancies. To date, no single biomarker exists for definitive diagnosis of MPM due to the lack of specificity and sensitivity; therefore, there is ongoing research and development in order to identify alternative biomarkers for this purpose. In this study, we utilized primary MPM cell lines and tested the expression of clinically used biomarker panels, including CK8/18, Calretinin, CK 5/6, CD141, HBME-1, WT-1, D2-40, EMA, CEA, TAG72, BG8, CD15, TTF-1, BAP1, and Ber-Ep4. The genomic alteration of CDNK2A and BAP1 is common in MPM and has potential diagnostic value. Changes in CDKN2A and BAP1 genomic expression were confirmed in MPM samples in the current study using Fluorescence In situ Hybridization (FISH) analysis or copy number variation (CNV) analysis with digital droplet PCR (ddPCR). To determine whether MPM tissue and cell lines were comparable in terms of molecular alterations, IHC marker expression was analyzed in both sample types. The percentage of MPM biomarker levels showed variation between original tissue and matched cells established in culture. Genomic deletions of BAP1 and CDKN2A, however, showed consistent levels between the two. The data from this study suggest that genomic deletion analysis may provide more accurate biomarker options for MPM diagnosis.