Construction of immune-related LncRNAs classifier to predict prognosis and immunotherapy response in thymic epithelial tumors

The primary objective of this study was to construct an immune-related long noncoding RNAs (IRLs) classifier to precisely predict the prognosis and immunotherapy response of patients with thymic epithelial tumors (TET). Based on univariable Cox regression analysis and Lasso regression, six prognosis-related IRLs (AC004466.3, {"type":"entrez-nucleotide","attrs":{"text":"AC138207.2","term_id":"29165571","term_text":"AC138207.2"}}AC138207.2, {"type":"entrez-nucleotide","attrs":{"text":"AC148477.2","term_id":"45504179","term_text":"AC148477.2"}}AC148477.2, {"type":"entrez-nucleotide","attrs":{"text":"AL450270.1","term_id":"11244554","term_text":"AL450270.1"}}AL450270.1, HOXB-AS1 and SNHG8) were selected to build an IRL classifier. Importantly, results of qRT-PCR validated that higher expression levels of {"type":"entrez-nucleotide","attrs":{"text":"AC138207.2","term_id":"29165571","term_text":"AC138207.2"}}AC138207.2, {"type":"entrez-nucleotide","attrs":{"text":"AC148477.2","term_id":"45504179","term_text":"AC148477.2"}}AC148477.2, {"type":"entrez-nucleotide","attrs":{"text":"AL450270.1","term_id":"11244554","term_text":"AL450270.1"}}AL450270.1 and SNHG8 as well as lower expression levels of AC004466.3, and HOXB-AS1 in TETs samples compared with normal controls. The IRL classifier could effectively classify patients into the low-risk and high-risk groups based on the different survival parameters. In terms of predictive ability and clinical utility, the IRL classifier was superior to Masaoka staging system. Additionally, IRL classifier is significantly associated with immune cells infiltration (dendritic cells, activated CD4 memory T cells and tumor-infiltrating lymphocyte (TIL), T cell subsets in particular), immune microenvironment (immune score and immune checkpoint inhibitors) and immunogenicity (TMB) in TETs, which hints that IRL classifier is tightly correlated with immune characteristics and might guide more effective immunotherapy strategies for TETs patients. Encouragingly, according to TIDE algorithm, there were more immunotherapy responders in the low-risk IRL subgroup and the IRL score was robustly negatively linked to the immunotherapeutic response. To sum up, the IRL classifier was established, which can be used to predict the prognosis, immune infiltration status, immunotherapy response in TETs patients, and may facilitate personalized counseling for immunotherapy.     

Clinical and prognostic effects of CDKN2A,CDKN2B and CDH13 promoter methylation in ovarian cancer: a study using meta-analysis and TCGA data

Abstract

Background: Promoter methylation of tumour suppressor genes (TSGs) CDKN2A, CDKN2B and CDH13 has been reported in ovarian cancer. However, the clinicopathological characteristics and prognostic role of CDKN2A, CDKN2B and CDH13 promoter methylation in ovarian carcinoma remained unclear.

Methods: The pooled odds ratio (OR) or hazard ratios (HRs) with their 95% confidence intervals (95% CIs) were calculated in this meta-analysis. The Cancer Genome Atlas data were obtained to confirm the role of CDKN2A, CDKN2B and CDH13 methylation in ovarian cancer.

Results: CDKN2A, CDKN2B and CDH13 promoter methylation was higher in ovarian cancer than in normal ovarian tissues. CDH13 promoter methylation was correlated with tumour histology (serous vs. non-serous type: OR?=?0.33, p?=?0.031). CDKN2A promoter methylation was not linked to overall survival (OS), but it was correlated with a poor prognosis in progression-free survival (HR?=?1.55, p?=?0.004). TCGA data showed no correlation between CDKN2A, CDKN2B and CDH13 methylation and OS as well as disease-free survival (DFS).

Conclusions: CDKN2A, CDKN2B and CDH13 promoter methylation may correlate with the increased risk of ovarian cancer. CDKN2A promoter methylation may be an independent prognostic biomarker for predicting progression-free survival.     

A genetic variant in CDKN2A/2B locus was associated with poor prognosis in patients with esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is among the leading causes of cancer related death. Despite of extensive efforts in identifying valid cancer prognostic biomarkers, only a very small number of markers have been identified. Several genetic variants in the 9p21 region have been identified that are associated with the risk of multiple cancers. Here, we explored the association of two genetic variants in the 9p21 region, CDKN2A/B, rs10811661, and rs1333049 for the first time in 273 subjects with, or without ESCC. We observed that the patients with ESCC had a higher frequency of a TT genotype for rs10811661 than individuals in the control group, and this polymorphism was also associated with tumor size. Moreover, a CC genotype for the rs1333049 polymorphism was associated with a reduced overall survival (OS) of patients with ESCC. In particular, patients with a CC (rs1333049) genotype had a significantly shorter OS (CC genotype: 34.5 ± 8.9 months vs. CG+GG: 47.7 ± 5.9 months; p value = 0.03). We have also shown the association of a novel genetic variant in CDKN2B gene with clinical outcome of patients with ESCC. Further investigations are warranted in a larger population to explore the value of emerging markers as a risk stratification marker in ESCC.     

Evaluation of the two polymorphisms rs1801133 in MTHFR and rs10811661 in CDKN2A/B in breast cancer

The 5,10-Methylenetetrahydrofolate reductase (MTHFR) was the rate-limiting enzyme in the methyl cycle, which was encoded by the MTHFR gene. MTHFR played a key role in homocysteine plasma level and was associated with the risk of breast cancer. The cyclin-dependent kinase (CDK) inhibitor (CDKN2A/B) was the tumor suppressor in the cell cycle regulation. The single-nucleotide polymorphism was thought to be associated with the predisposition of breast cancer and in subsequent immune response in different populations. The current study was conducted on a peripheral blood sample of 100 Iranian women with breast carcinoma and 142 cancer-free healthy female volunteers. The TaqMan real-time polymerase chain reaction technique was applied for genotyping of participants. The correlation of both variants and demographic data were investigated with the risk of breast cancer. Our data showed that the MTHFR allele T and TT genotype had the higher prevalence in patients (P < 0.0001) than the control group. The frequency of risk C allele into the CDKN2A/B rs10811661 was 72%. The correlations of menarche and underlying hormonal disorder with the risk of breast cancer were investigated; also our results showed that the menopause status was statistically significant between patients and controls (P = 0.036). Our investigations demonstrated that the MTHFR rs180113 and CDKN2A/B rs10811661 had a significant correlation with the elevated risk of breast cancer and they might be potentially valuable to apply as a prognostic factor for individual health care.     

Inhibition of breast cancer cell growth by the combination of clofarabine and sulforaphane involves epigenetically mediated CDKN2A upregulation

Many antineoplastic nucleoside analogue-based combinatorial strategies focused on remodelling aberrant DNA methylation patterns have been developed. The number of studies demonstrate high efficacy of bioactive phytochemicals in support of conventional chemotherapy. Our recent discoveries of the epigenetic effects of clofarabine (2′-deoxyadenosine analogue, antileukaemic drug) and clofarabine-based combinations with dietary bioactive compounds in breast cancer cells led us to look for more DNA methylation targets of these cancer-preventive agents. In the present study, using methylation-sensitive restriction analysis (MSRA) and qPCR, we showed that clofarabine in combination with sulforaphane, a phytochemical from cruciferous vegetables, significantly reactivates DNA methylation-silenced CDKN2A tumour suppressor and inhibits cancer cell growth at a non-invasive breast cancer stage.     

Expression Analyses Revealed Thymic Stromal Co-Transporter/Slc46A2 Is in Stem Cell Populations and Is a Putative Tumor Suppressor

By combining conventional single cell analysis with flow cytometry and public database searches with bioinformatics tools, we extended the expression profiling of thymic stromal cotransporter (TSCOT), Slc46A2/Ly110, that was shown to be expressed in bipotent precursor and cortical thymic epithelial cells. Genome scale analysis verified TSCOT expression in thymic tissue- and cell type- specific fashion and is also expressed in some other epithelial tissues including skin and lung. Coexpression profiling with genes, Foxn1 and Hoxa3, revealed the role of TSCOT during the organogenesis. TSCOT expression was detected in all thymic epithelial cells (TECs), but not in the CD31⁺ endothelial cell lineage in fetal thymus. In addition, ABC transporter-dependent side population and Sca-1⁺ fetal TEC populations both contain TSCOT-expressing cells, indicating TEC stem cells express TSCOT. TSCOT expression was identified as early as in differentiating embryonic stem cells. TSCOT expression is not under the control of Foxn1 since TSCOT is present in the thymic rudiment of nude mice. By searching variations in the expression levels, TSCOT is positively associated with Grhl3 and Irf6. Cytokines such as IL1b, IL22 and IL24 are the potential regulators of the TSCOT expression. Surprisingly, we found TSCOT expression in the lung is diminished in lung cancers, suggesting TSCOT may be involved in the suppression of lung tumor development. Based on these results, a model for TEC differentiation from the stem cells was proposed in context of multiple epithelial organ formation.     

In silico analysis of structural and functional consequences in p16INK4A by deleterious nsSNPs associated CDKN2A gene in malignant melanoma

In this study, we identified the most deleterious nsSNP in CDKN2A gene through structural and functional properties of its protein (p16INK4A) and investigated its binding affinity with cdk6. Out of 118 SNPs, 14 are nsSNPs in the coding region and 17 SNPs were found in the untranslated region (UTR). FastSNP suggested that 7 SNPs in the 5' UTR might change the protein expression levels. Sixty-four percent of nsSNPs are found to be damaged in PolyPhen server among the 14 nsSNPs investigated. With this effort, we modeled the mutant p16INK4A proteins based on these deleterious nsSNPs, out of which three nsSNPs associated p16INK4A had RMSD values of greater than 3.00 A with native protein. From a comparison of total energy of these three mutant proteins, we identified that the major mutation is from Aspartic acid to Tyrosine at the residue position of 84 of p16INK4A. Further, we compared the binding efficiency of both native and mutant p16INK4A with cdk6. We found that mutant p16INK4A has less binding affinity with cdk6 compared to native type. This is due to ten hydrogen bonds and eight salt bridges which exist between the native type and cdk6, whereas the mutant type makes only nine hydrogen bonds and five salt bridges with cdk6. Based on our investigation, we propose that the SNP with the ID rs11552822 could be the most deleterious nsSNP in CDKN2A gene, causing malignant melanoma, as it was well correlated with experimental studies carried out elsewhere.     

The beta-isoform of the BRCA2 and CDKN1A(p21)-interacting protein (BCCIP) stabilizes nuclear RPL23/uL14

BRCA2 and CDKN1A(p21,CIP1)-interacting protein (BCCIP) is an evolutionary conserved protein implicated in maintenance of genome stability and cell cycle progression. Two isoforms of BCCIP with distinct C-terminal domains exist in humans. We show that mammalian BCCIPβ, but not BCCIPα, forms a ternary complex with the ribosomal protein RPL23/uL14 and the pre-60S trans-acting factor eIF6. Complex formation is dependent on an intact C-terminal domain of BCCIPβ. Depletion of BCCIPβ reduces the pool of free RPL23, and decreases eIF6 levels in nucleoli. Overexpression of BCCIPβ leads to nucleoplasmic accumulation of extra-ribosomal RPL23 and stabilizes overexpressed RPL23, suggesting that BCCIPβ functions as nuclear chaperone for RPL23.     

Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines

We have investigated the potential for the p16‐cyclin D‐CDK4/6‐retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three‐quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma‐specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16^INK4A) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.     

The relationship between five widely-evaluated variants in CDKN2A/B and CDKAL1 genes and the risk of type 2 diabetes: A meta-analysis

The genes encoding two cyclin-dependent kinases-inhibitor-2A/B (CDKN2A/B) and 5 regulatory subunit-associated protein-like 1 (CDKAL1) have been investigated extensively in associations with type 2 diabetes; the results, however, are often irreproducible. We therefore sought to evaluate these associations by performing a meta-analysis on five widely-evaluated variants from the two genes. There were 38 studies (patients/controls: 51,940/52,234) for rs10811661, 16 studies (20,029/24,419) for rs564398 in CDKN2A/B gene, and 27 studies (28,383/47,635) for rs7756992, 26 studies (28,816/31,713) for rs7754840, 21 studies (29,260/38,400) for rs10946398 in CDKAL1 gene. Overall risk estimates for type 2 diabetes conferred by rs10811661-T, rs564398-A, rs7754840-C, rs7756992-G, and rs10946398-C alleles were 1.17 (95% CI: 1.10–1.23; P < 0.0005; I² = 83.9%), 1.1 (95% CI: 1.0–1.21; P = 0.051; I² = 88.3%), 1.24 (95% CI: 1.18–1.3; P < 0.0005; I² = 74.3%), 1.2 (95% CI: 1.11–1.3; P < 0.0005; I² = 92.0%), and 1.19 (95% CI: 1.1–1.29; P < 0.0005; I² = 90.8%), respectively. There was evident publication bias for rs564398 and rs7754840. Subgroup analyses by ethnicity showed remarkable divergences in risk estimate for rs564398 between Asians (odds ratio [OR] = 1.01; 95% CI: 0.86–1.19; P = 0.868) and Caucasians (OR = 1.19; 95% CI: 1.03–1.35; P = 0.012) (P < 0.05). For all variants examined, the results of studies in retrospective design or with population-based controls were comparative with that of overall studies. In meta-regression analyses, age was found to exert a significant influence on the association between rs10811661 and type 2 diabetes (P = 0.003), as well as between rs7754840 and gender (P = 0.034). Taken together, our findings provide evidence for a significant contribution of CDKN2A/B gene rs10811661 and CDKAL1 gene rs7756992 and rs10946398 to type 2 diabetes.     

Monoclonal antibody recognizing a determinant on type 2 chain blood group A and B oligosaccharides detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and human colon cancer cell lines

Altered expression of ABH blood group substances is a common feature of human colorectal carcinoma, yet it remains unclear how these structural changes influence the biological properties of tumor cells. Azoxymethane-induced rat colon tumors display many features of the human disease, thereby providing a potentially useful model to study the role of blood group substances in colon cancer progression. We have prepared monoclonal antibodies to a microsomal fraction isolated from an azoxymethane-induced rat colon tumor and selected an antibody that detects cancer-associated changes. Monoclonal antibody (mAb) 3A7 recognizes a determinant on type 2 chain blood group A (GalNAcα1–3[Fucα1–2]Galβ1–4GlcNAc-R) and B (Galα1–3[Fucα1–2]Galβ1–4GlcNAc-R) oligosaccharides. Expression of the epitope detected by this antibody was developmentally regulated in rat colon, with maximal expression from day 4–21 after birth. Immunohistochemical staining and Western blotting analyses of azoxymethane-induced colon tumors revealed increased expression of the epitope in all of the 21 colonic tumors examined, including preneoplastic glands within transitional mucosa. Conventional and signet-ring adenocarcinomas that had invaded through the muscularis propria (Duke's B2) consistently showed the most intense staining with mAb 3A7, including regions depicting angioinvasion. Some of the lymph node metastases (Duke's C2) stained poorly with the antibody. The epitope was also expressed in blood group A positive human colon carcinoma cell lines, including HT29 and SW480 but not by SW620, a cell line derived from a lymph node metastasis isolated in vivo from the SW480 primary tumor, or in the blood group B cell line SW1417. The glycoproteins detected by mAb 3A7 in rat colon tumors and HT29 cells ranged in size between 50 and 200 kd, including a major species of 140 kd. Affinity chromatography of detergent lysates of normal rat colon on the blood group A specific lectin Dolichos biflorus (DBA)-agarose resulted in nearly quantitative binding of glycoprotein species detected by the antibody. By contrast, immunoreactive glycoproteins from rat colon tumors or HT29 cells bound poorly to DBA-agarose but were retained by another blood group A-binding lectin, Helix-pomatia (HPA)-agarose. These results indicate that colon carcinogenesis results in quantitative as well as qualitative changes in oligosaccharides detected by mAb 3A7 and suggest that the combined use of mAb 3A7 and blood group A-specific lectins may provide a useful tool for early detection of colon cancer.     

Altered modulation of lamin A/C‐HDAC2 interaction and p21 expression during oxidative stress response in HGPS

Defects in stress response are main determinants of cellular senescence and organism aging. In fibroblasts from patients affected by Hutchinson–Gilford progeria, a severe LMNA‐linked syndrome associated with bone resorption, cardiovascular disorders, and premature aging, we found altered modulation of CDKN1A, encoding p21, upon oxidative stress induction, and accumulation of senescence markers during stress recovery. In this context, we unraveled a dynamic interaction of lamin A/C with HDAC2, an histone deacetylase that regulates CDKN1A expression. In control skin fibroblasts, lamin A/C is part of a protein complex including HDAC2 and its histone substrates; protein interaction is reduced at the onset of DNA damage response and recovered after completion of DNA repair. This interplay parallels modulation of p21 expression and global histone acetylation, and it is disrupted by LMNAmutations leading to progeroid phenotypes. In fact, HGPS cells show impaired lamin A/C‐HDAC2 interplay and accumulation of p21 upon stress recovery. Collectively, these results link altered physical interaction between lamin A/C and HDAC2 to cellular and organism aging. The lamin A/C‐HDAC2 complex may be a novel therapeutic target to slow down progression of progeria symptoms.     

A submicroscopic deletion involving part of the CREBBP gene detected by array-CGH in a patient with Rubinstein-Taybi syndrome

We report a girl with Rubinstein-Taybi syndrome (RSTS) who was found to have copy number loss on 16p13.3 by array-CGH. She has developmental delay and other features of RSTS including downslanting palpebral fissures, a prominent nose with the nasal septum extending below the alae nasi, broad thumbs and big toes, postaxial polydactyly of the right foot and constipation from birth. We report the junction sequence across the breakpoint region for a microdeletion in RSTS. The sequencing results also showed that the deletion was 81.4kb involving three genes DNASE 1, TRAP 1, and CREBBP.     

Comparison of Aberrant Methylation of CpG Islands in the p16/CDKN2A and p14/ARF Promoters in Non-Small Cell Lung Cancer and Acute Lymphoblastic Leukemia

Multiplex methylation-sensitive (MSe-PCR) and methylation-specific (MSp-PCR) PCRs were used to detect aberrant methylation of CpG islands in the promoter regions and first exons of p16/CDKN2A and p14/ARF in non-small cell lung cancer (NSCLC, 54 specimens) and B-cell acute lymphoblastic leukemia (B-ALL, 61 specimens). A difference in CpG methylation was observed for individual specimens and for the two malignancies. A high methylation frequency of the first exon of p16/CDKN2A was detected both in NSCLC (68%) and in B-ALL (55%). The CpG island of the p14/ARF first exon proved to be nonmethylated in both malignancies. Particular CpG-rich fragments were examined in the p16/CDKN2A and p14/ARF promoters. It was shown that methylation frequency can differ between the 5 regions of one promoter. The sensitivity was compared for MSe-PCR and MSp-PCR, which are commonly employed in methylation analysis.     

Role of Serotonin2A and Serotonin2B/2C Receptor Subtypes in the Control of Accumbal and Striatal Dopamine Release Elicited In Vivo by Dorsal Raphe Nucleus Electrical Stimulation

This study investigates, using in vivo microdialysis, the role of serotonin2A (5-HT2A) and 5-HT(2B/2C) receptors in the effect of dorsal raphe nucleus (DRN) electrical stimulation on dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) extracellular levels monitored in the nucleus accumbens (NAC) and the striatum of halothane-anesthetized rats. Following DRN stimulation (300 microA, 1 ms, 20 Hz, 15 min) DA release was enhanced in the NAC and reduced in the striatum. The 5-HT2A antagonist SR 46349B (0.5 mg/kg) and the mixed 5-HT(2A/2B/2C) antagonist ritanserin (0.63 mg/kg) significantly reduced the effect of DRN stimulation on DA release in the NAC but not in the striatum. DA responses to DRN stimulation were not affected by the 5-HT(2B/2C) antagonist SB 206553 (5 mg/kg) in either region. None of these compounds was able to modify the enhancement of DOPAC and 5-HIAA outflow induced by DRN stimulation in either the NAC or the striatum. Finally, in both brain regions basal DA release was significantly increased only by SB 206553. These results indicate that 5-HT2A but not 5-HT(2B/2C) receptors participate in the facilitatory control exerted by endogenous 5-HT on accumbal DA release. Conversely, 5-HT(2B/2C) receptors tonically inhibit basal DA release in both brain regions.