Prognostic Value of Immunohistochemical Markers for Locally Advanced Rectal Cancer

The aim of this study is to reveal the potential roles of apoptosis markers (Bcl2 and p53), proliferation markers (Ki-67 and CyclD1), and the neuroendocrine marker Chromogranin A as markers for the radioresistance of rectal cancer. Statistically significant differences were found in the expression of p53, Ki-67, and Chromogranin A in groups of patients with and without a favorable prognosis after radiotherapy. The survival analysis revealed that the marker of neuroendocrine differentiation, Chromogranin A, also demonstrated a high prognostic significance, indicating a poor prognosis. Markers of proliferation and apoptosis had no prognostic value for patients who received preoperative radiotherapy. Higher Chromogranin A values were predictors of poor prognosis. The results obtained from studying the Chromogranin A expression suggest that the secretion of biologically active substances by neuroendocrine cells causes an increase in tumor aggressiveness.     

PAK1 as a Potential Therapeutic Target in Male Smokers with EGFR-Mutant Non-Small Cell Lung Cancer

P21-activated kinases (PAKs) are serine/threonine protein kinases that contribute to several cellular processes. Here, we aimed to determine the prognostic value of PAK1 and its correlation with the clinicopathological characteristics and five-year survival rates in patients with non-small cell lung cancer (NSCLC). We evaluated PAK1 mRNA and protein expression in NSCLC cells and resected tumor specimens, as well as in healthy human bronchial epithelial cells and adjacent healthy lung tissues, respectively, for effective comparison. Immunohistochemical tissue microarray analysis of 201 NSCLC specimens showed the correlation of PAK1 expression with clinicopathological characteristics. The mRNA and protein expression of PAK1 were 2.9- and 4.3-fold higher in six of seven NSCLC cell types and human tumors (both, p < 0.001) than in healthy human bronchial epithelial BEAS-2B cells and adjacent healthy lung tissues, respectively. Decreased survival was significantly associated with PAK1 overexpression in the entire cohort (χ² = 8.48, p = 0.0036), men (χ² = 17.1, p < 0.0001), and current and former smokers (χ² = 19.2, p < 0.0001). Notably, epidermal growth factor receptor (EGFR) mutation-positive lung cancer patients with high PAK1 expression showed higher mortality rates than those with low PAK1 expression (91.3% vs. 62.5%, p = 0.02). Therefore, PAK1 overexpression could serve as a molecular target for the treatment of EGFR mutation-positive lung cancer, especially among male patients and current/former smokers.     

Ribosomal protein mutations in Korean patients with Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by hypoproliferative anemia, associated physical malformations and a predisposition to cancer. DBA has been associated with mutations and deletions in the large and small ribosomal protein genes, and genetic aberrations have been detected in ∼50–60% of patients. In this study, nine Korean DBA patients were screened for mutations in eight known DBA genes (RPS19, RPS24, RPS17, RPS10, RPS26, RPL35A, RPL5 and RPL11) using the direct sequencing method. Mutations in RPS19, RPS26 and RPS17 were detected in four, two and one patient, respectively. Among the mutations detected in RPS19, two mutations were novel (c.26T>A, c.357-2A>G). For the mutation-negative cases, array-CGH analysis was performed to identify copy-number variations, and no deletions involving the known DBA gene regions were identified. The relative mRNA expression of RPS19 estimated using real-time quantitative PCR analysis revealed two- to fourfold reductions in RPS19 mRNA expression in three patients with RPS19 mutations, and p53 protein expression analysis by immunohistochemistry showed variable but significant nuclear staining in the DBA patients. In conclusion, heterozygous mutations in the known DBA genes RPS19, RPS26 and RPS17 were detected in seven out of nine Korean DBA patients. Among these patients, RPS19 was the most frequently mutated gene. In addition, decreased RPS19 mRNA expression and p53 overexpression were observed in the Korean DBA patients, which supports the hypothesis that haploinsufficiency and p53 hyperactivation represent a central pathway underlying the pathogenesis of DBA.     

Clinicopathological values of NBS1 and DNA damage response genes in epithelial ovarian cancers

Epithelial ovarian cancers (EOCs) are highly lethal gynecological malignancies with a high recurrence rate. Therefore, developing prognostic markers for recurrence after chemotherapy is crucial for the treatment of ovarian cancers. As ovarian cancers frequently respond to DNA-damaging agents, we assessed the clinicopathological significance of key double-strand DNA break (DSB) repair genes, including BRCA1, BRCA2, BARD1, ATM, RAD51 and NBS1 in EOC cell lines and paraffin-embedded tissue sections from 140 EOC patients treated with cytoreductive surgery, followed by platinum-based chemotherapy. These samples were analyzed for the clinicopathological impact of DSB genes by western blot analysis, immunohistochemistry and quantitative real-time PCR. Of the DSB repair genes, BRCA1, ATM and NBS1, which are involved in the homologous recombination-mediated repair pathway, were related to aggressive parameters in EOC. When survival analysis was performed, NBS1 expression exhibited an association with EOC recurrence. Specifically, increased NBS1 expression was found in 107 out of 140 cases (76.0%) and correlated with advanced stage (P=0.001), high grade (P=0.001) and serous histology (P=0.008). The median recurrence-free survival in patients with positive and negative expression of NBS1 was 30 and 78 months, respectively (P=0.0068). In multivariate analysis, NBS1 was an independent prognostic factor for the recurrence of EOC. Together, these results suggest that NBS1 is a marker of poor prognosis for the recurrence of EOC and is associated with aggressive clinicopathological parameters.     

DERL3 functions as a tumor suppressor in gastric cancer

BackgroundGastric cancer is a common malignant tumor in the clinic with a high mortality rate, ranking the first among malignant tumors of the digestive system. Early gastric cancer exhibits no specific clinical symptoms and signs, and most of the patients were diagnosed as advanced gastric cancer. The prognosis is poor, and the 5-year overall survival rate is still lower than 30%, seriously threatening people’s life and health. However, the pathogenesis of gastric cancer is still unclear.MethodsThis study aimed to identify methylated differentially expressed genes in gastric cancer and to study the cellular functions and pathways that may be involved in its regulation, as well as the biological functions of key methylated differentially expressed genes. The gene expression data set and methylation data set of gastric cancer genes based on TCGA were analyzed to identify prognostic methylated genes.ResultsThis study showed that the methylation of the DERL3 promoter was correlated with the clinical analysis of tumors. Further studies were conducted on genes co-expressed with DERL3, whose functions and pathways to inhibit gastric cancer were adaptive immune response, T cell activation, immune response-regulating pathway, cell surface on molecules, and natural killer cell-mediated cytotoxicity. Finally, cell proliferation assay, cell scratch assay, and cell invasion assay confirmed that DERL3 as a tumor suppressor gene inhibited the malignant evolution of gastric cancer.ConclusionsThe analysis of key methylated differentially expressed genes helped elucidate the epigenetic regulation mechanism in the development of gastric cancer. DERL3, as a methylation biomarker, has a predictive and prognostic value in the accurate diagnosis and treatment of gastric cancer and provides potential targets for the precision treatment of gastric cancer.Trial RegistrationNot applicable.     

Anti-neoplastic effects of gallic acid, a major component of Toona sinensis leaf extract, on oral squamous carcinoma cells

Extract of Toona sinensis (TS) has been reported to have various effects on cultured cell lines, including anti-proliferative activity in cancer cells. We have studied the effects of TS on various human oral squamous carcinoma cell lines (HOSCC), including UM1, UM2, SCC-4, and SCC-9. These cell lines were treated with TS leaf extract and screened for viability, apoptosis, necrosis, and apoptotic gene expression. Normal human oral keratinocytes (NHOK) served as a control for cytotoxic assays. Viability of TS-treated HOSCC was reduced, whereas that of NHOK was not affected. FACScan analysis revealed that the leaf extract induced apoptosis or a combination of apoptosis and necrosis, depending on cell type. Microarray and semi-quantitative RT-PCR analysis for apoptotic-related gene expression revealed that 3,4,5-trihydroxybenzoic acid (gallic acid, one of the major bioactive compounds purified from TS extract) up-regulated pro-apoptotic genes such TNF-α, TP53BP2, and GADD45A, and down-regulated the anti-apoptotic genes Survivin and cIAP1, resulting in cell death. This study suggests that gallic acid, the major bioactive compound present, is responsible for the anti-neoplastic effect of Toona sinensis leaf extract.     

SET8 suppression mediates high glucose-induced vascular endothelial inflammation via the upregulation of PTEN

Hyperglycemia-mediated endothelial inflammation participates in the pathogenesis of cardiovascular complications in subjects with diabetes. Previous studies reported that phosphatase and tensin homolog deleted on chromosome ten (PTEN) and SET8 participate in high glucose-mediated endothelial inflammation. In this study, we hypothesize that SET8 regulates PTEN expression, thus contributing to high glucose-mediated vascular endothelial inflammation. Our data indicated that plasma soluble intercellular adhesion molecule-1 (sICAM-1) and endothelial selectin (e-selectin) were increased in patients with diabetes and diabetic rats. PTEN expression was augmented in the peripheral blood mononuclear cells of patients with diabetes and in the aortic tissues of diabetic rats. Our in vitro study indicated that high glucose increased monocyte/endothelial adhesion, endothelial adhesion molecule expression and p65 phosphorylation in human umbilical vein endothelial cells (HUVECs). Moreover, high glucose led to endothelial inflammation via upregulation of PTEN. Furthermore, high glucose inhibited SET8 expression and histone H4 lysine 20 methylation (H4K20me1), a downstream target of SET8. SET8 overexpression reversed the effects of high-glucose treatment. shSET8-mediated endothelial inflammation was counteracted by siPTEN. Furthermore, SET8 was found to interact with FOXO1. siFOXO1 attenuated high glucose-mediated endothelial inflammation. FOXO1 overexpression-mediated endothelial inflammation was counteracted by siPTEN. H4K20me1 and FOXO1 were enriched in the PTEN promoter region. shSET8 increased PTEN promoter activity and augmented the positive effect of FOXO1 overexpression on PTEN promoter activity. Our in vivo study indicated that SET8 was downregulated and FOXO1 was upregulated in the peripheral blood mononuclear cells of patients with diabetes and the aortic tissues of diabetic rats. In conclusion, SET8 interacted with FOXO1 to modulate PTEN expression in vascular endothelial cells, thus contributing to hyperglycemia-mediated endothelial inflammation.Subject terms: Diabetes complications, Vascular diseases     

Differential promoter methylation may be a key molecular mechanism in regulating BubR1 expression in cancer cells

The BubR1 mitotic-checkpoint protein monitors proper attachment of microtubules to kinetochores, and links regulation of chromosome-spindle attachment to mitotic-checkpoint signaling. Thus, disruption of BubR1 activity results in a loss of checkpoint control, chromosomal instability caused by a premature anaphase, and/or the early onset of tumorigenesis. The mechanisms by which deregulation and/or abnormalities of BubR1 expression operate, however, remain to be elucidated. In this study, we demonstrate that levels of BubR1 expression are significantly increased by demethylation. Bisulfite sequencing analysis revealed that the methylation status of two CpG sites in the essential BubR1 promoter appear to be associated with BubR1 expression levels. Associations of MBD2 and HDAC1 with the BubR1 promoter were significantly relieved by addition of 5-aza-2'-deoxycytidine, an irreversible DNA methyltransferase inhibitor. However, genomic DNA isolated from 31 patients with colorectal carcinomas exhibited a +84A/G polymorphic change in approximately 60% of patients, but this polymorphism had no effect on promoter activity. Our findings indicate that differential regulation of BubR1 expression is associated with changes in BubR1 promoter hypermethylation patterns, but not with promoter polymorphisms, thus providing a novel insight into the molecular regulation of BubR1 expression in human cancer cells.     

miR-638 is a new biomarker for outcome prediction of non-small cell lung cancer patients receiving chemotherapy

MicroRNAs (miRNAs), a class of small non-coding RNAs, mediate gene expression by either cleaving target mRNAs or inhibiting their translation. They have key roles in the tumorigenesis of several cancers, including non-small cell lung cancer (NSCLC). The aim of this study was to investigate the clinical significance of miR-638 in the evaluation of NSCLC patient prognosis in response to chemotherapy. First, we detected miR-638 expression levels in vitro in the culture supernatants of the NSCLC cell line SPC-A1 treated with cisplatin, as well as the apoptosis rates of SPC-A1. Second, serum miR-638 expression levels were detected in vivo by using nude mice xenograft models bearing SPC-A1 with and without cisplatin treatment. In the clinic, the serum miR-638 levels of 200 cases of NSCLC patients before and after chemotherapy were determined by quantitative real-time PCR, and the associations of clinicopathological features with miR-638 expression patterns after chemotherapy were analyzed. Our data helped in demonstrating that cisplatin induced apoptosis of the SPC-A1 cells in a dose- and time-dependent manner accompanied by increased miR-638 expression levels in the culture supernatants. In vivo data further revealed that cisplatin induced miR-638 upregulation in the serum derived from mice xenograft models, and in NSCLC patient sera, miR-638 expression patterns after chemotherapy significantly correlated with lymph node metastasis. Moreover, survival analyses revealed that patients who had increased miR-638 levels after chemotherapy showed significantly longer survival time than those who had decreased miR-638 levels. Our findings suggest that serum miR-638 levels are associated with the survival of NSCLC patients and may be considered a potential independent predictor for NSCLC prognosis.     

Identification of key classification features of early cervical squamous cell carcinoma

Despite the tremendous progress in molecular analysis of pan-cancer, little is known regarding molecular classification of cervical squamous cell carcinoma. In this study, we adopted a multi-omics approach to identify potential key classification features of cervical squamous cell carcinoma. Specifically, we analyzed mRNA, and microRNA (miRNA) expression data, as well as DNA methylation and copy number variation in cervical squamous cell carcinoma cases, using datasets obtained from The Cancer Genome Atlas (TCGA). Moreover, we identified molecules in each dimension, as well as integrated and clustered filtered classification features, and used them to distinguish different subtypes. The resulting key classification features were used to establish a classification model for cervical squamous cell carcinoma. Our results revealed two cervical squamous cell carcinoma subtypes, with significant differences across clinical survival levels, as well as 8 key classification features of cervical squamous cell carcinomas. These findings are expected to provide important references for early classification of cervical squamous cell carcinoma and identification of classification markers.     

Hypermethylation of PDX1, EN2, and MSX1 predicts the prognosis of colorectal cancer

Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.Subject terms: Prognostic markers, Methylation analysis     

The mitogen-activated protein kinase phosphatase-1 (MKP-1) gene is a potential methylation biomarker for malignancy of breast cancer

The mitogen-activated protein kinase (MAPK) phosphatase- 1 (MKP-1) belongs to the MAPK cascades which are central to cell proliferation and apoptosis. The carcinogenic role of MKP-1 has been reported in many types of cancer but it has rarely been investigated in breast cancer. The present study was designed to evaluate the MKP-1 mRNA expression and its possible regulation by methylation of MKP-1 promoter in the model of several breast cancer cell lines and tissues as well as controls. Our data demonstrate MKP-1 mRNA expression significantly decreased in five breast cancer cell lines compared to breast controls (P<0.01). Using the methylation-specific PCR (MSP) analysis, the unmethylated reaction (U) is dominant in both normal cell lines and benign breast tumors (100% vs. 86.2%), whereas the methylated reaction (M) is dominant in both breast cancer cell lines and invasive breast tumors (100% vs. 57.2%). In terms of methylation ratio (M/M+U), methylation level in MKP-1 promoter is significantly higher in the invasive breast tumor tissues (n = 152) than in benign breast tumor tissues (n = 29) (P<0.0001). Assessing the methylation ratio of the promoter of MKP-1 gene to diagnose the breast malignancy (invasive vs. benign), the area under the receiver- operating characteristic (ROC) curve was 0.809 (95% CI: 0.711-0.906, P<0.001). The best performance for this prediction has a sensitivity of 76.32% and a specificity of 82.76% at the cutoff value of 0.38. Taken together, we firstly demonstrated that the promoter methylation of MKP-1 gene is a potential breast cancer biomarker for breast malignancy.     

High mutation frequency at Ha-ras exons 1-4 in squamous cell carcinomas from PUVA-treated psoriasis patients

Clinical follow-up studies have revealed that PUVA-treated patients are at increased risk of skin cancer, particularly squamous cell carcinoma (SCC). However, since psoralen and UVA (PUVA) is not only a potent mutagen and carcinogen but also an immunosuppressor, and since other (co)carcinogenic factors often exist in psoriasis patients, the exact causes and mechanisms of PUVA-associated SCC are still not completely understood. In order to fill this gap the tools of molecular epidemiology are being used to study the SCC mutational spectra of p53 and Ha-ras, two of the most commonly mutated genes in human cancers. A previous mutation analysis revealed that SCC in PUVA-treated patients often carried mutated p53 genes and that many of the mutations had the UV fingerprint (i.e. C-->T or CC-->TT transitions at dipyrimidine sites). In the present study DNA-sequencing analysis revealed a total of 18 Ha-ras missense or nonsense mutations at exons 1-4 in 13 of 17 SCC (76%) from 8 of 11 (73%) PUVA-treated psoriasis patients. Six of the 18 mutations (33%) were of UV-fingerprint type (C-->T transitions), five (28%) were at 5'-TpG sites (i.e. potential psoralen-binding sites and thus potentially caused by PUVA) and seven were of other type (39%), including six G:C-->T:A transversions at hotspot codon 12. In addition, in the case of 6 of the 11 subjects (55%) both tumor and normal skin samples contained a T:A-->C:G base change at codon 27 (a 5'-ATT site), a change previously hypothesized to be a possible silent Ha-ras polymorphism at one allele. When we compared the present Ha-ras mutation spectrum with the p53 mutation spectrum from a previous study of the samples, we found that approximately half of the tumors harbored mutations in both Ha-ras and p53. Together, our results indicate that Ha-ras mutations are present in a large proportion of PUVA-associated SCC and that UVB, PUVA and other agents may induce Ha-ras mutations and act together with p53 in the formation of SCC in psoriasis patients.