Concomitant hypermethylation of multiple genes in non-small cell lung cancer (NSCLC)

Primary lung cancer remains the leading cause of cancer death worldwide. Promoter hypermethylation is a major inactivation mechanism of tumor-related genes, and increasingly appears to play an important role in carcinogenesis. In the present study, we used quantitative methylation-specific PCR (Q-MSP) assays to analyze promoter hypermethylation of nine genes in a large cohort of well-characterized non-small cell lung cancer (NSCLC) and explore their associations with the clinicopathological features of tumor. We found that there were significant differences in methylation levels for six of nine gene promoters between cancerous and noncancerous lung tissues. More importantly, with 100% diagnostic specificity, high sensitivity, ranging from 44.9% to 84.1%, was found for each of the nine genes. Interestingly, promoter hypermethylation of most genes was closely associated with histologic type, which was more frequent in squamous cell carcinomas (SCC) than in adenocarcinomas (ADC). In addition, highly frequent concomitant methylation of multiple genes was found in NSCLC, particularly in SCC. Our data showed that multiple genes were aberrantly methylated in lung tumorigenesis, and that they were closely associated with certain clinicopathological features of NSCLC, particularly of the histologic type, suggesting that these hypermethylated genes could be potential biomarkers in early detection of NSCLC in high-risk individuals, as well as in evaluating the prognosis of NSCLC patients.     

Different expression of cyclooxygenase-2 (COX-2) in selected nonmelanocytic human cutaneous lesions

The aim of our study was to elucidate the possible involvement of COX-2 in the development and/or progression of nonmelanocytic skin lesions. To evaluate the usefulness of that enzyme as a potential molecular marker, we examined the intensity and spatial distribution of COX-2 expression in selected types of such tumors using the same immunohistochemical procedure as in our earlier studies of melanocytic cancers. We examined 20 benign epithelial lesions, 11 precancerous lesions, 21 basal cell carcinomas (BCC), 14 squamous cell carcinomas (SCC) and eight fibromas. The levels of COX-2 expression detected in benign lesions and in normal skin were comparable. Elevated expression of this protein may play a role in the development of SCC, as indicated by strong immunostaining both in SCCs and precancerous lesions. Significantly stronger staining in SCCs compared to BCCs may indicate a role of COX-2 in cancer malignancy and serve as an indicator useful for differential diagnostics of the two types of cancer. Strong staining in all skin layers of SCC may help in detecting cancer cells infiltrating surrounding skin layers.     

Cyclooxygenase-2 is involved in S100A2-mediated tumor suppression in squamous cell carcinoma

S100A2 is considered a putative tumor suppressor due to its loss or down-regulation in several cancer types. However, no mechanism has been described for the tumor suppressor role of S100A2. In this study, ectopic expression of S100A2 in the human malignant squamous cell carcinoma cell line KB resulted in a significant inhibition of proliferation, migration, and invasion. Moreover, S100A2 significantly reduced the number of colonies (>or=0.5 mm) formed in semisolid agar and decreased tumor growth and burden in nude mice. cDNA microarray analysis was used to compare mRNA expression profiles of vector- and S100A2-expressing isogenic cells. Among the genes deregulated by S100A2, the expression of cyclooxygenase-2 (COX-2) mRNA was significantly suppressed by S100A2 (2.4-fold). Western blot analysis confirmed that S100A2 reduced the expression of COX-2 protein in stably and transiently transfected KB and RPMI-2650 cells. COX-2 is frequently overexpressed in various types of cancer and plays an important role in tumor progression. Partial restoration of COX-2 expression attenuated the antitumor effect of S100A2 both in vitro and in vivo. Although the interplay between S100A2 and COX-2 remains to be clarified, these findings first showed a potent antitumor role of S100A2 in squamous cell carcinoma partly via reduced expression of COX-2.     

Downregulation of RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2 in non-small cell lung cancer

Chromosomal and genome abnormalities of 3p are frequent in many epithelial tumors, including lung cancer. Several critical regions with a high frequency of hemi-and homozygous deletions in tumors are known for 3p, and more than 20 cancer-related genes occur in 3p21.3. Quantitative real-time PCR was used to measure the mRNA level for tumor-suppressor and candidate genes of 3p21.3 (RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2) in major types of non-small cell lung cancer (NSCLC): squamous cell lung cancer (SCC) and lung adenocarcinoma (AC). A significant (2-to 100-fold) and frequent (44–100%) decrease in mRNA levels was observed in NSCLC. The mRNA level decrease and its frequency depended on the histological type of NSCLC for all genes. The downregulation of RASSF1A and ITGA9 was significantly associated with AC progression; the same tendency was observed for RBSP3/CTDSPL, NPRL2/G21, HYAL1, and HYAL2. In SCC, the downregulation of all genes was not associated with the clinical stage, tumor cells differentiation, and metastasis in lymph nodes. The RBSP3/CTDSPL, NPRL2/G21, ITGA9, HYAL1, and HYAL2 mRNA levels significantly (5-to 13-fold on average) decreased at a high frequency (83–100%) as early as SCC stage I. Simultaneous downregulation of all six genes was observed in some tumor samples and was independent of the gene position in 3p21.3 and the functions of the protein products. The Spearman correlation coefficient r _s was 0.63–0.91, p < 0.001. The highest r _s values were obtained for gene pairs ITGA9-HYAL2 and HYAL1-HYAL2, whose products mediate cell-cell adhesion and cell-matrix interactions; coregulation of the genes was assumed on this basis. Both genetic and epigenetic mechanisms proved to be important for downregulation of RBSP3/CTDSPL and ITGA9. This finding supported the hypothesis that the cluster of cancerrelated genes in the extended 3p21.3 locus is simultaneously inactivated during the development and progression of lung cancer and other epithelial tumors. A significant and frequent decrease in the mRNA level of the six genes in SCC could be important for developing specific biomarker sets for early SCC diagnosis and new approaches to gene therapy of NSCLC.     

Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays

This study aimed to clarify genetic and epigenetic alterations that occur during lung carcinogenesis and to design perspective sets of newly identified biomarkers. The original method includes chromosome 3 specific NotI-microarrays containing 180 NotI clones associated with genes for hybridization with 40 paired normal/tumor DNA samples of primary lung tumors: 28 squamous cell carcinomas (SCC) and 12 adenocarcinomas (ADC). The NotI-microarray data were confirmed by qPCR and bisulfite sequencing analyses. Forty-four genes showed methylation and/or deletions in more than 15% of non–small cell lung cancer (NSCLC) samples. In general, SCC samples were more frequently methylated/deleted than ADC. Moreover, the SCC alterations were observed already at stage I of tumor development, whereas in ADC many genes showed tumor progression specific methylation/deletions. Among genes frequently methylated/deleted in NSCLC, only a few were already known tumor suppressor genes: RBSP3 (CTDSPL), VHL and THRB. The RPL32, LOC285205, FGD5 and other genes were previously not shown to be involved in lung carcinogenesis. Ten methylated genes, i.e., IQSEC1, RBSP3, ITGA9, FOXP1, LRRN1, GNAI2, VHL, FGD5, ALDH1L1 and BCL6 were tested for expression by qPCR and were found downregulated in the majority of cases. Three genes (RBSP3, FBLN2 and ITGA9) demonstrated strong cell growth inhibition activity. A comprehensive statistical analysis suggested the set of 19 gene markers, ANKRD28, BHLHE40, CGGBP1, RBSP3, EPHB1, FGD5, FOXP1, GORASP1/TTC21, IQSEC1, ITGA9, LOC285375, LRRC3B, LRRN1, MITF, NKIRAS1/RPL15, TRH, UBE2E2, VHL, WNT7A, to allow early detection, tumor progression, metastases and to discriminate between SCC and ADC with sensitivity and specificity of 80–100%.     

The Characteristics and Function of S100A7 Induction in Squamous Cell Carcinoma: Heterogeneity,Promotion of Cell Proliferation and Suppression of Differentiation

S100A7 is highly expressed in squamous cell carcinomas (SCC) and is related to the terminal differentiation of keratinocytes. However, its characteristic and function in SCC is not very known. In this present study, we used immunohistochemistry to examine the expression of S100A7 in 452 SCC specimens, including the lung, esophagus, oral cavity, skin, cervix, bladder, and three SCC cell lines. We found that S100A7-positive staining showed significant heterogeneity in six types of SCC specimen and three SCC cell lines. Further examination found that S100A7-positive cells and its expression at mRNA and protein levels could be induced in HCC94, FaDu, and A-431 cells both in vitro and in vivo using immunohistochemistry, real-time PCR, and Western blotting. Notably, the upregulation of squamous differentiation markers, including keratin-4, keratin-13, TG-1, and involucrin, also accompanied S100A7 induction, and a similar staining pattern of S100A7 and keratin-13 was found in HCC94 cells both in vitro and in vivo. Further study revealed that the overexpression of S100A7 significantly increased proliferation and inhibited squamous differentiation in A-431 cells both in vitro and in vivo. Conversely, silencing S100A7 inhibited cell growth and survival and increased the expression of keratin-4, keratin-13, TG-1, and involucrin in HCC94 cells. Therefore, these results demonstrate that S100A7 displays heterogeneous and inducible characteristic in SCC and also provide novel evidence that S100A7 acts as a dual regulator in promoting proliferation and suppressing squamous differentiation of SCC.     

Immunohistochemical analysis of selected molecular markers in esophagus precancerous,adenocarcinoma and squamous cell carcinoma in Iranian subjects

Background: The molecular and cellular mechanisms linking chronic inflammation and gastrointestinal malignancy are not known with certainty. Aim: To investigate changes in potential causative factors during progression of esophagus cancer in a population living in high-risk area in Iran. Subjects: Formalin-fixed, paraffin-embedded esophageal specimens (n = 87) from patients with gastroesophageal reflux disease (GERD), Barrett's metaplasia, adenocarcinoma (ADC) and squamous cells carcinoma (SCC) were collected based on their pathological diagnosis. Methods: Immunohistochemical (IHC) technique was used to study tissue accumulation of P53, P21, cyclooxygenase-2 (COX-2), glutathione S-transferase-P (GST-Pi) and nitrotyrosine (NT) in patients and controls. Results: P53 expression was not detected in esophageal tissues from normal and GERD samples, whereas it was found positive in Barrett's, ADC, and SCC samples. P21 positive sample was relatively higher in ADC patients as compared to that in SCC (ADC: 52.6%; SCC: 25%). GST-Pi expression was equally accumulated in all the samples. NT was predominantly expressed in ADC (72.7%). COX-2 expression was significantly higher in Barrett's (60.0%) and ADC (66.6%) as compared to that in GERD, SCC and normal. These data were further confirmed by detecting the scores of immunostainings in all the positive samples. Conclusion: The pathological changes in ADC and SCC samples which were associated with increasing frequency of NT and COX-2 provides further evidence for involvement of these inflammatory factors in progression of esophagus cancer.     

Expression of cyclooxygenase-2 in invasive breast carcinomas and its prognostic impact

Representative tumour sections from 468 patients with invasive breast cancer were immunostained for cyclooxygenase-2 (COX-2) and evaluated. The relationships between COX-2 expression, clinical outcome and various clinicopathological variables, including tumour vascularity and disseminated tumour cells (DTC) in the bone marrow were examined. COX-2 expression in invasive breast carcinoma cells was positively associated with oestrogen receptor and/or progesterone receptor positivity (p<0.001). Triple-negative tumours showed no/low COX-2 expression more frequently than other tumour types (p<0.001). Expression of COX-2 was not associated with breast cancer-specific survival (p=0.49, log-rank) or distant disease-free survival (p=0.67, log-rank) for all patients, including lymph node-negative, untreated patients (p>0.14, log-rank). There was also no significant association between COX-2 expression and histological grade, tumour size, nodal status, DTC in bone marrow, p53, HER2, or tumour vascularity. In conclusion, COX-2 expression in this series was associated with the presence of hormone receptors. Low COX-2 expression was observed in triple-negative breast carcinomas.     

Differential expression of an ensemble of the key genes involved in cell-cycle regulation in lung cancer

Targeted cancer therapy directed at individual targets is often accompanied by the rapid development of drug resistance. The development of a new generation of antitumor drugs involves the search for many targets simultaneously to block or, conversely, restore their activity. In this regard, simultaneous analysis of gene expression in a complex network of interactions, primarily cell cycle control elements, is relevant for the search of specific molecular markers for the differential diagnosis of adenocarcinoma (ADC) and squamous cell lung cancer (SCC), as well as new targets for therapy. In this paper we performed an extended quantitative analysis of the expression of two suppressor genes, CTDSPL and its target RB1, as well as 84 genes of the main participants of the p16^INK4A-Cdk/cyclin D1-Rb and p53/p21^Waf1 signaling pathways in the histological types of non-small-cell lung cancer (NSCLC), i.e., ADC and SCC, using the special panel of the Human Cell Cycle Regulation Panel. The expression profile of some genes shows the specificity to the histological type of NSCLC and the presence of metastases. The genes with a significantly increased expression that affect the activity of Rb (cyclins, cyclin-dependent kinases, their activators, inhibitors, etc.) can serve as potential targets for combined therapy of both ADC and SCC.     

PTEN和MDM2在非小细胞肺癌中的表达及临床意义

目的：研究PTEN和MDM2在非小细胞肺癌（NSCLC）发生发展中的临床意义。方法：采用免疫组化SP法检测56例非小细胞肺癌组织及20例正常肺组织中PTEN和MDM2蛋白的表达。结果：56例肺癌组织中PTEN表达率为48.21%（27/56）,20例正常肺组织中PTEN表达率为95%（19/20）,二者有统计学意义（p〈0.05）。PTEN表达率与肿瘤的分化程度、临床分期、淋巴结转移有关（p〈0.05）。56例肺癌组织中MDM2表达率为53.57%（30/56）,20例正常肺组织MDM2表达率为10%（2/20）,二者有统计学意义（p〈0.05）,MDM2表达率与肿瘤的分化程度、临床分期、淋巴结转移有关（p〈0.05）。在NSCLC中,PTEN和MDM2的表达呈负相关（p〈0.05）。结论：PTEN和MDM2的异常表达在非小细胞肺癌的发生发展过程中起着重要的作用。     

Research on circadian clock genes in non-small-cell lung carcinoma

Circadian clock genes have become a hot topic in cancer research in recent years, and more and more studies are showing that clock genes are involved in regulating cell proliferation cycle and apoptosis of malignant tumors, neuroendocrine and immune function, and other processes. Lung cancer is a malignant tumor with increasing incidence worldwide. The pathogenesis of lung cancer is extremely complicated and includes genetic factors, living environment, and smoking, and the occurrence of lung cancer is related to the regulation of many oncogenes and tumor suppressor genes. But there are few studies on clock genes in lung cancer. Studies on clock genes may help to better understand the mechanism of lung cancer development for an improved treatment. The expressions of all 14 kinds of clock genes in adenocarcinoma (ADC) and squamous cell carcinoma (SCC), two main kinds of non-small-cell lung cancer (NSCLC), were studied based on integration and analysis of data from The Cancer Genome Atlas (TCGA) to show the association between clock gene expression and prognosis of cancer patients. Analysis of TCGA data indicated that overexpression of Cry2, BMAL1, and RORA with underexpression of Timeless and NPAS2 was associated with a favorable prognosis of ADC, and the expression of NPAS2 was associated with the time of patient survival. Additionally, the expression of Cry2 was related to TNM stage. In SCC, high expression of DEC1 was correlated with poor overall survival in patients and the expression of Timeless was associated with the time of patient survival. In NSCLC, circadian clock genes constitute cancer circadian rhythm by interacting with each other, showing that asynchrony with normal tissues, which collectively controlling the occurrence and development of NSCLC.     

大肠癌组织及癌旁组织中c—myc，COX-2和CD44v6基因表达量差异的检测

目的：通过检测大肠癌组织和癌旁组织中c-myc,COX-2以及CD44v6的表达水平,探讨这三种基因在大肠癌发生和发展中的意义。方法：应用实时荧光定量PCR技术检测了10例大肠癌组织和相应癌旁组织中c-myc,COX-2以及CD44v6基因表达水平的差异,并探讨了各基因在癌纽织中的表达水平与大肠癌临床病理指标之间的关系。结果：c-myc,COX-2以及CD44v6在大肠癌组织和癌旁组织中的表达均有非常显著性差异（P〈0．01）;癌组织中COX-2和CD44v6的表达与淋巴结转移、分化程度及Dukes分期有关（P〈0．05）。结论：c—myc,COX-2和CD44v6的异常表达均与大肠癌密切相关,三者从不同方面对大肠癌的发生和发展起到了重要作用,可作为早期诊断和预后的参考指标。     

Distinct roles of heterogeneous nuclear ribonuclear protein K and microRNA-16 in cyclooxygenase-2 RNA stability induced by S100b, a ligand of the receptor for advanced glycation end products

Advanced glycation end products play major roles in diabetic complications. They act via their receptor RAGE to induce inflammatory genes such as cyclooxygenase-2 (COX-2). We examined the molecular mechanisms by which the RAGE ligand, S100b, induces COX-2 in monocytes. S100b significantly increased COX-2 mRNA accumulation in THP-1 monocytes at 2 h via mRNA stability. This was further confirmed by showing that S100b increased stability of luciferase-COX-2 3'-UTR mRNA. Chromatin immunoprecipitation and RNA immunoprecipitation revealed that S100b decreased occupancy of the DNA/RNA-binding protein, heterogeneous nuclear ribonuclear protein K (hnRNPK), at the COX-2 promoter but simultaneously increased its binding to the COX-2 3'-UTR. S100b treatment promoted the translocation of nuclear hnRNPK to cytoplasm, whereas a cytoplasmic translocation-deficient hnRNPK mutant inhibited S100b-induced COX-2 mRNA stability. Small interfering RNA-mediated specific knockdown of hnRNPK blocked S100b-induced COX-2 mRNA stability, whereas on the other hand, overexpression of hnRNPK increased S100b-induced COX-2 mRNA stability. S100b promoted the release of entrapped COX-2 mRNA from cytoplasmic processing bodies, sites of mRNA degradation. Furthermore, S100b significantly down-regulated the expression of a key microRNA, miR-16, which can destabilize COX-2 mRNA by binding to its 3'-UTR. MiR-16 inhibitor oligonucleotides increased, whereas, conversely, miR-16 mimic oligonucleotides decreased COX-2 mRNA stability in monocytes, further supporting the inhibitory effects of miR-16. Interestingly, hnRNPK knockdown increased miR-16 binding to COX-2 3'-UTR, indicating a cross-talk between them. These new results demonstrate that diabetic stimuli can efficiently stabilize inflammatory genes via opposing actions of key RNA-binding proteins and miRs.