Improved Classification of Lung Cancer Tumors Based on Structural and Physicochemical Properties of Proteins Using Data Mining Models

Detecting divergence between oncogenic tumors plays a pivotal role in cancer diagnosis and therapy. This research work was focused on designing a computational strategy to predict the class of lung cancer tumors from the structural and physicochemical properties (1497 attributes) of protein sequences obtained from genes defined by microarray analysis. The proposed methodology involved the use of hybrid feature selection techniques (gain ratio and correlation based subset evaluators with Incremental Feature Selection) followed by Bayesian Network prediction to discriminate lung cancer tumors as Small Cell Lung Cancer (SCLC), Non-Small Cell Lung Cancer (NSCLC) and the COMMON classes. Moreover, this methodology eliminated the need for extensive data cleansing strategies on the protein properties and revealed the optimal and minimal set of features that contributed to lung cancer tumor classification with an improved accuracy compared to previous work. We also attempted to predict via supervised clustering the possible clusters in the lung tumor data. Our results revealed that supervised clustering algorithms exhibited poor performance in differentiating the lung tumor classes. Hybrid feature selection identified the distribution of solvent accessibility, polarizability and hydrophobicity as the highest ranked features with Incremental feature selection and Bayesian Network prediction generating the optimal Jack-knife cross validation accuracy of 87.6%. Precise categorization of oncogenic genes causing SCLC and NSCLC based on the structural and physicochemical properties of their protein sequences is expected to unravel the functionality of proteins that are essential in maintaining the genomic integrity of a cell and also act as an informative source for drug design, targeting essential protein properties and their composition that are found to exist in lung cancer tumors.     

Prediction of thermostability from amino acid attributes by combination of clustering with attribute weighting: a new vista in engineering enzymes

The engineering of thermostable enzymes is receiving increased attention. The paper, detergent, and biofuel industries, in particular, seek to use environmentally friendly enzymes instead of toxic chlorine chemicals. Enzymes typically function at temperatures below 60°C and denature if exposed to higher temperatures. In contrast, a small portion of enzymes can withstand higher temperatures as a result of various structural adaptations. Understanding the protein attributes that are involved in this adaptation is the first step toward engineering thermostable enzymes. We employed various supervised and unsupervised machine learning algorithms as well as attribute weighting approaches to find amino acid composition attributes that contribute to enzyme thermostability. Specifically, we compared two groups of enzymes: mesostable and thermostable enzymes. Furthermore, a combination of attribute weighting with supervised and unsupervised clustering algorithms was used for prediction and modelling of protein thermostability from amino acid composition properties. Mining a large number of protein sequences (2090) through a variety of machine learning algorithms, which were based on the analysis of more than 800 amino acid attributes, increased the accuracy of this study. Moreover, these models were successful in predicting thermostability from the primary structure of proteins. The results showed that expectation maximization clustering in combination with uncertainly and correlation attribute weighting algorithms can effectively (100%) classify thermostable and mesostable proteins. Seventy per cent of the weighting methods selected Gln content and frequency of hydrophilic residues as the most important protein attributes. On the dipeptide level, the frequency of Asn-Glu was the key factor in distinguishing mesostable from thermostable enzymes. This study demonstrates the feasibility of predicting thermostability irrespective of sequence similarity and will serve as a basis for engineering thermostable enzymes in the laboratory.     

Cytofluorimetric evaluation of DNA ploidy in lung cancer: a bronchoscopic study

The study of the biological characteristics of lung cancer is gaining more and more interest both because of their potential role as prognostic indicators and for therapeutic reasons. The DNA content estimated by flow cytometry in surgical samples of non-small cell lung cancer (NSCLC) has already been demonstrated to be correlated with survival in these patients. From July 1990 to February 1992 we analyzed the DNA distribution of bronchoscopic biopsies from 88 patients with lung cancer (18 small cell lung cancer, SCLC, and 68 NSCLC, two unspecified histology). Twenty-eight tumors (34.6%) had a diploid DNA distribution, while 53 were aneuploid (65.4%). A correlation was found between DNA ploidy and survival. Evaluation of the DNA content in bronchoscopic samples in a large series of patients could determine the role of this analysis prior to surgery in NSCLC and its value as a marker with respect to prognosis and response to therapy in SCLC.     

Expression of inhibitor of apoptosis proteins in small- and non-small-cell lung carcinoma cells

Small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cells both initiate apoptotic signaling, resulting in caspase activation, after treatment with anti-cancer agents. However, in contrast to SCLC cells, NSCLC cells do not fully execute apoptosis. The apoptotic process in NSCLC cells seems to be blocked downstream of caspase activation, thus the failure of NSCLC cells to execute apoptosis could result from inhibition of active caspases by inhibitor of apoptosis proteins (IAPs). Here we investigate the mRNA and protein expression of IAPs in a panel of SCLC and NSCLC cell lines. The NSCLC cell lines had a stronger cIAP-2 expression at both mRNA and protein levels, while the SCLC cell lines had a higher level of XIAP protein. Expression of cIAP-1, cIAP-2, and XIAP, the most potent caspase inhibitors, was further investigated in three lung carcinoma cell lines after treatment with 8 Gy of ionizing radiation or etoposide (VP16). In response to treatment, the level of IAPs was not altered in a way that explained the differences in cellular chemo- and radiosensitivity. The intracellular localization of IAPs was analyzed in untreated and treated lung cancer cells. Surprisingly, we found that cIAP-2 was mainly detected in the mitochondrial fraction, although the function of this protein in mitochondria is unknown. No major relocalization of IAPs was observed after treatment. Taken together, these results indicate that IAPs alone are not the main factor responsible for the resistance of NSCLC cells to treatment.     

Towards a genetic-based classification of human lung cancer.

Lung cancer is a highly aggressive neoplasm which is reflected by a multitude of genetic aberrations being detectable on the chromosomal and molecular level. In order to understand this seemingly genetic chaos, we performed Comparative Genomic Hybridisation (CGH) in a large collective of human lung carcinomas investigating different tumor entities as well as multiple individual tumour specimens of single patients. Despite the considerable genetic instability being reflected by the well known morphological heterogeneity of lung cancer the comparison of different tumour groups using custom made computer software revealed recurrent aberration patterns and highlighted chromosomal imbalances that were significantly associated with morphological histotypes and biological phenotypes. Specifically we identified imbalances in NSCLC being associated with metastasis formation which are typically present in SCLC thus explaining why the latter is such an aggressive neoplasm characterized by widespread tumor dissemination. Based on the genetic data a new model for the development of SCLC is presented. It suggests that SCLC evolving from the same stem cell as NSCLC should be differentiated into primary and secondary tumors. Primary SCLC corresponding to the classical type evolved directly from an epithelial precursor cell. In contrast, secondary SCLC correlating with the combined SCLC develops via an NSCLC intermediate. In addition, we established libraries of differentially expressed genes from different human lung cancer types to identify new candidate genes for several of the chromosomal subregions identified by CGH. In this review, we summarise the status of our results aiming at a refined classification of lung cancer based on the pattern of genetic aberrations.     

Silencing of death receptor and caspase-8 expression in small cell lung carcinoma cell lines and tumors by DNA methylation

Small cell lung cancer cell lines were resistant to FasL and TRAIL-induced apoptosis, which could be explained by an absence of Fas and TRAIL-R1 mRNA expression and a deficiency of surface TRAIL-R2 protein. In addition, caspase-8 expression was absent, whereas FADD, FLIP and caspases-3, -7, -9 and -10 could be detected. Analysis of SCLC tumors revealed reduced levels of Fas, TRAIL-R1 and caspase-8 mRNA compared to non-small cell lung cancer (NSCLC) tumors. Methylation-specific PCR demonstrated methylation of CpG islands of the Fas, TRAIL-R1 and caspase-8 genes in SCLC cell lines and tumor samples, whereas NSCLC samples were not methylated. Cotreatment of SCLC cells with the demethylating agent 5'-aza-2-deoxycytidine and IFNgamma partially restored Fas, TRAIL-R1 and caspase-8 expression and increased sensitivity to FasL and TRAIL-induced death. These results suggest that SCLC cells are highly resistant to apoptosis mediated by death receptors and that this resistance can be reduced by a combination of demethylation and treatment with IFNgamma.     

VEGF and IL-18 in induced sputum of lung cancer patients

Cytokines are key players in the biological processes of malignant tumors and special interest has been focused on cytokines exerting tumor and anti-tumor properties, such as vascular endothelial growth factor (VEGF) and Interleukin-18 (IL-18). Aim of this study was to assess IL-18 and VEGF levels in induced sputum of lung cancer patients at diagnosis, and assess their possible association with the histological type of cancer, the stage and the overall patient survival. Seventy six patients with a diagnosis of lung cancer were recruited and were followed up for 48months. Thirteen healthy smokers and 16 healthy non-smokers were used as control groups. VEGF and IL-18 were measured by ELISA in sputum supernatants at the time of diagnosis. Lung cancer patients had significantly higher baseline IL-18 and VEGF levels compared to healthy controls (p<0.001). No difference was found in IL-18 and VEGF levels between the various stages in non-small cell lung cancer (NSCLC) and between limited and extended small cell lung cancer (SCLC). However, the ratio of VEGF/IL-18 was significantly higher in NSCLC compared to SCLC patients (p=0.018). In extended SCLC overall survival was inversely associated with baseline sputum VEGF levels (p=0.034) and estimated mortality risk was 1.14 (95% CI 1.006-1.283) for an increase of 100pg/ml in VEGF levels. Such association was not found regarding baseline IL-18 levels. VEGF levels in induced sputum may have a prognostic role in the survival of SCLC. The ratio VEGF/IL-18 in induced sputum differs between NSCLC and SCLC, indicating differences in angiogenesis mechanisms and/or immunological response in these two major histological types of lung cancer.     

Spectral histopathology of the lung: A review of two large studies

This paper summarizes results from two large lung cancer studies comprising over 700 samples that demonstrate the ability of spectral histopathology (SHP) to distinguish cancerous tissue regions from normal tissue, to differentiate benign lesions from normal tissue and cancerous lesions, and to classify lung cancer types. Furthermore, malignancy‐associated changes can be identified in cancer‐adjacent normal tissue. The ability to differentiate a multitude of normal cells and tissue types allow SHP to identify tumor margins and immune cell infiltration. Finally, SHP easily distinguishes small cell lung cancer (SCLC) from non‐SCLC (NSCLC) and provides a further differentiation of NSCLC into adenocarcinomas and squamous cell carcinomas with an accuracy comparable of classical histopathology combined with immunohistochemistry. Case studies are presented that demonstrates that SHP can resolve interobserver discrepancies in standard histopathology.     

Detection of cytoskeletal proteins in small cell lung carcinoma

Small cell lung carcinoma (SCLC) is the most aggressive of lung tumors, metastasize widely and are virtually incurable by surgical means. Therefore, the classification of lung cancer into SCLC and non-small cell lung carcinoma is essential for disease prognosis and treatment. For this purpose we have compared the immunohistochemical distribution of different cytoskeletal proteins as tumor markers. Analysis was performed by using of monoclonal antibodies directed against cytokeratins, neurofilaments, betaIII-tubulin, epithelial membrane antigen and neuron-specific enolase. Our results indicate that keratin and epithelial membrane antigen are reliable epithelial markers for SCLC. In addition, the positive staining with monoclonal antibodies TU-20 against betaIII-tubulin and neuron-specific enolase was found in some cases of SCLC. We suggest, that these antibodies could be a useful tool for complex immunohistochemical diagnosis of SCLC.     

Role of cell-cycle regulators in lung cancer

Lung cancer is the leading cause of cancer death worldwide. Histologically, 80% of lung cancers are classified as non-small-cell lung cancer (NSCLC), and the remaining 20% as small-cell lung cancer (SCLC). Lung carcinoma is the result of molecular changes in the cell, resulting in the deregulation of pathways controlling normal cellular growth, differentiation, and apoptosis. This review summarizes some of the most recent findings about the role of cell-cycle proteins in lung cancer pathogenesis and progression.     

Serum NSE, CEA, CT, CA 15-3 levels in human lung cancer

The significance of neuron specific enolase (NSE) was investigated in comparison with other tumor markers (CEA, CT, CA 15-3) used in the diagnosis and treatment monitoring of lung cancer. As previously described, the calcitonin assay proved to have very low sensitivity for small cell lung cancer (SCLC). The serum NSE assay was, however, shown to be a useful diagnostic aid for discrimination between histologically different lung cancers, and therefore this assay may be a valuable tool for treatment monitoring in SCLC patients. CA 15-3, also an unspecific marker, showed similar sensitivity to the NSE assay in SCLC patients, the sensitivity being higher than CEA in non small cell lung cancer (NSCLC).     

Proliferating activity, DNA ploidy changes of lung cancer cells before and after chemotherapy

To assess proliferating activity, DNA ploidy changes of lung cancer cells before and after chemotherapy, we performed a flow cytometry analysis (FC) using fresh bronchoscopy specimens from 38 patients with lung cancer. Among 33 males and 5 females, squamous cell carcinoma (NSLC) was recognized in 12 males, 15 males had small cell lung cancer (SCLC) and 6 males had lung cancer with no histological type (LC) defined. Three women had SCLC, 1 had NSCLC and one had LC. Control consisted of 11 COPD patients. The percentage of diploid cells was significantly lower and cells with hypoploid cells were significantly higher in study group before treatment. High percentage of G2M cells characterised NSCLC and LC groups, whether high number of S phase cells characterised NSCLC and SCLC group before treatment. The treatment lowered percentage of G2M cells in NSCLC and CA group, whether diploid, hypoploid and S phase cells did not differ than those from before treatment.     

nm23、EGFR和Bcl-2蛋白在肺癌组织中的表达及临床意义

目的:观察nm23、EGFR和Bcl-2在人肺癌中的表达,探讨其与肺癌临床生物学行为的关系。方法:采用免疫组化检测了49例肺癌及癌旁组织中nm23、EGFR和Bcl-2蛋白的表达情况,并对其与肺癌临床生物学行为进行相关分析。结果:在49例肺癌中,EGFR和Bel-2蛋白的阳性表达率分别为51.0%和34.7%,均高于正常肺组织17.5%和12.5%(p<0.05),nm23蛋白的阳性表达率为55.1%,则显著低于正常肺组织87.5%(p<0.01)。在病理分级的高一中分化、低分化和未分化中nm23的阳性表达率分别为82.6%、41.2%和11.1%(p<0.01)。EGFR在非小细胞肺癌(NSCLC)组为61.5%,在小细胞肺癌(SCLC)组为10.0%(p<0.01)。在NSCLC TNM分期中,Ⅰ期 Ⅱ期与Ⅲ P<0.01)期中EGFR的阳性表达率分别为39.3%和81.8%(p<0.01);病理分级的高-中分化和低分化中EGFR的阳性表达率分别为39.1%和88.2%(p<0.01);在淋巴结转移组为57.9%,无淋巴结转移组为27.3%(p<0.05)。Bcl-2的阳性表达率在NSCLC组为25.6%,在SCLC组为70%,统计学分析差异有统计学意义(p<0.01);在NSCLC组病理分级的高分化、中分化和低分化中Bcl-2阳性表达率分别为62.5%、26.7%和5.89%(p<0.01)。结论:癌基因EGFR、凋亡抑制基因Bcl-2与抑癌基因nm23在肺癌的发生、发展中存在着相互促进作用。     

Lung carcinoid cell lines have bombesin-like peptides and EGF receptors

The biochemical properties of lung cancer cell lines were investigated. Bombesin-like peptides were present in three small cell lung cancer (SCLC) cell lines examined and three of four lung carcinoids but not in five non-small cell lung cancer (NSCLC) cell lines. Therefore SCLC and some lung carcinoids, but not NSCLC, are enriched in neuroendocrine properties. In contrast, 125I-EGF bound with high affinity to all five NSCLC cell lines and three of four lung carcinoids but not to the three SCLC cell lines examined. For lung carcinoid cell line NCI-H727, 125I-EGF bound with high affinity (Kd = 6 nM) to a single class of sites (Bmax = 110,000/cell). The 125I-EGF bound was rapidly internalized at 37 degrees C but not 4 degrees C. Using Western blot techniques and antiphosphotyrosine antibodies, EGF induced phosphorylation of a major 170 Kd protein. Using immunoprecipitation techniques and anti-EGF receptor antibodies a major 170 Kd protein was labeled. These data indicate that biologically active EGF receptors are present on NSCLC and lung carcinoid cell lines.     

Expression of Prostate-Specific Membrane Antigen in Lung Cancer Cells and Tumor Neovasculature Endothelial Cells and Its Clinical Significance

BackgroundProstate-specific membrane antigen (PSMA) has been found in tumor neovasculature endothelial cells (NECs) of non-prostate cancers and may become the most promising target for anti-tumor therapy. To study the value of PSMA as a potential new target for lung cancer treatment, PSMA expression in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) tissues and its relationship with clinicopathology were investigated in the current study.MethodsImmunohistochemistry was used to detect PSMA expression in a total of 150 lung specimens of patients with lung cancer. The data were analyzed using univariate and multivariate statistical analyses.ResultsThe percentages of NSCLC patients who had PSMA (+) tumor cells and PSMA (+) NECs were 54.02% and 85.06%, respectively. The percentage of patients younger than 60 years old who had PSMA (+) tumor cells was 69.05%, which was significantly greater than the percentage of patients aged 60 years or older (40.00%, p<0.05). A significant difference was observed in the percentage of NSCLC patients with PMSA (+) NECs and stage I or II cancer (92.98%) and those patients with stage III or IV cancer (76.77%). In the SCLC tissues, NEC PSMA expression (70.00%) did not differ significantly from NSCLC. SCLC tumor cells and normal lung tissues cells were all negative. There was no significant correlation between the presence of PSMA (+) NECs in SCLC patients and the observed clinicopathological parameters.ConclusionsPSMA is expressed not only in NECs of NSCLC and SCLC but also in tumor cells of most NSCLC patients. The presence of PSMA (+) tumor cells and PSMA (+) NECs in NSCLC was negatively correlated with age and the clinicopathological stage of the patients, respectively.     

MicroRNA expression and clinical outcome of small cell lung cancer

The role of microRNAs in small-cell lung carcinoma (SCLC) is largely unknown. miR-34a is known as a p53 regulated tumor suppressor microRNA in many cancer types. However, its therapeutic implication has never been studied in SCLC, a cancer type with frequent dysfunction of p53. We investigated the expression of a panel of 7 microRNAs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a) in 31 SCLC tumors, 14 SCLC cell lines, and 26 NSCLC cell lines. We observed significantly lower miR-21, miR-29b, and miR-34a expression in SCLC cell lines than in NSCLC cell lines. The expression of the 7 microRNAs was unrelated to SCLC patients' clinical characteristics and was neither prognostic in term of overall survival or progression-free survival nor predictive of treatment response. Overexpression or downregulation of miR-34a did not influence SCLC cell viability. The expression of these 7 microRNAs also did not predict in vitro sensitivity to cisplatin or etoposide in SCLC cell lines. Overexpression or downregulation of miR-34a did not influence sensitivity to cisplatin or etoposide in SCLC cell lines. In contrast to downregulation of the miR-34a target genes cMET and Axl by overexpression of miR-34a in NSCLC cell lines, the intrinsic expression of cMET and Axl was low in SCLC cell lines and was not influenced by overexpression of miR-34a. Our results suggest that the expression of the 7 selected microRNAs are not prognostic in SCLC patients, and miR-34a is unrelated to the malignant behavior of SCLC cells and is unlikely to be a therapeutic target.     

The substance P/neurokinin-1 receptor system in lung cancer: Focus on the antitumor action of neurokinin-1 receptor antagonists

The last decades have seen no significant progress in extending the survival of lung cancer patients and there is an urgent need to improve current therapies. The substance P (SP)/neurokinin-1 receptor (NK-1R) system plays an important role in the development of cancer: SP and NK-1R antagonists respectively induce cell proliferation and inhibition in human cancer cell lines. No study of the involvement of this system in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells has been carried out in depth. Here, we demonstrate the involvement of the SP/NK-1R system in human H-69 (SCLC) and COR-L23 (NSCLC) cell lines: (1) they express isoforms of the NK-1R and mRNA for the NK-1R; (2) they overexpress the tachykinin 1 gene; (3) the NK-1R is involved in their viability; (4) SP induces their proliferation; (5) NK-1R antagonists (Aprepitant (Emend), L-733,060, L-732,138) inhibit the growth of both cell lines in a concentration-dependent manner; (6) the specific antitumor action of these antagonists against such cells occurs through the NK-1R; and (7) lung cancer cell death is due to apoptosis. We also demonstrate the presence of NK-1Rs and SP in all the human SCLC and NSCLC samples studied. Our findings indicate that the NK-1R may be a promising new target in the treatment of lung cancer and that NK-1R antagonists could be new candidate antitumor drugs in the treatment of SCLC and NSCLC.     

SBF2-AS1: An oncogenic lncRNA in small-cell lung cancer

The long noncoding RNAs (lncRNAs) SBF2 antisense RNA 1 (SBF2-AS1) was found to act as an oncogenic lncRNA in non–small-cell lung cancer (NSCLC), but the role of SBF2-AS1 in small-cell lung cancer (SCLC) was still unclear. The purpose of this study was to provide the clinical significance and biological function of SBF2-AS1 in SCLC. In our results, SBF2-AS1 was found to be upregulated in SCLC tissues compared with NSCLC tissues or adjacent normal lung tissues. Besides, SBF2-AS1 expression was also elevated in SCLC cell lines compared with the normal bronchial epithelial cell line or NSCLC lines. Moreover, high expression of SBF2-AS1 was associated with clinical stage, tumor size, lymph node metastasis and distant metastasis in SCLC patients. Survival analysis showed SCLC patients with high expression of SBF2-AS1 had shorter overall survival than patients with low expression of SBF2-AS1, and high expression of SBF2-AS1 acted as an independent poor prognostic factor for overall survival in SCLC patients. The study in vitro suggested inhibition of SBF2-AS1 obviously depressed cell proliferation, migration, and invasion in SCLC. In conclusion, SBF2-AS1 acts as a novel oncogenic lncRNA in SCLC.