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The link between a lung tumor and the lung microbiome is a largely unexplored issue. To investigate the relationship between a lung microbiome and the phenotype of an inflammatory stromal infiltrate, we studied a cohort of 89 patients with non-small cell lung cancer. The microbiome was analyzed in tumor and adjacent normal tissue by 16S rRNA amplicon sequencing. Characterization of the tumor stroma was done using immunohistochemistry. We demonstrated that the bacterial load was higher in adjacent normal tissue than in a tumor (p = 0.0325) with similar patterns of taxonomic structure and alpha diversity. Lung adenocarcinomas did not differ in their alpha diversity from squamous cell carcinomas, although the content of Gram-positive bacteria increased significantly in the adenocarcinoma group (p = 0.0419). An analysis of an inflammatory infiltrate of tumor stroma showed a correlation of CD68, iNOS and FOXP3 with a histological type of tumor. For the first time we showed that high bacterial load in the tumor combined with increased iNOS expression is a favorable prognostic factor (HR = 0.1824, p = 0.0123), while high bacterial load combined with the increased number of FOXP3+ cells is a marker of poor prognosis (HR = 4.651, p = 0.0116). Thus, we established that bacterial load of the tumor has an opposite prognostic value depending on the status of local antitumor immunity.

Bronchopulmonary neuroendocrine tumors (NETs) refer to malignant epithelial neoplasms of neuroendocrine origin, which form highly heterogeneous group with respect to biological behavior and clinical manifestations. Three main categories of different grades of malignancy are distinguished in the diagnosis of lung NETs: typical carcinoids (TK), atypical carcinoids (AK) and the most aggressive low-differentiated tumors including small-cell and large-cell neuroendocrine lung carcinomas. These groups differ in terms of disease prognosis and therapeutic approaches, but the criteria currently used do not always allow clear boundaries between different histological variants. The search for additional diagnostic parameters and individual prognosis markers is currently actual for the grading and optimal classification of NETs. For the first time we studied the expression of Retinoic Acid Binding Protein-1 (CRABP1) in different variants of lung NETs. IHC analysis of 43 samples of lung NETs with various degrees of differentiation and grades revealed the statistically significant correlation between nuclear localization of CRABP1 and proliferation index «Ki-67» and tumor grade. The results pointed on the involvement of CRABP1 in the pathogenesis of lung NETs and indicated the need for further investigation of the relationship of the nuclear CRABP1 with clinical parameters and patient survival to determine whether this protein can be used as a marker for differential diagnosis and/or disease prognosis.

MicroRNA miR-155 is one of the most characterized and actively studied microRNAs that regulate processes closely related to carcinogenesis, including cell differentiation, adhesion, migration and invasion of the tumor cells, metastasis, apoptosis and immunosuppression. In addition, this miRNA is involved in differentiation of hematopoetic cells and developing of inflammation. The association between specific deregulation of the expression of miR-155 and carcinogenesis is confirmed by a number of both fundamental and clinical studies and is due to the posttranscriptional regulation of the most important genes of the tumor associated pathways. Modulation of the levels of expression of miR-155 is associated with the emergence of a number of leukemias and lymphomas, as well as some solid tumors. An increase of the level of cellular and/or circulating miR-155 in some cancers can serve as a marker for progression and drug resistance. In addition, inhibition of the expression of miR-155 can be a promising method for developing new approaches in antitumor therapy.

Recent research on cancer-associated microbial communities led to the accumulation of data on the interplay between bacteria, immune and tumor cells, the pathways of bacterial induction of carcinogenesis, and its meaningfulness for medicine. Microbial communities that have any kind of impact on tumor progression and microorganisms associated with tumors have been defined as oncobiome. Over the last decades, a number of studies were dedicated toHelicobacter pyloriand its role in the progression of stomach tumors, so this correlation can be regarded as proven. Involvement of bacteria in the induction of lung cancer has been largely ignored for a long time, though some correlations between this type of cancer and lung microbiome were established. Despite the fact that in the present the microbial impact on lung cancer progression has many confirmations, the underlying mechanisms are poorly understood. Microorganisms can contribute to tumor initiation and progression through production of bacteriotoxins and other proinflammatory factors. The purpose of this review is to organize the available data on lung cancer microbiome and its role in malignant tumor progression.

Targeted inhibitors of the epidermal growth factor receptor (EGFR) are used for the treatment of non-small cell lung cancer (NSCLC). Somatic mutations in the EGFR gene and key effectors of the EGFR-signaling pathway (KRAS, BRAF, PIK3CA) are associated with sensitivity to these drugs. We developed a highly sensitive LUNG CANCER (LC)-biochip approach for the detection of the most common EGFR, KRAS, PIK3CA, and BRAF gene mutations. The locked nucleic acid clamp PCR technique was used to increase the sensitivity of the assay, then allele-specific hybridization of a fluorescently labeled target on a biochip was performed. To prove the feasibility of the approach, clinical samples from 112 patients with NSCLC were analyzed. A total of 14 EGFR (12.5%) mutations, 21 (18.8%) KRAS mutations, 12 (10.7%) PIK3CA mutations, and 1 BRAF mutation (0.9%) were found. We compared the results with those from direct sequencing. We detected 50 different mutations by the LC-biochip assay and only 33 of them were found by direct sequencing. To demonstrate that the LC-biochip assay did not give false-positive results, the 17 specimens with discordant results were subjected to locked nucleic acid clamp PCR followed by sequencing. The results of this analysis were identical to the results obtained by the LC-biochip assay indicating that the biochip-based assay was both accurate and reliable. This approach was able to detect approximately 0.5% of mutated alleles in wild-type DNA background. The biochip-based assay is a reliable and inexpensive method for the identification of NSCLC patients, who may respond to a specific targeted therapy.

The impairment of retinoic acid (RA)-dependent signaling is a frequent event during carcinogenesis. Cellular retinoic acid-binding proteins (CRABP1 and CRABP2) are important modulators of RA activity. Up to date, the role of these proteins in cancer progression remains poorly investigated. Here, we studied for the first time the simultaneous messenger RNA (mRNA) and protein expression of CRABPs in non-small cell lung cancer (NSCLC) samples. CRABP1 and CRABP2 mRNA levels were elevated in 42 and 56 % of NSCLC samples, respectively. Decrease of CRABP2 mRNA expression was significantly associated with the presence of lymph node metastases. Protein expression of CRABP1 and CRABP2 was detected in 50 and 56 % of tumor samples, respectively. We also found a positive correlation between CRABP1 and CRABP2 expression. Taken together, we demonstrated significant changes in CRABP expression in NSCLC samples. Importantly, the presented data provide the first evidence of potential involvement of CRABP2 in lung cancer metastasis.

CRABP1 (cellular retinoic acid binding protein 1) belongs to the family of fatty acid binding proteins. Retinoic acid binding is the only known functional activity of this protein. The role of CRABP1 in human carcinogenesis remains poorly understood. Here, for the first time we demonstrated pro-metastatic and pro-tumorigenic activity of CRABP1 in mesenchymal tumors. Further functional analysis revealed that the pro-tumorigenic effect of CRABP1 does not depend on retinoic acid binding activity. These results suggest that CRABP1 could have an alternative intracellular functional activity that contributes to the high malignancy of transformed mesenchymal cells. Microarray analysis detected CRABP1-mediated alterations in the expression of about 100 genes, including those encoding key regulatory proteins. CRABP1 is ubiquitously expressed in monophasic synovial sarcomas, while in biphasic synovial sarcomas it is expressed uniquely by the spindle cells of the aggressive mesenchymal component. High level of CRABP1 expression is associated with lymph node metastasis and poor differentiation/high grade of pancreatic neuroendocrine tumors (pNETs). Presented data suggest CRABP1 as a promising biomarker of pNETs’ clinical behavior. Our results give the first evidence of pro-tumorigenic and pro-metastatic activity of CRABP1 in mesenchymal and neuroendocrine tumors.

Transduction of many cellular signals is mediated by special plasma membrane microdomains that are called lipid rafts. Lipid rafts are dynamic and transient structures; however, they can be stabilized by lipid raft proteins, including the family of flotillins which is represented by flotillin-1 and flotillin-2. Flotillins are expressed in different tissues and may regulate many signalling pathways. However, their role in carcinogenesis remains unclear. The aim of this work was to investigate variations of flotillin-2 expression on mRNA and protein level in lung adenocarcinoma specimens. We also studied the influence of flotillin-2 expression on the characteristics of A549 lung cancer cell line. The level of flotillin-2 mRNA was reduced in the vast majority of investigated adenocarcinoma specimens in comparison to corresponding normal tissues. However, the amount of protein varied widely and was preferentially increased (40%) than decreased (15%). Flotillin-2 overexpression in A549 cell line did not change proliferation but stimulated migration of cultivated cells. Conversely, knockdown of flotillin-2 using small hairpin RNA (shRNA) downregulated proliferation as well as migration of tumor cells. These results indicate that the expression of flotillin-2 changes in human lung adenocarcinoma and that this protein may influence the key characteristics of tumor cells.

ISSN 16076729, Doklady Biochemistry and Biophysics, 2011, Vol. 438, pp. 147–150. © Pleiades Publishing, Ltd., 2011.Original Russian Text © K.N. Kashkin, E.A. Musatkina, A.V. Komelkov, E.A. Tonevitsky, D.A. Sakharov, T.V. Vinogradova, E.P. Kopantsev, M.V. Zinovyeva, I.A. Favorskaya,Ya.A. Kainov, V.N. Aushev, I.B. Zborovskaya, A.G. Tonevitsky, E.D. Sverdlov, 2011, published in Doklady Akademii Nauk, 2011, Vol. 438, No. 6, pp. 829–833.

Evaluation of tumor marker expression pattern that determines individual progression parameters is one of the major topics in molecular oncopathology research. This work represents research on expression analysis of several Ras-Ral associated signal transduction pathway proteins (Arf6, RalA, and BIRC5) in accordance with clinical criteria in nonsmall cell lung cancer patients. Using Western-blot analysis and RT-PCR Arf6, RalA, and BIRC5, expression has been analyzed in 53 nonsmall cell lung cancer samples of different origin. Arf6 protein expression was increased in 55% nonsmall cell lung cancer tumor samples in comparison with normal tissue. In the group of squamous cell lung cancer, increase of Arf6 expression was observed more often. RalA protein expression was decreased in comparison to normal tissue samples in 64% of nonsmall cell lung cancer regardless of morphological structure. Correlation between the decrease of RalA protein expression and the absence of regional metastases was revealed for squamous cell lung cancer. BIRC5 protein expression in tumor samples versus corresponding normal tissue was 1.3 times more often elevated in the squamous cell lung cancer group (in 76% tumor samples). At the same time, increase of BIRC5 expression was detected only in 63% of adenocarcinoma tumor samples. A statistically significant decrease (p = 0.015) of RalA protein expression and the increase (p = 0.049) of Arf6 protein expression in comparison with normal tissue was found in T1–2N0M0 and T1–2N1–2M0 groups of squamous cell lung cancer, respectively.

Objectives: Lysosomal proteases cathepsins B and D (CB and CD) play a significant part in cancer progression. For many oncological diseases protein expression levels of CB and CD have been investigated and correlations with tumour characteristics revealed. Meanwhile, there is very little information concerning mRNA expression level.Methods: In the present work, data about mRNA levels of CB and CD in human lung cancer was obtained using reverse transcription followed by real-time polymerase chain reaction.Results: For the first time CD and CB mRNA in human lung cancer tumours was quantified. It was shown that CB and CD mRNA levels do not correlate with any tumour characteristics. However, in most analysed tumours, expression of CD mRNA was downregulated compared with adjacent normal tissue (p

Numerous human cancers are accompanied with the increase of ferritin content in blood serum. Ferritin is composed of light and heavy chains, encoded by FTL and FTH genes, respectively. The analysis of EST database showed that the expression of FTL and FTH genes in lung tumors is decreased compared to normal tissues, and is not altered in renal cancer cells. The alteration of mRNA corresponding to FTL and FTH genes was estimated by real-time PCR in primary lung and kidney tumors. A significant and frequent inhibition of FTL and FTH gene expression (on average by 11 and 9 times, in 83% (33/40) and 73% (11/15) of cases, respectively) was detected in primary squamous lung carcinoma. The expression of these genes was not altered so significantly (by 6 and 3 times, in 58% and 27% of samples) in clear cell renal cell carcinoma. Our work reports for the first time the down-regulation of FTL gene expression at the first stage of lung cancer (10/10), and proposes this gene as a potential oncomarker for early diagnosis. The FTL mRNA content may be quantified by non-competitive hybridization on expression DNA microarrays. The possible causes of a serum ferritin increase in lung squamous cell carcinoma and clear cell renal cell carcinoma are discussed.

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.

An analysis was made of the change in the expression of the extracellular inhibitor of the Wnt-signal pathway WIF1 in non-small cell lung carcinomas. The results of the analysis revealed a frequent (67% of the cases) suppression of the WIF1 transcript in non-small cell lung carcinomas. On the basis of the data obtained and the results of previously published investigations, it was suggested that inhibition of WIF1 expression is typical of squamous cell carcinoma of the lungs and only to a lesser degree of adenocarcinoma. An analysis of the content of the WIF1 protein in tumor lysates with a suppressed level of the WIF1 transcript also revealed an accompanying decrease in the level of the WIF1 protein in the tumors. The results of the investigation confirm the frequent suppression both of the transcript and of the WIF protein in lung tumors, which may cause a disregulation of the Wnt-signal pathway and may contribute to the development of the tumor.

Lung cancer is among the most common neoplasms in Russia, the United States, and in Western Europe and is accompanied by changes in the functional activity of many genes. The transcription levels of TIMP3, DAPK1, and AKR1B10 were compared for normal and tumor lung tissues of patients with squamous-cell cancer (SCC) by RT-PCR. A substantial increase in AKR1B10 transcription level was observed in 80% of the tumors. The transcription levels of TIMP3 and DAPK1 were significantly decreased in 76 and 72% of the tumors, respectively. The results implicated the genes in carcinogenesis in SCC, AKR1B10 acting as a potential oncogene, and TIMP3 and DAPK1 acting as potential tumor suppressor genes. It was assumed that dramatic changes in their transcription levels could be used for early diagnosis of SCC.

// Anna Zagryazhskaya 1 , Olga Surova 1, 2 , Nadeem S. Akbar 1 , Giulia Allavena 1, 3 , Katarina Gyuraszova 1, 4 , Irina B. Zborovskaya 5, 6 , Elena M. Tchevkina 5, 6 , Boris Zhivotovsky 1, 6 1 Institute of Environmental Medicine, Division of Toxicology, Stockholm, Sweden 2 Ludwig Institute for Cancer Research Ltd, Karolinska Institutet, Stockholm, Sweden 3 Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy 4 Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Safarik University in Kosice, Kosice, Slovakia 5 NN Blokhin Russian Cancer Research Center, Moscow, Russia 6 Faculty of Fundamental Medicine, ML Lomonosov State University, Moscow, Russia Correspondence to: Boris Zhivotovsky, e-mail: Boris.Zhivotovsky@ki.se Keywords: tudor staphylococcal nuclease, S100A11, phospholipase A 2 , non-small cell lung cancer, apoptosis Received: December 19, 2014 Accepted: March 07, 2015 Published: March 26, 2015 ABSTRACT Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC), the major lung cancer subtype, is characterized by high resistance to chemotherapy. Here we demonstrate that Tudor staphylococcal nuclease (SND1 or TSN) is overexpressed in NSCLC cell lines and tissues, and is important for maintaining NSCLC chemoresistance. Downregulation of TSN by RNAi in NSCLC cells led to strong potentiation of cell death in response to cisplatin. Silencing of TSN was accompanied by a significant decrease in S100A11 expression at both mRNA and protein level. Downregulation of S100A11 by RNAi resulted in enhanced sensitivity of NSCLC cells to cisplatin, oxaliplatin and 5-fluouracil. AACOCF 3 , a phospholipase A 2 (PLA 2 ) inhibitor, strongly abrogated chemosensitization upon silencing of S100A11 suggesting that PLA 2 inhibition by S100A11 governs the chemoresistance of NSCLC. Moreover, silencing of S100A11 stimulated mitochondrial superoxide production, which was decreased by AACOCF 3 , as well as N -acetyl- L -cysteine, which also mimicked the effect of PLA 2 inhibitor on NSCLC chemosensitization upon S100A11 silencing. Thus, we present the novel TSN-S100A11-PLA 2 axis regulating superoxide-dependent apoptosis, triggered by platinum-based chemotherapeutic agents in NSCLC that may be targeted by innovative cancer therapies.

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.

Studies performed in the recent decade in the Laboratory of the Regulation of Cell and Virus Oncogenes associated structural and functional defects of several oncogenes and tumor suppressor genes with various stages of non-small-cell lung cancer. High risk of lung cancer was established for carriers of rare alleles of the Hras1 minisatellite, the hypermethylated p16 INK4A promoter, and microsatellite defects in chromosome regions 3p12, 3p14.2, 3p22–24, 3p21, 3p25, 9p21, and 17p13. Analysis of the Hras1 minisatellite and microsatellites located in two minimal deletion overlap regions of 1p36 was shown to allow a more reliable prognosis in lung cancer. The results testified again that panels of molecular markers are useful for individual risk assessment, early and differential diagnosis, and prognosis in cancer.

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, HIC1, and N33 5′ regions in non-small cell lung cancer (51 tumors). Methylation was observed for the two suppressor genes involved in controlling the cell cycle through the Cdk–Rb–E2F signaling pathway, RB1 (10/51, 19%) and p16 (20/51, 39%). The highest methylation frequencies were established for CDH1 (72%) and HIC1 (82%). The CpG islands of p14 and p15 proved to be nonmethylated. At least one gene was methylated in 90% (46/51) tumors and no gene, in 10% (5/51) tumors. In addition, the genes were tested for methylation in peripheral blood lymphocytes of healthy subjects. Methylation frequency significantly differed between tumors and normal cells in the case of RB1, p16, CDH1, HIC1, and N33. Gene methylation frequency was tested for association with histological type of the tumor and stage of tumor progression. Methylation index of a panel of tumor suppressor genes was established for groups of tumors varying in clinical and morphological parameters.

Allele distribution at a highly polymorphic minisatellite adjacent to the c-Hras1 gene as well as deletions of microsatellite markers, D3S966, D3S1298, D9S171, and a microsatellite within p53 gene, were examined in bronchial epithelium specimens obtained from 53 chronic obstructive pulmonary disease (COPD) patients and healthy donors. A higher frequency of rare Hras1minisatellite alleles in COPD patients than in the individuals without pulmonary pathology (6.6% versus 2.2%; P < 0.05) was shown. This difference was most pronounced in the group of ten COPD patients with idiopathic pulmonary fibrosis. Three of these patients had rare Hras1 minisatellite allele (P < 0.02 in comparison with healthy controls). Alterations in at least one of the microsatellite markers (deletions or microsatellite instability) were detected in bronchial epithelium samples obtained from: 4 of 10 COPD patients with pneumofibrosis (40%); 15 of 43 COPD patients (34.9%) without pneumofibrosis; and 8 of 20 tobacco smokers (40%) without pulmonary pathology. These defects were not observed in the analogous samples obtained from healthy nonsmoking individuals. No statistically significant differences were revealed between COPD patients and healthy smokers upon comparison of both the total number of molecular defects and the number of defects in the individual chromosomal loci. The total number of molecular defects revealed in bronchial epithelium samples from the individuals of two groups examined correlated with the intensity of exposure to tobacco smoke carcinogens (r = 0.28; P < 0.05). These findings suggest that rare alleles at theHras1 locus may be associated with hereditary predisposition to COPD and the development of pneumofibrosis, while mutations in microsatellite markers result from exposure to tobacco smoke carcinogens and are not associated with the appearance of these pathologies.

Ionizing radiation is a potent lung carcinogen, but the precise molecular damage associated with it is still unknown. In this study we investigated cancer-related molecular abnormalities including K-ras (codon 12) mutation, p16(INK4A) promoter hypermethylation and microsatellite alterations at seven chromosomal regions in successive biopsies obtained from former Chernobyl cleanup workers in comparison with smokers and nonsmokers who have never had radiation exposure. Our results indicate that prolonged persistence of inhaled radioactive particles is associated with appearance of allelic loss at 3p12, 3p14.2 (FHIT), 3p21, 3p22-24 (hMLH1) and 9p21 (p16INK4A) in bronchial epithelium of former Chernobyl clean-up workers. The prevalence of 3p14.2 allelic loss was associated with decreased expression of the FHIT mRNA in their bronchial epithelium in comparison with control group of smokers. During several years of our monitoring samples of epithelium were collected from the same area of bronchial tree. In epithelium exposed to carcinogens (tobacco smoke and/or radioactivity) the total number of molecular abnormalities was significantly higher in dysplasia and in morphologically normal foci progressed later to dysplasia than in these samples which never showed evidence of such progression. Our findings indicate that extensive cancer-related molecular abnormalities sequentially occur in radiation damaged bronchial epithelium of former Chernobyl clean-up workers.

Background At the present time, there is a lack of data about the involvement of flotillins and stomatin in the development of non-small cell lung cancer (NSCLC) and soft tissue sarcomas (STS). Moreover, changes in expression of members of different families of the microdomain-forming proteins (caveolins and SPFH-domain containing family) are usually investigated independently of each other. In this study we performed a combined analysis of flotillins, stomatin, and caveolin-1 expression in these pathologies and evaluated correlations between generated data and clinicopathological characteristics of the specimens. Methods The protein and mRNA expression was analyzed by Western blotting and real-time PCR, respectively, in tissue specimens of patients undergoing surgery for non-small cell lung cancer and soft tissue sarcomas. Association between expression of studied proteins and patient clinicopathological characteristics or outcome was evaluated. Results Stomatin protein expression was down-regulated in 80% of NSCLC samples and this decrease significantly associated with presence of lymph node metastases. Flotillin-2 protein expression was up-regulated in the majority of NSCLC samples whereas caveolin-1α expression was decreased. We revealed a strong correlation between STOM and FLOT-1 mRNA expression in both pathologies, although the gene expression changes were diverse. Conclusions Our data demonstrate for the first time that expression of stomatin, a poorly studied microdomain-forming protein, significantly changes in human tumors, thus pointing to its importance in the progression of NSCLC. We also suggest the existence of some relationship between the expression of these proteins.

Allelic losses at 1p32-pter have been reported as frequent events in human non-small cell lung cancer (NSCLC). To further characterize the region of deletions, we studied loss of heterozygosity on a panel of 102 microdissected NSCLC samples with 20 polymorphic markers spanning 1p32-pter. Two shortest regions of the overlap of the deletions (SROs) were found: SRO 2a (D1S417--D1S57) and SRO 2b (D1S450--D1S243). Allelic losses at either region correlated independently with advanced stage of disease and with postoperative metastasis and relapse (P < 0.05), suggesting that crucial genes in these regions are involved in NSCLC progression. Mol. Carcinog. 30:151--158, 2001.

We studied the relation between genetic anomalies in thep73 gene encoding a product structurally and functionally similar to the protein p53 and the pathogenesis of non-small-cell lung carcinoma (NSCLC; 83 patients). Loss of one of thep73 alleles was revealed in 44% (15/34) informative cases. Presence of deletions correlated with the features common for tumor development: metastatic affection of regional lymph nodes (p=0.045), large tumor size (p=0.037), advanced stage of the disease (p=0.017). Allele expression of the genep73 was studied basing on analysis of the polymorphic C/T site in the second exon. All ten studied samples of normal bronchogenic epithelium showed monoallelic expression ofp73, contrary to the six NSCLC samples which preserved both of thep73 alleles and showed biallelic expression. Enhanced expression of p73 mRNA in tumor tissue compared with normal bronchogenic epithelium was found in 28 of 34 (82.4%) NSCLC patients. Expression of p73 in NSCLC cells showed correlation neither with deletions of one of the alleles, nor with any parameter reflecting clinical pathology. The results suggest thatp73 is not a classical tumor suppressor gene in NSCLC. However, alterations ofp73 expression are common for NSCLC. This may be important for our understanding of the NSCLC origin and development.

ISSN 16076729, Doklady Biochemistry and Biophysics, 2011, Vol. 437, pp. 105–108. © Pleiades Publishing, Ltd., 2011.Original Russian Text © K.N. Kashkin, E.A. Musatkina, A.V. Komelkov, D.A. Sakharov, E.V. Trushkin, E.A. Tonevitsky, T.V. Vinogradova, E.P. Kopantzev, M.V. Zinovyeva,O.V. Kovaleva, K.A. Arkhipova, I.B. Zborovskaya, A.G. Tonevitsky, E.D. Sverdlov, 2011, published in Doklady Akademii Nauk, 2011, Vol. 437, No. 6, pp. 837–841.

Loss of heterozygosity (LOH) affects a number of chromosome regions in ovarian cancer, pointing to the possible involvement of tumour-suppressor genes in ovarian tumorigenesis. We performed comparative analysis of allelic loss at 6 frequently affected chromosome regions in a panel of 53 benign, borderline and malignant ovarian tumours. Precursor lesions could provide evidence that an accumulation of genetic events is required for normal ovarian epithelium to generate malignant tumours. LOH on chromosome 1p was relatively common in benign, borderline and malignant tumours, while at 11p and 7q it was observed not only in invasive but also in borderline tumours. Moreover, 17q and 18q were affected mainly in advanced malignant tumours and revealed a high frequency of clonal intratumoral heterogeneity. We encountered different spectra of genetic alterations in primary tumours and their metastasis, which may be the results of intratumoral heterogeneity leading to dissemination in only some sub-clones.

The mapping of allelic loss on the short arm of chromosome 1 has been performed in non-small-cell lung cancer. We used a set of 11 microsatellite loci spanning 1p to examine the frequency of allelic imbalance in a panel of 58 tumours. Fifty-one of 58 (87.9%) cases have shown somatic allelic loss at one or more loci tested. The two shortest regions of the overlap (SRO) of the deletions have been identified: SRO 1 at 1p13.1 and SRO 2 at 1p32-pter. Allelic losses at these regions have been compared among adenocarcinoma and squamous cell carcinoma and no difference has been found. In contrast to SRO 1, deletions at SRO 2 significantly correlated with advanced stage of the disease as well as post-operative metastasizing and relapse. These data may suggest that SRO 1 and SRO 2 can harbour tumour-supressor genes (TSGs) involved in different stages of NSCLC development. SRO 2 is still quite large and its refined mapping should help attempts to clone and identify the putative TSG(s). Microsatellite instability (replication errors) affecting only 6 (10.3%) of 58 tumour samples is an infrequent genetic alteration at the loci tested. Images Figure 2

Lung cancer is the major human malignancy, accounting for 30% of all cancer-related deaths worldwide. Poor survival of lung cancer patients, together with late diagnosis and resistance to classic chemotherapy, highlights the need for identification of new biomarkers for early detection. Among different cancer biomarkers, small non-coding RNAs called microRNAs (miRNAs) are considered the most promising, owing to their remarkable stability, their cancer-type specificity, and their presence in body fluids. However, results of multiple previous attempts to identify circulating miRNAs specific for lung cancer are inconsistent, likely due to two main reasons: prominent variability in blood miRNA content among individuals and difficulties in distinguishing tumor-relevant miRNAs in the blood from their non-tumor counterparts. To overcome these impediments, we compared circulating miRNA profiles in patients with lung squamous cell carcinoma (SCC) before and after tumor removal, assuming that the levels of all tumor-relevant miRNAs would drop after the surgery. Our results revealed a specific panel of the miRNAs (miR-205, -19a, -19b, -30b, and -20a) whose levels decreased strikingly in the blood of patients after lung SCC surgery. Interestingly, miRNA profiling of plasma fractions of lung SCC patients revealed high levels of these miRNA species in tumor-specific exosomes; additionally, some of these miRNAs were also found to be selectively secreted to the medium by cultivated lung cancer cells. These results strengthen the notion that tumor cells secrete miRNA-containing exosomes into circulation, and that miRNA profiling of the exosomal plasma fraction may reveal powerful cancer biomarkers.

Proprotein convertases (PCs) is a protein family which includes nine highly specific subtilisin-like serine endopeptidases in mammals. The system of PCs is involved in carcinogenesis and levels of PC mRNAs alter in cancer, which suggests expression status of PCs as a possible marker for cancer typing and prognosis. The goal of this work was to assess the information value of expression profiling of PC genes. Quantitative polymerase chain reaction was used for the first time to analyze mRNA levels of all PC genes as well as matrix metalloproteinase genes MMP2 and MMP14, which are substrates of PCs, in 30 matched pairs of samples of human lung cancer tumor and adjacent tissues without pathology. Significant changes in the expression of PCs have been revealed in tumor tissues: increased FURIN mRNA level (p

The restriction fragment length polymorphism of c-Ha-ras-1 and L-myc genes and expression of cell surface effector molecules were studied to determine their potential utility as markers for assessing risk of metastasis in 84 lung cancer patients. We performed a comparative study of primary lung carcinomas, metastases, adjacent tissues and blood samples in a group of patients with lung cancer of different histological types, grade of differentiation and presence of regional and distant metastasis. No differences in the frequency of c-Ha-ras-1 rare alleles were found between lung cancer patients and unaffected controls. The detection of common a4-allele seems to be associated with metastasis and low differentiation of lung carcinomas. S-allele of L-myc was observed in 82.6% of patients with metastatic lesions. Homozygosity of L-allele patients was not evidence for distant metastasis and only 17.4% of these patients have metastatic lesions of the lymph nodes. The expression of HLA class I and receptor of transferrin (TrRec) were tested immunohistochemically in the same patients. In the group of squamous cell carcinomas with regional metastases the expression of HLA class I antigens was decreased [7/21 (33.3%) positive staining tumors versus 13/20 (65.0%) in the group without metastases]. The opposite situation was observed for TrRec. The data of restriction fragment length polymorphism of oncogenes and expression of two cell surface effector molecules, identified in the same patients, were combined. The registration of more than one poor marker, tested in individuals with squamous cell carcinoma, closely correlated with dissemination and advanced stage of the disease. Nearly 90% (20/22) of patients with well and moderately differentiated tumor revealed metastatic lesions versus 6.6% (1/15) of patients with manifestation of a single poor marker. Finally, proposals could be made for the development of a risk group that incorporates both clinical and molecular biology features in the prediction of metastasis.

The expression of 9 oncogenes in primary tumors and in human tumors passaged in nude mice was tested: a total of 28 tumor types was analyzed. Oncogenes src, fps, and mos were not expressed in any of the tumors tested but oncogene myc was transcribed in most of the tumors and myc was over-expressed in 3 tumors passaged in nude mice (Ewing sarcoma, large intestine carcinoma and kidney carcinoma) and in primary fibrous histiocytoma. Enhanced transcription of ras and fos genes was observed nonspecifically in different tumors. Oncogene sis was activated specifically in metastases of different tumors in lymph nodes.