I. V. Pronina

Differential Expression of CHL1 Gene during Development of Major Human Cancers

Background CHL1 gene (also known as CALL) on 3p26.3 encodes a one-pass trans-membrane cell adhesion molecule (CAM). Previously CAMs of this type, including L1, were shown to be involved in cancer growth and metastasis. Methodology/Principal Findings We used Clontech Cancer Profiling Arrays (19 different types of cancers, 395 samples) to analyze expression of the CHL1 gene. The results were further validated by RT-qPCR for breast, renal and lung cancer. Cancer Profiling Arrays revealed differential expression of the gene: down-regulation/silencing in a majority of primary tumors and up-regulation associated with invasive/metastatic growth. Frequent down-regulation (>40% of cases) was detected in 11 types of cancer (breast, kidney, rectum, colon, thyroid, stomach, skin, small intestine, bladder, vulva and pancreatic cancer) and frequent up-regulation (>40% of cases) – in 5 types (lung, ovary, uterus, liver and trachea) of cancer. Using real-time quantitative PCR (RT-qPCR) we found that CHL1 expression was decreased in 61% of breast, 60% of lung, 87% of clear cell and 89% papillary renal cancer specimens (P<0.03 for all the cases). There was a higher frequency of CHL1 mRNA decrease in lung squamous cell carcinoma compared to adenocarcinoma (81% vs. 38%, P = 0.02) without association with tumor progression. Conclusions/Significance Our results suggested that CHL1 is involved in the development of different human cancers. Initially, during the primary tumor growth CHL1 could act as a putative tumor suppressor and is silenced to facilitate in situ tumor growth for 11 cancer types. We also suggested that re-expression of the gene on the edge of tumor mass might promote local invasive growth and enable further metastatic spread in ovary, colon and breast cancer. Our data also supported the role of CHL1 as a potentially novel specific biomarker in the early pathogenesis of two major histological types of renal cancer.

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%.

Tumor Suppressor Function of the SEMA3B Gene in Human Lung and Renal Cancers

The SEMA3B gene is located in the 3p21.3 LUCA region, which is frequently affected in different types of cancer. The objective of our study was to expand our knowledge of the SEMA3B gene as a tumor suppressor and the mechanisms of its inactivation. In this study, several experimental approaches were used: tumor growth analyses and apoptosis assays in vitro and in SCID mice, expression and methylation assays and other. With the use of the small cell lung cancer cell line U2020 we confirmed the function of SEMA3B as a tumor suppressor, and showed that the suppression can be realized through the induction of apoptosis and, possibly, associated with the inhibition of angiogenesis. In addition, for the first time, high methylation frequencies have been observed in both intronic (32-39%) and promoter (44-52%) CpG-islands in 38 non-small cell lung carcinomas, including 16 squamous cell carcinomas (SCC) and 22 adenocarcinomas (ADC), and in 83 clear cell renal cell carcinomas (ccRCC). Correlations between the methylation frequencies of the promoter and the intronic CpG-islands of SEMA3B with tumor stage and grade have been revealed for SCC, ADC and ccRCC. The association between the decrease of the SEMA3B mRNA level and hypermethylation of the promoter and the intronic CpG-islands has been estimated in renal primary tumors (P < 0.01). Using qPCR, we observed on the average 10- and 14-fold decrease of the SEMA3B mRNA level in SCC and ADC, respectively, and a 4-fold decrease in ccRCC. The frequency of this effect was high in both lung (92-95%) and renal (84%) tumor samples. Moreover, we showed a clear difference (P < 0.05) of the SEMA3B relative mRNA levels in ADC with and without lymph node metastases. We conclude that aberrant expression and methylation of SEMA3B could be suggested as markers of lung and renal cancer progression.

DNA methylation contributes to deregulation of 12 cancer-associated microRNAs and breast cancer progression.

The methylation of promoter CpG islands and the interaction between microRNAs (miRNAs) and messenger RNAs (mRNAs) of target genes are considered two crucial mechanisms for gene and pathway deregulation in malignant tumors. The aim of this study was to analyze the role of promoter methylation in altering the expression of 13 miRNAs that are associated with breast cancer (BC): miR-124, -125b, -127, -132, -137, -148a, -191, -193a, -203, -212, -34b, -375, -9. The role of methylation in the deregulation of these miRNAs has not been previously assessed in the representative set of BC samples. We used a set of 58 paired (tumor/normal) breast tissue samples and methylation-specific PCR to demonstrate significant aberrations in the methylation patterns of 9 miRNA genes. In particular, we observed hypermethylation of MIR-127, -132, and -193a, and hypomethylation of MIR-191 for the first time. Using quantitative PCR, we established a strong correlation between promoter methylation and expression levels for 12 miRNA genes (all except MIR-212); this finding demonstrates the functional importance of altered methylation patterns. We also performed a correlation analysis between expression levels of the 13 miRNAs and 5 cancer-associated genes, namely RASSF1(A), CHL1, APAF1, DAPK1, and BCL2, which were predicted as targets for these miRNAs, to investigate the impact of these miRNAs on these genes with key cellular functions in BC. Significant negative correlation was revealed for the following miRNA-mRNA pairs: miR-127-5p and DAPK1, miR-375 and RASSF1(A), and miR-124-3p and BCL2. Additionally, we also found a strong association between hypermethylation of MIR-127 and MIR-125b-1 and BC progression, particularly metastasis. Thus, our findings provide evidence for the significant role of methylation in the deregulation of 12 miRNA genes in BC, identify putative novel functional miRNA-mRNA pairs, and suggest MIR-127 and MIR-125b-1 hypermethylation to be potential biomarkers of BC metastasis.

Use of NotI microarrays in analysis of epigenetic and structural changes in epithelial tumor genomes by the example of human chromosome 3

A new comparative genome hybridization technology using NotI microarrays is described (Karolinska Institute International Patent WO02/086163). The method is based on comparative genome hybridization of NotI-enriched probes from tumor and normal genomic DNA with radically new NotI microarrays. A total of 181 NotI-binding loci of human chromosome 3 were assayed in 200 human malignant tissue samples from various organs: kidney, lung, breast, ovary, cervix, and prostate. The most significant portion (above 30%) of aberrations (deletions and methylation) were detected in NotI sites located in the MINT24, BHLHB2, RPL15, RARbeta1, ITGA9, RBSP3, VHL, and ZIC4 genes. This indicates that they may be associated with cancer development. Methylation of these genomic loci was confirmed by methylation-specific PCR and bisulfite sequencing. The results confirm that the proposed method can contribute to cancer genomics.

[Methylation of the putative tumor suppressor gene, RASSF1A, in primary cervical tumors].

Methylation-sensitive restriction endonuclease analysis (MSRA) followed by polymerase chain reaction (PCR) have been used to estimate the methylation level of 13 CpG dinucleotides in the promoter region of the putative suppressor gene RASSF1A (3p21.31) in squamous cell carcinomas of the uterine cervix (SCCs) carrying human papillomavirus (HPV) types 16, 18, and related types. Methylation of 3 to 13 CpG pairs has been found in 64% (25 out of 39) tumor DNA samples, 22% (2 out of 9) DNA samples from morphologically normal tissues adjacent to the tumor (P = 0.0306), and two out of three DNA samples from peripheral blood leukocytes of carcinoma patients. These CpG pairs are not methylated in the DNA of leukocytes of healthy donors (0 out of 10). The methylation level of the RASSF1A promoter region studied in tumors of the patients with regional lymph node metastases is significantly higher than in tumors of the patient that have no metastases (P = 8.5 × 10–12). The methylation frequency of gene RASSF1A is two times higher than the frequency of hemi- and homozygous deletions in the chromosome 3 region where the gene is located. The data obtained indicate that methylation is one of the main mechanisms of the RASSF1A gene inactivation in HPV-positive human cervical tumors. The methylation of this gene may be an early event in the genesis of cervical tumors, the methylation level increasing with tumor progression.

Methylation profile of group of miRNA genes in clear cell renal cell carcinoma and their involvement in cancer progression

MicroRNA regulates gene expression, is involved in many cellular processes, and plays an important role in the development of cancer. The regulation of the expression of miRNA genes can be achieved by methylating their CpG islands, which is shown in different types of tumors. The methylation of miRNA genes in clear cell renal cell carcinoma (CCRCC) has mainly been studied for the miR-9 and miR-34 families. The methylation of six miRNA genes (miR-124a-2, -124a-3, -9-1, -9-3, -34b/c, -129-2) was analyzed with using a representative sample (46 cases). Methylation of three genes miR -124a-2, -124a-3, and -129-2 was studied in kidney tumors for the first time. Methylation analysis was performed using methyl specific PCR. It is shown that the frequency of methylation of six genes was changed from 37% to 65% in tumor samples and significantly higher in tumor samples than in samples of histologically normal tissue (P ≤ 3 × 10−5 by Fisher’s exact test). These results suggest the properties of tumor suppressors for the six miRNA genes indicated in CCRCC. We also found correlations between the methylation frequency of some miRNA genes and signs of the progression of CCRCC (tumor size, clinical stage, loss of differentiation, and metastasis).

Altered Expression of the SEMA3B gene in Epithelial Tumors

Tumor-specific expression downregulation may be indicative of a gene’s involvement in tumor suppression. For instance, SEMA3B mRNA levels are decreased in many cell lines of small-cell and non-small cell lung cancer, and SEMA3B was shown to suppress the growth of the NSCLC cell line NCI-H1299 and tumor formation in immunodeficient mice. In this work, SEMA3B expression levels were determined in epithelial tumors of different localizations. In cell lines of renal, breast, and ovarian cancer, SEMA3B mRNA levels were frequently (4/11, 36%) decreased as much as 10–250-fold according to semiquantitative RT-PCR assay. SEMA3B expression levels were also determined in primary tumor extracts of kidney, lung, breast, ovarian, and colorectal cancer. In clear cell renal cell carcinoma, SEMA3B expression was decreased 5–1000-fold in 25 of 51 extracts (49%) compared to 5/51 (10%) extracts with increased mRNA levels; the result was highly significant: P < 0.0001 by Fisher’s exact test. SEMA3B was frequently downregulated in ovarian (5/16, 31% vs. 2/16, 12%) and colorectal cancer (6/11, 54% vs. 2/11, 18%). These results suggest that SEMA3B is involved in the suppression of kidney, ovarian, and colon tumor growth.

Activation of RHOA transcription in epithelial tumors may be caused by gene amplification and/or demethylation of the promoter region

RHOA is a small GTPase involved in morphogenesis, cell adhesion, and cell cycle control. RHOA (3p21.31) is a potential oncogene, causing cell malignant transformation. A study was made of the mechanisms activating RHOA transcription in several epithelial tumors. Semiquantitative RT-PCR revealed elevated transcription of RHOA in tumors (45 cases of breast cancer, renal cell carcinoma, and epithelial ovarian carcinoma; p < 10−4). For the first time transcriptional activation of RHOA in tumors was reliably associated with its amplification (p = 10−7). Digestion with four methyl-sensitive restriction enzymes (HpaII, HhaI, AciI, and Bsh1236I) and subsequent PCR revealed a change in methylation of the RHOA promoter region in 23 out of 45 tumors, hypomethylation being two times more frequent than hypermethylation. Demethylation of the RHOA promoter region was associated with a two-to tenfold increase in the transcription level of the gene. Thus, two mechanisms—amplification and demethylation of the promoter region—were shown to change the level of RHOA transcription in epithelial tumors.

Expression and DNA methylation alterations of seven cancer-associated 3p genes and their predicted regulator miRNAs (miR-129-2, miR-9-1) in breast and ovarian cancers

The methylation of promoter CpG islands and interactions between microRNAs (miRNAs) and messenger RNAs (mRNAs) of target genes are considered two crucial epigenetic mechanisms for inducing gene and pathway deregulation in tumors. Here, the expression levels of seven cancer-associated 3p genes (RASSF1(isoform A), RARB(isoform 2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and their predicted regulator miRNAs (miR-129-2, miR-9-1) were analyzed in breast (BC, 40 samples) and ovarian (OC, 14 samples) cancers using RT-PCR and qPCR. We first revealed a negative correlation between the level of the miR-129-2 precursor and RASSF1(A) and GPX1 mRNA levels in BC (Spearmans correlation coefficient (rs) was − 0.26 in both cases). Similar results were observed for the miR-129-2 precursor and the RASSF1(A), GPX1, RARB(2), and CHL1 genes in OC (rs was in the range − 0.48 to − 0.54). Using methylation-specific PCR, a significant correlation was shown between promoter hypermethylation and the down-regulation of the RASSF1(A), GPX1, RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 genes in BC (rs = 0.41 to 0.75) and of the RASSF1(A) gene in OC (rs = 0.67). We first demonstrated a high hypermethylation frequency of MIR-129-2 and SEMA3B (up to 45 to 48%) in both BC (69 samples) and OC (41 samples). Moreover, we observed a positive correlation between the hypermethylation of MIR-129-2 and the up-regulation of the RASSF1(A) and GPX1 genes in BC (rs = 0.38 and 0.42, respectively). QPCR analysis of the expression of RASSF1(A) and mature miR-129-2 in additional BC sample set (24 samples) revealed a negative correlation between them (rs = − 0.41) that strengthened the results obtained during the analysis of miR-129-2 precursor level. In summary, the obtained data indicate the involvement of methylation in the down-regulation of the studied coding and miRNA genes and suggest the involvement of miR-129-2 in the deregulation of RASSF1(A) via a direct interaction or/and mediators in common pathways (according to KEGG, Gene Ontology (FDR < 0.01), and GeneCards data) in the examined gynecological tumors.