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

Inhibition of tumor growth by <i>SEMA3B</i> re-expression.

<p>The growth rate of U2020 cells (U7111 clone) in SCID mice: blue line—U2020 cells without <i>SEMA3B</i> expression (+ doxycycline, 4 mice), red and yellow line—U2020 cells with <i>SEMA3B</i> expression (- doxycycline, 4 mice and 1 mouse respectively). *—no expression of <i>SEMA3B</i> gene according to the Northern blot (data not shown). One +dox and one—dox mice were withdrawn from the study after one month.</p

Relative mRNA level of the <i>SEMA3B</i> gene in NSCLC (A) and ccRCC (B).

<p>QPCR data, additional samplings. Light grey columns—samples without metastases, dark grey columns—samples with lymph node or distant metastases. The numbers of primary tumors correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123369#pone.0123369.s001" target="_blank">S1 Table</a>. Mean values ± standard deviations for 3 replicates are represented.</p

<i>SEMA3B</i> gene expression level (A), copy number (C) and methylation status of its two CpG-islands (B) in the same ccRCC samples.

<p>Semi-quantitative PCR (A, C) and MSP (B) data. Numbers of primary tumors correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123369#pone.0123369.s001" target="_blank">S1 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123369#pone.0123369.g005" target="_blank">Fig 5B</a>. (A) Light grey columns—samples without metastases, dark grey columns—samples with lymph node or distant metastases. (B) 1-st CpG—promoter CpG-island, 2-nd CpG—intronic CpG-island. Grey squares show methylated CpG-islands, white squares—unmethylated. (C) Grey squares show hemi- or homozygous deletions of the 5’Sema5 marker, black—amplification, white squares—retention. Assessed mean values ± error bars are represented in the “A” part.</p

Absence of <i>SEMA3B</i> expression in tumors grown <i>in vivo</i>.

<p>Electropherogram of multiplex PCR from plasmids, clones and SCID mice tumors of three genes. M—marker, 1—PCR from plasmid pETE/<i>SEMA3B</i>, 2—PCR from plasmid pETE/<i>TUSC2</i>, 3—PCR from plasmid pETE/<i>ZMYND10</i>, 4—PCR from U7111/<i>SEMA3B</i> cell clone 1, 5—PCR from U7111/<i>TUSC2</i> cell clone 3, 6—PCR from U7111/<i>ZMYND10</i> cell clone 4, 7—mixed cell clones, 8—PCR from tumor 1, 9—PCR from tumor 2, 10—PCR from tumor 3, 11—negative control.</p

Methylation profile of the promoter CpG-island of the <i>SEMA3B</i> gene in lung (A) and renal (B) cancer cell lines and primary tumors.

<p>Bisulfite sequencing data, 16 CpG-dinucleotides (2–17) of the CpG-island are given. Grey squares show methylated CpG-dinucleotides, white squares—unmethylated. Numbers of primary tumors correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123369#pone.0123369.s001" target="_blank">S1 Table</a>. The bold numbers of CpG-dinucleotides (3–4 and 9–12) indicate the location of the primers that were used for MSP method.</p

Correlations between the frequency of CpG-island methylation and tumor stage or grade.

<p>Note: Based on MSP data. 1-st CpG—promoter CpG-island; 2-nd CpG—intronic CpG-island; <i>r</i>_<i>s</i>—Spearman’s rank correlation coefficient; <i>P</i>—p-value.</p><p>Correlations between the frequency of CpG-island methylation and tumor stage or grade.</p

Pathological and histological characteristics of the tumors.

<p>Note: The slash separates the number of samples used in the methylation studies and the expression or copy number studies by semi-quantitative RT-PCR and the number of samples used in the qPCR expression studies.</p><p>Pathological and histological characteristics of the tumors.</p

<i>In vitro</i> growth of U2020 cells (U7111 clone) depends on the expression of <i>SEMA3B</i>.

<p>A—The growth rate of U2020 cells: dashed line with squares—U7111 clone with pETE/<i>SEMA3B</i>, solid line with circles—U2020 cells with pETE (control); B—colony formation assay; C—PI-FACS analysis of cells with and without expression of <i>SEMA3B</i>. Mean values ± standard deviations for 4 replicates are represented in each case.</p

Frequency and mRNA level changes of the <i>SEMA3B</i> gene in NSCLC (ADC and SCC) and ccRCC in groups of samples with different pathological and histological characteristics.

<p>Note: QPCR data. <i>R</i>—relative mRNA level.</p><p>*—in parentheses a range of mRNA level changes is shown.</p><p>Frequency and mRNA level changes of the <i>SEMA3B</i> gene in NSCLC (ADC and SCC) and ccRCC in groups of samples with different pathological and histological characteristics.</p