Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21 and RASSF1A in primary non-small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>The short arm of human chromosome 3 is involved in the development of many cancers including lung cancer. Three bona fide lung cancer tumor suppressor genes namely <it>RBSP3 </it>(AP20 region),<it>NPRL2 </it>and <it>RASSF1A </it>(LUCA region) were identified in the 3p21.3 region. We have shown previously that homozygous deletions in AP20 and LUCA sub-regions often occurred in the same tumor (P < 10^-6).</p> <p>Methods</p> <p>We estimated the quantity of <it>RBSP3, NPRL2, RASSF1A, GAPDH, RPN1 </it>mRNA and <it>RBSP3 </it>DNA copy number in 59 primary non-small cell lung cancers, including 41 squamous cell and 18 adenocarcinomas by real-time reverse transcription-polymerase chain reaction based on TaqMan technology and relative quantification.</p> <p>Results</p> <p>We evaluated the relationship between mRNA level and clinicopathologic characteristics in non-small cell lung cancer. A significant expression decrease (≥2) was found for all three genes early in tumor development: in 85% of cases for <it>RBSP3</it>; 73% for <it>NPRL2 </it>and 67% for <it>RASSF1A </it>(P < 0.001), more strongly pronounced in squamous cell than in adenocarcinomas. Strong suppression of both, <it>NPRL2 </it>and <it>RBSP3 </it>was seen in 100% of cases already at Stage I of squamous cell carcinomas. Deregulation of <it>RASSF1A </it>correlated with tumor progression of squamous cell (P = 0.196) and adenocarcinomas (P < 0.05). Most likely, genetic and epigenetic mechanisms might be responsible for transcriptional inactivation of <it>RBSP3 </it>in non-small cell lung cancers as promoter methylation of <it>RBSP3 </it>according to NotI microarrays data was detected in 80% of squamous cell and in 38% of adenocarcinomas. With NotI microarrays we tested how often LUCA (<it>NPRL2, RASSF1A</it>) and AP20 (<it>RBSP3</it>) regions were deleted or methylated in the same tumor sample and found that this occured in 39% of all studied samples (P < 0.05).</p> <p>Conclusion</p> <p>Our data support the hypothesis that these TSG are involved in tumorigenesis of NSCLC. Both genetic and epigenetic mechanisms contribute to down-regulation of these three genes representing two tumor suppressor clusters in 3p21.3. Most importantly expression of <it>RBSP3, NPRL2 </it>and <it>RASSF1A </it>was simultaneously decreased in the same sample of primary NSCLC: in 39% of cases all these three genes showed reduced expression (P < 0.05).</p

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

Tumor Suppressor Properties of Small C-Terminal Domain Phosphatases in Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) accounts for 80–90% of kidney cancers worldwide. Small C-terminal domain phosphatases CTDSP1, CTDSP2, and CTDSPL (also known as SCP1, 2, 3) are involved in the regulation of several important pathways associated with carcinogenesis. In various cancer types, these phosphatases may demonstrate either antitumor or oncogenic activity. Tumor-suppressive activity of these phosphatases in kidney cancer has been shown previously, but in general case, the antitumor activity may be dependent on the choice of cell line. In the present work, transfection of the Caki-1 cell line (ccRCC morphologic phenotype) with expression constructs containing the coding regions of these genes resulted in inhibition of cell growth in vitro in the case of CTDSP1 (p CTDSPL (p CTDSP2. The analysis of The Cancer Genome Atlas (TCGA) data showed differential expression of some of CTDSP genes and of their target, RB1. These results were confirmed by quantitative RT-PCR using an independent sample of primary ccRCC tumors (n = 52). We observed CTDSPL downregulation and found a positive correlation of expression for two gene pairs: CTDSP1 and CTDSP2 (rs = 0.76; p CTDSPL and RB1 (rs = 0.38; p CTDSP1, CTDSP2, CTDSPL, and RB1 with poor survival of ccRCC patients (p CTDSP1, CTDSP2, and RB1 were differently expressed in two subtypes of ccRCC—ccA and ccB, characterized by different survival rates. These results confirm that CTDSP1 and CTDSPL have tumor suppressor properties in ccRCC and reflect their association with the more aggressive ccRCC phenotype

HYAL1 and HYAL2 inhibit tumour growth in vivo but not in vitro.

BACKGROUND: We identified two 3p21.3 regions (LUCA and AP20) as most frequently affected in lung, breast and other carcinomas and reported their fine physical and gene maps. It is becoming increasingly clear that each of these two regions contains several TSGs. Until now TSGs which were isolated from AP20 and LUCA regions (e.g.G21/NPRL2, RASSF1A, RASSF1C, SEMA3B, SEMA3F, RBSP3) were shown to inhibit tumour cell growth both in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: The effect of expression HYAL1 and HYAL2 was studied by colony formation inhibition, growth curve and cell proliferation tests in vitro and tumour growth assay in vivo. Very modest growth inhibition was detected in vitro in U2020 lung and KRC/Y renal carcinoma cell lines. In the in vivo experiment stably transfected KRC/Y cells expressing HYAL1 or HYAL2 were inoculated into SCID mice (10 and 12 mice respectively). Tumours grew in eight mice inoculated with HYAL1. Ectopic HYAL1 was deleted in all of them. HYAL2 was inoculated into 12 mice and only four tumours were obtained. In 3 of them the gene was deleted. In one tumour it was present but not expressed. As expected for tumour suppressor genes HYAL1 and HYAL2 were down-expressed in 15 fresh lung squamous cell carcinomas (100%) and clear cell RCC tumours (60-67%). CONCLUSIONS/SIGNIFICANCE: The results suggest that the expression of either gene has led to inhibition of tumour growth in vivo without noticeable effect on growth in vitro. HYAL1 and HYAL2 thus differ in this aspect from other tumour suppressors like P53 or RASSF1A that inhibit growth both in vitro and in vivo. Targeting the microenvironment of cancer cells is one of the most promising venues of cancer therapeutics. As major hyaluronidases in human cells, HYAL1 and HYAL2 may control intercellular interactions and microenvironment of tumour cells providing excellent targets for cancer treatment