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

High Mutability of the Tumor Suppressor Genes RASSF1 and RBSP3 (CTDSPL) in Cancer

BACKGROUND:Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas. METHODOLOGY/PRINCIPAL FINDINGS:We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1-2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3-5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines. CONCLUSIONS/SIGNIFICANCE:This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread

Positional cloning of tumor suppressor genes from 3p21.3 involved in major human cancers

We have performed a comprehensive deletion survey of 3p on more than 400 lung, renal, breast, cervical and ovarian carcinomas (major epithelial cancers) using a defined set of markers, combining conventional LOH with quantitative real-time PCR (QPCR), NotI linking and jumping libraries, comparative genomic and NotI microarrays hybridizations. We identified two most frequently affected 3p21.3 regions, LUCA (LUng CAncer) at the centromeric and AP20 at the telomeric border of 3p21.3. Aberrations of either region were detected in more than 90% of the studied tumors suggesting they harbor multiple tumor suppressor genes (TSG). Homozygous deletions (HD) were frequently detected in all tumors in both the LUCA and AP20 regions. To facilitate the identification of tumor suppressor genes (TSGs) in the chromosome 3p21.3 AP20 and LUCA sub-regions, we constructed physical and gene map of these segments. More than 30 genes were localized in these two regions and among them at least 12 TSGs were identified: RBSP3, ITGA9, MLH1, VILL, APRG1, RASSF1, HYAL1, HYAL2, SEMA3B, SEMA3F, NPRL2 and CACNA2D2. Among these TSGs were representatives of new types of TSGs: e.g. HYAL1 showing growth inhibiting activity only in vivo and RASSF1A and RASSF1C that are alternative forms of the same gene showing different tissue specificity. We found that several tumor suppressor genes in AP20 and LUCA 3p21.3 regions were co-regulated in tumors. These results supported the hypothesis on simultaneous inactivation of clusters cancer-causing genes in AP20 and LUCA regions during the development and progression of lung cancer and other epithelial tumors. Moreover we found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) in major epithelial tumors. These mutations functionally inactivated tumor suppressor activity of these genes. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread. The data could be important for development of specific biomarker sets for early cancer diagnosis and new therapeutic approaches/strategies for cancer treatment. For example we selected a set of 23 markers (BHLHB2, FBLN2, EPHB1, GATA2, GORASP1, PRICKLE2, Hmm61490, ITGA9, LOC285205, LRRC3B, MINA, MITF, MRPS17P3, NKIRAS1, PLCL2, TRH, UBE2E2, WNT7A, RARB2, p20-CGGBP, GNAI2, RPL32, THRB) that would discriminate/diagnose the majority of NSCLC cases with P > 95% and most complicated cases with a probability of more than 80%

Epigenetic analysis of childhood acute lymphoblastic leukemia

We used a chromosome 3 wide NotI microarray for identification of epigenetically inactivated genes in childhood acute lymphoblastic leukemia (ALL). Three novel genes demonstrated frequent methylation in childhood ALL. PPP2R3A (protein phosphatase 2, regulatory subunit B'', a) was frequently methylated in T (69%) and B (82%)-ALL. Whilst FBLN2 (fibulin 2) and THRB (thyroid hormone receptor, beta) showed frequent methylation in B-ALL (58%; 56% respectively), but were less frequently methylated in T-ALL (17% for both genes). Recently it was demonstrated that BNC1 (Basonuclin 1) and MSX1 (msh homeobox 1) were frequently methylated across common epithelial cancers. In our series of childhood ALL BNC1 was frequently methylated in both T (77%) and B-ALL (79%), whilst MSX1 showed T-ALL (25%) specific methylation. The methylation of the above five genes was cancer specific and expression of the genes could be restored in methylated leukemia cell lines treated with 5-aza-2'-deoxycytidine. This is the first report demonstrating frequent epigenetic inactivation of PPP2R3A, FBLN2, THRB, BNC1 and MSX1 in leukemia. The identification of frequently methylated genes showing cancer specific methylation will be useful in developing early cancer detection screens and for targeted epigenetic therapies

NotI linking/jumping clones of human chromosome 3: mapping of the TFRC, RAB7 and HAUSP genes to regions rearranged in leukemia and deleted in solid tumors

AbstractBy applying the `recognition mask' strategy to 300 mammalian sequences containing NotI sites we demonstrated that 5′ ends of genes are highly enriched in NotI sites. A NotI linking clone NL2-252 (D3S1678) containing transferrin receptor (TFRC) gene was used as an initial point for chromosomal jumping. One of the jumping clones, J21-045 traverses 210 kbp and links NL2-252 to NL26 (D3S1632), a NotI linking clone containing highly polymorphic sequences. The TFRC gene was mapped to 3q29, close to the telomeric marker D3S2344, by linkage analysis, a panel of hybrid cell lines, GeneBridge 4 panel and FISH. Clone NLM-007 (D3S4302) was found to contain ras-homologous gene RAB7. By FISH and a panel of hybrid cell lines this gene was mapped to 3q21. This region is of particular interest due to frequent rearrangements in different types of leukemia. Clone L2-081 (D3S4283) containing new member of ubiquitin-specific proteases (HAUSP gene) was localized in 3p21 inspiring further investigation of involvement of this gene in development of lung and renal carcinomas

NotI clones in the analysis of the human genome

NotI linking clones contain sequences flanking NotI recognition sites and were previously shown to be tightly associated with CpG islands and genes. To directly assess the value of NotI clones in genome research, high density grids with 50 000 NotI linking clones originating from six representative NotI linking libraries were constructed. Altogether, these libraries contained nearly 100 times the total number of NotI sites in the human genome. A total of 3437 sequences flanking NotI sites were generated. Analysis of 3265 unique sequences demonstrated that 51% of the clones displayed significant protein similarity to SWISSPROT and TREMBL database proteins based on MSPcrunch filtering with stringent parameters. Of the 3265 sequences, 1868 (57.2%) were new sequences, not present in the EMBL and EST databases (similarity ≤ 90%). Among these new sequences, 795 (24.3%) showed similarity to known proteins and 712 (21.8%) displayed an identity of >75% at the nucleotide level to sequences from EMBL or EST databases. The remaining 361 (11.1%) sequences were completely new, i.e. <75% identical. The work also showed tight, specific association of NotI sites with the first exon and suggest that the so-called 3′ ESTs can actually be generated from 5′-ends of genes that contain NotI sites in their first exon