7869

SEMA3B

LUCA-1|SEMA5|SEMAA|SemA|semaV;sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3B;SEMA3B;sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3B

sema A(V)|sema V|semaphorin A|semaphorin V|semaphorin-3B|semaphorin-V

3p21.3

protein-coding

Count: 2; Pubmed_search,Generif

The critical region on human chromosome 3p21.3 harboring a putative lung cancer tumor suppressor gene (TSG) was previously defined by allelotyping and recently refined by overlapping homozygous deletions. We report the construction of a 700-kb (cosmid and one P1 phage) clone contig covering the deletion overlap and its flanks. The minimal set of 23 cosmids comprises 600 kb and is extended by one P1 phage to 700 kb to cover the distal breakpoint of the overlap. The clone contig was extensively characterized by restriction and expression mapping to produce high resolution physical and transcription maps of the cloned region. Potential transcribed fragments were detected by hybridization with PCR-amplified cDNA libraries, direct cDNA selection "zoo" blotting, cDNA screening, and identification of 24 CpG islands. Thus far, 15 new genes represented by partial or full-length cDNAs were isolated, characterized, and precisely positioned on the contig. Two previously cloned genes, namely GNAI-2 and GNAT-1, were also positioned. In addition, the telomeric breakpoint of the NCI H740 deletion and centromeric breakpoint of the overlapping GLC20 deletion were discovered and mapped to define precisely the candidate TSG region. This large cosmid clone contig and high resolution maps will prove crucial in the identification of the lung cancer TSG(s).

SEMA3B, a member of class 3 semaphorins, is a tumor suppressor. Competition with vascular endothelial growth factor (VEGF)165 explains a portion of the activity, whereas the VEGF-independent mechanism was not elucidated. We employed a microarray and screened for the genes whose expression was increased by SEMA3B in NCI-H1299 cells. Insulin-like growth factor-binding protein-6 (IGFBP-6), a tumor suppressor, showed greatest difference in the expression level. Introduction of IGFBP-6 cDNA reduced colony formation both on the dish surface and in soft agar. Insulin-like growth factor II, which antagonizes IGFBP-6, partly abrogated the effect. Inhibition of IGFBP-6 by small interfering RNA diminished the sub-G0/G1 population that was induced by SEMA3B and abrogated the growth suppressive effect of SEMA3B. We concluded that IGFBP-6 is the effector of tumor suppressor activity of SEMA3B in NCI-H1299 cells. It has been reported that beta-catenin suppresses the expression of IGFBP-6. Introduction of beta-catenin into the cells partly abrogated the growth suppressive effect of SEMA3B. Our result indicates that semaphorin signaling and beta-catenin signaling converge on IGFBP-6 and antithetically affect their functions.

Semaphorin 3B (SEMA3B), located at 3p21.3, is a secreted member of the semaphorin family important in axonal guidance. SEMA3B undergoes allele and expression loss in lung and breast cancer and can function as a tumor suppressor. Previously, we found that SEMA3B induces apoptosis in tumor cells either by reexpression or when applied as a soluble ligand. SEMA3B-induced apoptosis was mediated, in part, by blocking vascular endothelial growth factor autocrine activity in tumor cells. In the current study, treatment of lung and breast cancer cells with picomolar concentrations of soluble SEMA3B inhibited their growth; induced apoptosis; and was associated with decreased Akt phosphorylation, increase in cytochrome c release and caspase-3 cleavage, as well as increased phosphorylation of several proapoptotic proteins, including glycogen synthase kinase-3beta, FKHR, and MDM-2. Lung and breast cancer lines resistant to SEMA3B did not show these signaling changes and a tumor-derived missense SEMA3B mutant was inactive in this regard, providing specificity. SEMA3B-mediated inhibition of proliferation and induction of apoptosis in cancer cells were blocked by expressing a constitutively active Akt mutant and are linked to tumor cell expression of neuropilin-1 (Np-1). SEMA3B-insensitive Np-1-negative tumor cells acquired sensitivity to SEMA3B after forced expression of Np-1, whereas SEMA3B-sensitive Np-1-positive tumor cells lost sensitivity to SEMA3B after knockdown of Np-1 by small interfering RNA. We conclude that SEMA3B is a potential tumor suppressor that induces apoptosis in SEMA3B-inactivated tumor cells through the Np-1 receptor by inactivating the Akt signaling pathway. CA118384

Semaphorins are a large family of evolutionarily conserved morphogenetic molecules originally identified for their repelling role in axonal guidance. Intriguingly, semaphorins have recently been implicated in cancer progression (Neufeld, G., T. Lange, A. Varshavsky, and O. Kessler. 2007. Adv. Exp. Med. Biol. 600:118-131). In particular, semaphorin 3B (SEMA3B) is considered a putative tumor suppressor, and yet we found that it is expressed at high levels in many invasive and metastatic human cancers. By investigating experimental tumor models, we confirmed that SEMA3B expression inhibited tumor growth, whereas metastatic dissemination was surprisingly increased. We found that SEMA3B induced the production of interleukin (IL) 8 by tumor cells by activating the p38-mitogen-activated protein kinase pathway in a neuropilin 1-dependent manner. Silencing the expression of endogenous SEMA3B in tumor cells impaired IL-8 transcription. The release of IL-8, in turn, induced the recruitment of tumor-associated macrophages and metastatic dissemination to the lung, which could be rescued by blocking IL-8 with neutralizing antibodies. In conclusion, we report that SEMA3B exerts unexpected functions in cancer progression by fostering a prometastatic environment through elevated IL-8 secretion and recruitment of macrophages coupled to the suppression of tumor growth.

Large-scale hemizygous loss of chromosome 3p is a common event in neuroblastoma, occurring preferentially in tumors that exhibit loss of chromosome 11q and lack MYCN amplification. Although numerous tumor suppressor genes (TSG) have been mapped to the 3p region, the gene or genes contributing to neuroblastoma pathogenesis have remained elusive. High-resolution oligonucleotide array CGH mapping of chromosome 3p breakpoints relative to the positions of known TSGs indicates that more than one gene may contribute to neuroblastoma pathogenesis. We evaluated the methylation status of semaphorin 3B (SEMA3B), one of the chromosome 3p TSGs, in neuroblastoma tumors with (n = 12) and without (n = 32) 3p deletions. A significantly higher percentage of methylated CpG sites in the SEMA3B promoter was detected in tumors exhibiting 3p loss (95%), relative to tumors without loss (52%), suggestive of a two-hit mechanism of allele inactivation. The involvement of methylation in the control of SEMA3B expression was confirmed by treatment of neuroblastoma cell lines with the demethylating agent 5-aza-2-deoxycytidine. Transcriptional regulation of this locus is complex, however; low levels of SEMA3B expression were also seen in tumors with unmethylated SEMA3B promoters (n = 4). SEMA3B is known to play an important role in the development of normal sympathetic neurons, and interestingly, we found higher levels of SEMA3B expression in differentiated tumors with favorable histopathology (n = 19) than in tumors with unfavorable histology (n = 22). Furthermore, SEMA3B was upregulated in the SK-N-BE neuroblastoma cell line following induction of differentiation with retinoic acid. The association of SEMA3B expression with neuroblastoma differentiation suggests that this TSG may play a role in neuroblastoma pathobiology.

Semaphorins SEMA3B and its homologue SEMA3F are 3p21.3 candidate tumor suppressor genes (TSGs), the expression of which is frequently lost in lung cancers. To test the TSG candidacy of SEMA3B and SEMA3F, we transfected them into lung cancer NCI-H1299 cells, which do not express either gene. Colony formation of H1299 cells was reduced 90% after transfection with wild-type SEMA3B compared with the control vector. By contrast, only 30-40% reduction in colony formation was seen after the transfection of SEMA3F or SEMA3B variants carrying lung cancer-associated single amino acid missense mutations. H1299 cells transfected with wild-type but not mutant SEMA3B underwent apoptosis. We found that lung cancers (n = 34) always express the neuropilin-1 receptor for secreted semaphorins, whereas 82% expressed the neuropilin-2 receptor. Because SEMA3B and SEMA3F are secreted proteins, we tested conditioned medium from COS-7 cells transfected with SEMA3B and SEMA3F and found that medium from wild-type SEMA3B transfectants reduced the growth of several lung cancer lines 30-90%, whereas SEMA3B mutants or SEMA3F had little effect in the same assay. Sequencing of sodium bisulfite-treated DNA showed dense methylation of CpG sites in the SEMA3B 5' region of lung cancers not expressing SEMA3B but no methylation in SEMA3B-expressing tumors. These results are consistent with SEMA3B functioning as a TSG, the expression of which is inactivated frequently in lung cancers by allele loss and promoter region methylation.

We used overlapping and nested homozygous deletions, contig building, genomic sequencing, and physical and transcript mapping to further define a approximately 630-kb lung cancer homozygous deletion region harboring one or more tumor suppressor genes (TSGs) on chromosome 3p21.3. This location was identified through somatic genetic mapping in tumors, cancer cell lines, and premalignant lesions of the lung and breast, including the discovery of several homozygous deletions. The combination of molecular manual methods and computational predictions permitted us to detect, isolate, characterize, and annotate a set of 25 genes that likely constitute the complete set of protein-coding genes residing in this approximately 630-kb sequence. A subset of 19 of these genes was found within the deleted overlap region of approximately 370-kb. This region was further subdivided by a nesting 200-kb breast cancer homozygous deletion into two gene sets: 8 genes lying in the proximal approximately 120-kb segment and 11 genes lying in the distal approximately 250-kb segment. These 19 genes were analyzed extensively by computational methods and were tested by manual methods for loss of expression and mutations in lung cancers to identify candidate TSGs from within this group. Four genes showed loss-of-expression or reduced mRNA levels in non-small cell lung cancer (CACNA2D2/alpha2delta-2, SEMA3B [formerly SEMA(V), BLU, and HYAL1] or small cell lung cancer (SEMA3B, BLU, and HYAL1) cell lines. We found six of the genes to have two or more amino acid sequence-altering mutations including BLU, NPRL2/Gene21, FUS1, HYAL1, FUS2, and SEMA3B. However, none of the 19 genes tested for mutation showed a frequent (>10%) mutation rate in lung cancer samples. This led us to exclude several of the genes in the region as classical tumor suppressors for sporadic lung cancer. On the other hand, the putative lung cancer TSG in this location may either be inactivated by tumor-acquired promoter hypermethylation or belong to the novel class of haploinsufficient genes that predispose to cancer in a hemizygous (+/-) state but do not show a second mutation in the remaining wild-type allele in the tumor. We discuss the data in the context of novel and classic cancer gene models as applied to lung carcinogenesis. Further functional testing of the critical genes by gene transfer and gene disruption strategies should permit the identification of the putative lung cancer TSG(s), LUCA, Analysis of the approximately 630-kb sequence also provides an opportunity to probe and understand the genomic structure, evolution, and functional organization of this relatively gene-rich region.