Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor SEMA3B

Yoshio Tomizawa*†, Yoshitaka Sekido*‡, Masashi Kondo*, Boning Gao*, Jun Yokota†, Joe¨ lle Roche§, Harry Drabkin¶, Michael I. Lermanʈ, Adi F. Gazdar*, and John D. Minna*,**

*Hamon Center for Therapeutic Oncology Research, Departments of Internal Medicine Pharmacology or Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390; †Biology Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan; ‡Department of Clinical Preventive Medicine, Nagoya University School of Medicine, Nagoya 466-8550, Japan; ʈLaboratory of Immunobiology, Center for Cancer Research, National Cancer Institute–Frederick, Frederick, MD 21702; §Institut de Biologie Moleculaire et d’Inge´nierie, Centre National de la Recherche Scientifique FRE 2224, Universite´de Poitiers, Poitiers 86022, France; and ¶The Division of Medical Oncology, University of Colorado Health Sciences Center, Denver, CO 80262

Communicated by Alfred G. Gilman, University of Texas Southwestern Medical Center, Dallas, TX, September 18, 2001 (received for review May 29, 2001) Semaphorins SEMA3B and its homologue SEMA3F are 3p21.3 lung, marking it as one of the first sites involved (6). Two of the candidate tumor suppressor (TSGs), the expression of which 19 genes are semaphorin family members (SEMA3B and is frequently lost in lung cancers. To test the TSG candidacy of SEMA3F) lying Ϸ70 kb apart (7, 8). In assessing the TSG SEMA3B and SEMA3F, we transfected them into lung cancer candidacy of SEMA3B and SEMA3F, we found only a few NCI-H1299 cells, which do not express either gene. Colony forma- mutations but frequent loss of expression of SEMA3B mRNA tion of H1299 cells was reduced 90% after transfection with (occurring in Ϸ80% of lung cancers). We and others found no wild-type SEMA3B compared with the control vector. By contrast, SEMA3F mutations, and loss of SEMA3F expression occurred in only 30–40% reduction in colony formation was seen after the 18% of these same lung cancers (8, 9). However, recent immu- transfection of SEMA3F or SEMA3B variants carrying lung cancer- nohistochemical studies of lung cancers, found reduction of associated single amino acid missense mutations. H1299 cells SEMA3F expression in higher stages of lung cancer, and a transfected with wild-type but not mutant SEMA3B underwent change in SEMA3F localization from the membrane to the always express the cytoplasm compared with normal lung epithelium (10). In (34 ؍ apoptosis. We found that lung cancers (n neuropilin-1 receptor for secreted semaphorins, whereas 82% addition, functional studies using a P1 clone containing SEMA3F expressed the neuropilin-2 receptor. Because SEMA3B and SEMA3F (and potentially SEMA3B) showed a tumor-suppressive effect are secreted , we tested conditioned medium from COS-7 for mouse A9 fibrosarcoma cells (11). Recent studies have cells transfected with SEMA3B and SEMA3F and found that me- implicated tumor-acquired promoter hypermethylation as a dium from wild-type SEMA3B transfectants reduced the growth of mechanism of inactivation of mRNA expression of TSGs in the several lung cancer lines 30–90%, whereas SEMA3B mutants or pathogenesis of several human cancers (12). In fact, we and SEMA3F had little effect in the same assay. Sequencing of sodium others have found that one isoform at the RASSF1 , bisulfite-treated DNA showed dense methylation of CpG sites in RASSF1A, located Ϸ60 kb centromeric of SEMA3B, underwent the SEMA3B 5؅ region of lung cancers not expressing SEMA3B but tumor-acquired promoter methylation, leading to inactivated no methylation in SEMA3B-expressing tumors. These results are expression in lung and breast tumors (13, 14). Because of the consistent with SEMA3B functioning as a TSG, the expression of mutations and the functional studies, we also needed to study the which is inactivated frequently in lung cancers by allele loss and semaphorin genes. Here we demonstrate that lung cancers promoter region methylation. express semaphorin receptors, exhibit 5Ј CpG island SEMA3B methylation, and reexpression of exogenous wild-type SEMA3B semaphorin ͉ neuropilin ͉ methylation but not SEMA3F or lung cancer-related SEMA3B mutations induce apoptosis in lung cancers. Furthermore, conditioned he semaphorin family comprised of secreted and membrane- medium from COS-7 cells transfected with wild-type but not Tassociated proteins contributes to axonal path-finding during mutant SEMA3B genes also suppresses lung cancer growth. neural development by repulsing axons, inhibiting growth cone These results suggests that SEMA3B can function as an epige- extension, and causing collapse of growth cones (1–3). The SEMA3 netically inactivated potent suppressor of lung cancer growth. family members encode secreted proteins that signal through Materials and Methods binding to neuropilin receptors (NP) interacting with plexins (1–3). ؅ Several semaphorins are expressed in adult nonneuronal tissues, Analysis of CpG Methylation of the SEMA3B 5 Region. Genomic suggesting other functions. For example, SEMA3A inhibited the DNAs from lung cancer cell lines not expressing SEMA3B motility of aortic endothelial cells expressing NP1, disrupted the (NCI-H209, H524, H1299, and H661) or expressing SEMA3B formation of lamellipodia, induced depolymerization of F-actin (4), (H2009 and H1666; ref. 8) were modified by sodium bisulfite treatment as described (15). Treated DNAs were PCR-amplified and inhibited branching morphogenesis in the fetal mouse lung (5). Ј However, the roles of SEMA3B and SEMA3F in nonneuronal cells with the primers M2AS (5 -TAACCCTAAAAATATACCCA- and human cancer are unknown. The loss of heterozygosity of 3p sequences is a Abbreviations: NP, neuropilin receptor; TSG, tumor suppressor gene; SCLC, small cell lung critical event in the pathogenesis of lung and other cancers and cancer; NSCLC, non-SCLC; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP directed a tumor suppressor gene (TSG) search to this region. nick end labeling; VEGF, vascular endothelial growth factor. Multiple distinct 3p regions are involved in human lung cancer **To whom reprint requests should be addressed at: Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines pathogenesis including one at 3p21.3 where we identified 19 Boulevard, NB8-206, Dallas, TX 75390-8593. E-mail: [email protected]. candidate TSGs (6, 7). This defined 3p21.3 region undergoes Ϸ Ϸ The publication costs of this article were defrayed in part by page charge payment. This allele loss in 80% of primary lung cancers and 40% of article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. preneoplastic or normal epithelial samples of smoking-damaged §1734 solely to indicate this fact.

13954–13959 ͉ PNAS ͉ November 20, 2001 ͉ vol. 98 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.231490898 Downloaded by guest on September 29, 2021 sequences confirmed through the PCR-manipulated regions, and all produced appropriately sized peptides detected in West- ern blotting by specific anti-SEMA3B or SEMA3F antibodies after transfection.

Transfection and Colony Formation Assays. Transfections with DM- RIE-C reagent (Life Technologies GIBCO) used 2 ␮g of each plasmid per 10-cm dish containing 5 ϫ 105 cells seeded 24 h before transfection. Transfections were terminated at 5 h; 48 h after transfection, 5 ϫ 104 transfected cells were seeded and maintained in RPMI medium 1640 10% fetal bovine serum supplemented with 800 ␮g͞ml G418 (Life Technologies GIBCO). Surviving colonies were counted 12–21 days later after staining with methylene blue.

Antibodies and Western-Blot Analysis. The peptides corresponding to amino acid residues Thr-732 to Trp-749 of human SEMA3B (U28369) and Pro-722 to Lys-742 of SEMA3F (U38276) were synthesized, and three rabbits were immunized with each peptide (Alpha Diagnostic, San Antonio, TX). SEMA3B antisera were Fig. 1. Methylation analysis of SEMA3B.(A) Sequence of the region that was purified on immunoaffinity columns in which the peptide was subjected to analysis. The positions of CpG sites are numbered 1–8. Primers covalently linked to an Amino Link column (Pierce). Anti-NP1 used for amplification are indicated by arrows. (B) Methylation status of each and -NP2 rabbit polyclonal antibodies were provided by Dr. A. CpG site (labeled 1–8inA) determined by sequencing sodium bisulfite-treated Kolodkin. Cellular proteins were extracted from 106 cells with 40 genomic DNA from tumor cells. White and black squares represent unmeth- ␮l of lysis buffer (40 mM Hepes-NaOH, pH 7.4, 1% Nonidet ylated and methylated CpGs, respectively. Partially filled squares represent partially methylated CpG. P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and 10 ␮g͞ml aprotinin). Fifty ␮g of total per lane was separated on an 8% SDS͞polyacrylamide gel and electroblotted 3Ј) and M1S (5Ј-TATTTTAGTAGTTTAGGGTG-3Ј) target- to nitrocellulose membranes (Bio-Rad). After blocking with 5% ing a 269-bp sequence with multiple CpG sites immediately 5Ј of nonfat dry milk and 0.1% Tween 20 in Tris-buffered saline, the SEMA3B ATG. Note that primers M2AS and M1S are membranes were incubated at 37°Cfor2hwithanti-NP1, designed to amplify the opposite strand of sodium bisulfite- anti-NP2 (18), anti-SEMA3B, or anti-SEMA3F antibodies. The treated DNA promoter sequence shown in Fig. 1. PCR cycling membranes then were developed with peroxidase-labeled anti- conditions consisted of 12 min at 95°C followed by 40 cycles of rabbit IgG (Amersham Pharmacia) by Super Signal chemilumi- 30 sec denaturation at 94°C, 30 sec of annealing at 50°C, 30 sec nescence substrate (Pierce). Equal loading of protein was con-

of extension at 72°C, with final extension at 72°C for 10 min. We firmed after detection by staining the membrane with amido MEDICAL SCIENCES reamplified and sequenced the PCR product to obtain CpG black 10B (Sigma). methylation levels (Fig. 1). Cell Cycle Analysis. Cells were harvested 48 h after the transfec- ͞ Analysis of Primary Lung Cancer Samples for SEMA3B Mutations. tion, fixed with 50% ethanol, treated with 5 mg ml RNase A (Roche Molecular Biochemicals), stained with 50 ␮g͞ml pro- Forty-six primary lung tumors [nine small cell lung cancers pidium iodide, and analyzed by flow cytometry for DNA syn- (SCLCs) and 37 non-SCLCs (NSCLCs) selected pathologically thesis and cell cycle status (FACSCaliber instrument, Becton to contain Ն90% tumor tissue] and corresponding noncancerous Dickinson, with CELLQUEST software). tissues were obtained from the National Cancer Center Hospital (Tokyo, Japan), and genomic DNA was prepared (16). Seven- Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick End La- teen genomic DNA fragments covering the entire coding region beling (TUNEL) and Caspase-3 Apoptosis Assays. Cells were fixed 24 h of SEMA3B were amplified by PCR with SEMA3B-specific ͞ after the transfection with 4% paraformaldehyde (Sigma) solu- oligonucleotide primers using exon intron information from tion in PBS for1hatroom temperature, treated with 0.3% cDNA (U28369) and genomic (U73167) sequences and sub- H2O2-methanol solution, and then permeabilized with 0.1% jected to single-stranded conformation polymorphism analysis, Triton X-100 in 0.1% sodium citrate solution for 2 min on ice. and abnormal bands were sequenced. (Primers and conditions The TUNEL assay (Roche Molecular Biochemicals) was carried are available on request.) out following the manufacturer’s instruction. Caspase-3 activity was measured with an Apoalert Caspase-3 colorimetric kit Cell Lines. Lung cancer cell lines (8, 17) were propagated in RPMI (CLONTECH). Cells (5 ϫ 105) were transfected with empty or medium 1640 (Life Technologies GIBCO) supplemented with SEMA3B plasmids by lipofection. After 20 h, cell lysates were ͞ 10% fetal bovine serum. Normal human bronchial tracheal prepared and preincubated with or without the caspase inhibitor epithelial cells (Clonetics, San Diego) were propagated in Clo- DEVD-fmk (CLONTECH) for 30 min. Then, DEVD-pNA, the netics growth medium. caspase-3 substrate, was added to the samples and incubated for 1 h, and the product was measured at 400 nm. Expression Plasmids. The expression plasmids pcDNA3-SEMA3B (pCB11, pSEMA3B) and pcDNA3-SEMA3B antisense (pCB14, Results pSEMA3B-Antisense) (8) and site-directed mutagenesis (Strat- Methylation Status of CpG Sites in SEMA3B Nonexpressing Lung agene) were used to make the mutant SEMA3B constructs Cancers. We determined the CpG methylation status in the 5Ј (pSEMA3B-R348C, pSEMA3B-D397H, pSEMA3B-T415I, and region of SEMA3B by sequencing sodium bisulfite-modified pSEMA3B-D561N) containing lung cancer single amino acid DNA from four lung cancer cell lines not expressing SEMA3B as missense mutations (8). A SEMA3F pcDNA3 expression con- well as two lung cancer lines expressing SEMA3B (Fig. 1). All the struct (pSEMA3F) was also made. All constructs had their SEMA3B nonexpressing tumor cell lines exhibited methylation

Tomizawa et al. PNAS ͉ November 20, 2001 ͉ vol. 98 ͉ no. 24 ͉ 13955 Downloaded by guest on September 29, 2021 Fig. 2. Expression of neuropilin peptides in lung cancer cell lines by Western blotting (50 ␮g of total cell lysate per lane). NCI-H1299, H23, and H322 express NP1 but not NP2, whereas H661, H2227, H358, and H2009 express both NP1 and NP2. Not shown are: SCLC lines expressing NP1 and NP2 NCI-H187, H209, H345, H378, H524, H526, H740, H865, H889, H1045, H1092, H1238, H1514, H1618, H1672, H2141, H2171, and H2227; and NSCLC lines expressing NP1 and NP2 H358, H838, H1437, H1792, H2009, H2077, H661, H2106, and H28 (me- sothelioma). Lung cancer lines expressing NP1 but not NP2 are H1666, H460, and H2052 (mesothelioma).

of almost all CpG dinucleotides in this region. The two tumor cell lines that did express SEMA3B were either not methylated at these CpG sites or else showed a single CpG site with a mixed methylation pattern.

Additional Mutations Found in SEMA3B in Primary Lung Tumors. Genomic DNA from 46 primary lung tumors were examined for mutations in SEMA3B by PCR͞single-strand conformational polymorphism analysis and direct DNA sequencing (data not shown). An acquired mutation in SEMA3B (nucleotide G1916A substitution leading to a D561N amino acid change in the semaphorin domain of SEMA3B) was detected in one of nine primary SCLCs (but not the normal tissue), and germline changes T415I, and a G-to-A nucleotide substitution at the intron side of the exon 15͞intron boundary were found in one NSCLC each. All three were associated with a loss of the wild-type alleles in tumors. As discussed below, T415I and D561N lead to loss of SEMA3B growth-suppressing function.

Expression of Neuropilins in Lung Cancer Cell Lines. Because SEMA3 family members act through NPs (and plexin coreceptors; refs. Fig. 3. The effect of exogenous expression of SEMA3B and SEMA3F on the 19 and 20), we examined the expression of NP1 and NP2 proteins colony formation of H1299 NSCLC cells. (A) Western-blot analysis of NSCLC H1299 cells transfected with various SEMA3B plasmids; (B) Western blot of in 18 SCLC, 14 NSCLC, and 2 mesothelioma cell lines in which H1299 cells transfected with SEMA3F; (C and D) H1299 colony formation after the expression of SEMA3B and SEMA3F mRNAs were also transfection and G418 selection. In A and B, plasmids were transfected into 5 ϫ known (Fig. 2; ref. 8). NP1 was expressed strongly in all 34 cancer 105 H1299 cells by lipofection, cells were harvested 48 h later, and 50 ␮gof cell lines, whereas NP2 was expressed to varying degrees in all total lysate was Western-blotted with anti-SEMA3B (A) and anti-SEMA3F SCLCs, nine NSCLCs, and one mesothelioma. We found no antisera (B). (C) colony formation after transfection and selection with G418 correlation of the expression patterns of SEMA3B, SEMA3F, stained with methylene blue. Vector Control, pcDNA3; AS, pcDNA3 with and the neuropilins. wild-type SEMA3B in antisense direction; SEMA3BWT, SEMA3B wild type; SEMA3F, SEMA3F wild type; R348C, D397H, and T415I, SEMA3B constructs with indicated mutations introduced; p53, pcDNA3 with wild-type p53. (D) Inhibition of Colony Formation by SEMA3B in Lung Cancer Cell Lines. Quantitation of the number of G418-selected H1299 colonies. The vector To test for the growth-suppressive effect of ectopically expressed control was set at 100%. The data represent the mean Ϯ SD of five indepen- semaphorins, we performed colony formation assays selecting dent experiments, each done in triplicate plates. The D561N data are not for the neo gene carried by our expression plasmids. Plasmids shown. containing the full open reading frame of SEMA3B, anti-sense SEMA3B, the full open reading frame of SEMA3F, and four SEMA3B constructs carrying lung cancer associated missense p53 control dramatically suppressed colony formation (Fig. 3 C mutations were transfected into NSCLC NCI-H1299 cells, which and D). In addition, the number of G418-resistant colonies after do not express endogenous SEMA3B or SEMA3F (8). A positive transfection with wild-type SEMA3B was reduced 90% com- control was wild-type p53, which is known to inhibit the growth pared with transfection with the control vector in five indepen- and induce apoptosis of H1299 cells, which contain a homozy- dent experiments by using three independent plasmid DNA gous deletion of p53 (21). The wild-type SEMA3B and SEMA3F preparations (Fig. 3 C and D). By contrast, four separate and mutant SEMA3B expression constructs all produced equiv- SEMA3B missense mutations had lost most of this colony- alent amounts of protein after transient transfection detected by suppressing activity despite robust protein expression (Fig. 3 C using affinity-purified anti-SEMA3B or anti-SEMA3F antibod- and D). As a further control, we moved SEMA3B from pcDNA3 ies (Fig. 3 A and B). Forty-eight hours after transfection, cells to the pcDNA3.1 vector and still saw the same degree of were selected with geneticin (G418), and resistant colonies suppression of colony formation. We also tested the growth- developing 12 days later were stained. As expected, the wild-type suppressive effect of SEMA3F and found the number of colonies

13956 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.231490898 Tomizawa et al. Downloaded by guest on September 29, 2021 Table 1. G418-resistant colony formation after transfection of a SEMA3B-neo expression plasmid into lung cancer cell lines with various SEMA3B, NP1, and NP2 expression patterns G418-resistant colony NCI lung Expression of: formation,* % of cancer cell vector control lines SEMA3B Npn1 Npn2 transfections

H23 (NSCLC) ϪϩϪ 71 Ϯ 5 H1299 (NSCLC) ϪϩϪ 10 Ϯ 1 H2227 (SCLC) Ϫϩϩ 10 Ϯ 4 H661 (NSCLC) Ϫϩϩ 4 Ϯ 4 H322 (NSCLC) ϪϩϪ 3 Ϯ 1 H2009 (NSCLC) ϩϩϩϩ ϩ ϩ 1 Ϯ 1 H358 (NSCLC) ϩϩ ϩ ϩ 0 Ϯ 0

*Values are the mean Ϯ SD of three separate experiments, each calculated from counting colonies on triplicate plates. In each case the pcDNA3.1 (neo) vector control-transfected number of G418-resistant colonies was set at 100%. SEMA3B expression is from Sekido et al. (8). Note that H358 carries a D397H SEMA3B mutation.

after transfection with SEMA3F was only slightly different (70 Ϯ 17%) than the vector control (Fig. 3 C and D). The numbers of colonies were reduced also after transfection with SEMA3B into many other NSCLC lines, whereas little reduction was found in NSCLC line NCI-H23 (Table 1). The growth-suppressive effect was seen in lung cancer lines both expressing and not expressing NP2 and in two lung cancers (H2009 and H358) expressing endogenous mRNA for SEMA3B (Table 1). In the case of H358, the expressed SEMA3B mRNA contains a D397H mutation that, as shown above, has very reduced growth-suppressing activity.

Induction of Apoptosis by Exogenous Expression of SEMA3B. When the wild-type SEMA3B expression plasmid was transfected into H1299 cells, the number of surviving cells was decreased at 48 h MEDICAL SCIENCES after transfection compared with the control plasmid (Fig. 4A). The amount of this decrease was impressive because the trans- fection efficiency for the overall population was Ϸ30%. Thus, it is likely a ‘‘bystander’’ effect may be active, potentially mediated by secretion of SEMA3B from transfected cells (see below). This decrease in tumor cell number was associated with the appear- ance of TUNEL-positive cells (Fig. 4B) and a 10-fold increase Fig. 4. SEMA3B transfection induces apoptosis in H1299 NSCLC cells. (A) ϫ 4 (2–24%) of cells with sub-G1 content DNA (Fig. 4C) compared H1299 cells (1 10 ) were seeded in 35-mm dishes. After 24 h, empty vector with the control vector, indicating that growth suppression by control or SEMA3B expression plasmids were transfected (Ϸ20–30% efficien- SEMA3B was caused by induction of apoptosis. Transfection of cy), and the number of cells was counted 48 h later. The data represent the Ϯ the mutant SEMA3B constructs did not result in apoptosis mean SD of five independent experiments. (B) The TUNEL assay 24 h after H1299 cells were transfected with a SEMA3B plasmid. The TUNEL-positive cells detected by the TUNEL assay (data not shown). We did not use (Ϸ20%) are indicated by arrows. Vector control-transfected cells (TUNEL an epitope-tagged version of SEMA3B in these experiments, positive Ͻ2%) are not shown. (C) Fluorescence-activated cell-sorting profiles because other studies showed that a SEMA3B C-terminal of H1299 transfected with empty vector (Control) or the SEMA3B expression FLAG-tagged construct was inactive in tumor growth suppres- plasmid. Cells were harvested 48 h later, stained with propidium iodide, and sion despite conferring similar levels of SEMA3B protein on the analyzed by using the flow cytometer. Horizontal and vertical axes represent transfected cells (data not shown). The caspase-3 activity was DNA content and cell number, respectively. The percentage of sub-G1 cells increased significantly (P Ͻ 0.05) after wild-type p53 and undergoing apoptosis is indicated. (D) Caspase-3 activity in H1299 cells 20 h SEMA3B transfection, and the activity was blocked in both cases after transfection with SEMA3B or p53 (positive control) expression plasmids. Ϯ by the caspase-3 inhibitor DEVD-fmk, indicating caspase in- The data represent the mean SD of three independent experiments. p53- and SEMA3B-transfected cells had significantly higher caspase-3 activity than volvement in SEMA3B induced apoptosis (Fig. 4D). vector controls, whereas DEVD-fmk-treated p53 and SEMA3B cells did not.

Growth Suppression by the Conditioned Medium from COS-7 Cells Transfected with SEMA3B. Because SEMA3B is a secreted protein and lung cancers express NP receptors, we analyzed the effect of cancer growth-suppressing effect was not found in conditioned conditioned medium harvested from COS-7 cells transfected medium from SEMA3F-transfected COS-7 cells (data not with SEMA3B on H1299 lung cancer cells. The growth rate of shown). To confirm the growth-suppressive effect of SEMA3B- H1299 cells treated with conditioned medium from COS-7 cells transfected COS-7 conditioned medium, this assay was per- transfected with SEMA3B was reduced compared with condi- formed in several other lung cancer cell lines that have various tioned medium vector control-transfected COS-7 cells, condi- expression patterns of SEMA3B, NP1, and NP2, and growth tioned medium harvested after transfection with SEMA3B- inhibition of 30–60% was seen compared with vector control- Antisense, or mutant SEMA3B constructs (Fig. 5). This lung transfected COS-7 cell conditioned medium (Table 2). By con-

Tomizawa et al. PNAS ͉ November 20, 2001 ͉ vol. 98 ͉ no. 24 ͉ 13957 Downloaded by guest on September 29, 2021 did show growth inhibition to SEMA3B COS-7 cell conditioned medium (Table 2). Discussion Recently it has become clear that tumor-acquired promoter hypermethylation is a common mechanism for the inactivation of TSG expression in lung and other cancers (7, 12, 22), and the current study shows that the loss of SEMA3B expression is associated with methylation of CpGs in the putative SEMA3B promoter region. After transfection and expression, wild-type SEMA3B induces growth inhibition of lung cancer cells, whereas lung cancer-associated SEMA3B missense mutations or the addition of a C-terminal FLAG tag cause a loss of this tumor growth-suppressive function. In addition, exogenous expression of SEMA3F caused only a slight decrease in colony formation. We note that these SEMA3B mutant constructs differ in only a single amino acid from the wild-type sequence and were ex- pressed at similar protein levels but were not toxic to lung cancer cells compared with wild-type SEMA3B. These mutants, tagged versions, and the resistant NCI-H23 tumor line provide impor- tant specificity controls. The regions of mutations in SEMA3B Fig. 5. The effect of COS-7 condition medium transfected with SEMA3B are located at codons 348, 397, 415, and 561. These sites are expression plasmids on H1299 NSCLC cells. (A) Each indicated plasmid was located in the semaphorin domain, which is necessary for transfected into 5 ϫ 105 COS-7 cells by lipofection. After 48 h of incubation, the semaphorin dimerization and biological activity (1–3). Thus, it is conditioned medium was collected and applied to 5 ϫ 103 H1299 cells seeded likely that the semaphorin domain in SEMA3B is important for in each well of 6-well plates. After 4 days, the cells in each indicated condi- the lung cancer growth-suppressive activity. Two lung cancers tioned medium were photographed (A; original magnification is ϫ40) or continuing to express SEMA3B mRNA (H2009 and H358) also counted (B). The number of H1299 cells in the cultures treated with condi- showed inhibition of colony formation after forced expression of tioned medium from empty vector controls were set at 100%. The data represent the mean Ϯ SD of three independent experiments. SEMA3B- SEMA3B. In the case of H358 this result was expected, because transfected COS-7 cell conditioned medium gave significantly fewer numbers the expressed SEMA3B mRNA contained a D397H mutation of H1299 cells compared with treatment with condition medium after trans- (8). However, the inhibition of colony formation of lung cancer fection with the SEMA3B mutants (R348C, D397H, T415I, and D561N; data not H2009 by SEMA3B was unexpected and potentially could shown) or the SEMA3B antisense (AS) constructs. (C) Growth curve of H1299 indicate haploinsufficiency of SEMA3B expression or post- NSCLC cells in the conditioned medium of COS-7 cells transfected with control translational mechanisms of inactivation of SEMA3B in this or SEMA3B expression plasmids. At each indicated time point, cell viability was tumor. determined and represented as the degree of absorbance 540 nm using the In the Knudson recessive oncogene model, one defective copy 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The of a gene is inherited in the germline with subsequent loss of the mean Ϯ SD absorbance (triplicate wells) for each time point is plotted as a function of the number of days after seeding. wild-type allele in tumors (23). Thus, it was of great interest to discover that the germline T415I alteration leads to a significant loss of wild-type SEMA3B lung cancer growth-suppressing ac- ͞ tivity. This suggests further studies to see whether germline trast, the growth rate of normal human bronchial tracheal T415I represents an inherited risk factor for lung cancer, because epithelial cells treated with SEMA3B COS-7 conditioned me- we found it associated with wild-type allele loss in tumors. dium was not significantly different from treatment with vector Because many lung cancers undergo 3p21.3 allele loss and the control-transfected COS-7 control medium (Table 2). We note large majority of lung cancers do not express SEMA3B, it seems that although NSCLC NCI-H23 colony formation was resistant that there is biallelic inactivation of SEMA3B in sporadic tumors to transfection-induced expression of SEMA3B, this tumor line also consistent with Knudson’s model. Whether other events besides promoter methylation can lead to loss of SEMA3B expression requires further study. Table 2. Lung cancer cell line growth after exposure to The mechanism of SEMA3B-induced apoptosis is unknown. conditioned medium from COS-7 cells transfected with SEMA3B Another secreted semaphorin, SEMA3A, induced apoptosis in sympathetic (24) and sensory neurons (25). Semaphorins con- NCI lung cancer cell lines Growth inhibition* tain a short sequence of homology to tarantula hanatoxin, and H1299 90 Ϯ 3 the sequence is required to induce growth cone collapse in H358 62 Ϯ 10 neuronal cells (26). However, we did not find growth suppression H661 56 Ϯ 13 after transfection of mutant SEMA3B constructs retaining the H23 55 Ϯ 12 hanatoxin sequence. The growth of several lung cancer cell lines H322 37 Ϯ 16 but not normal bronchial epithelial cultures was suppressed by H2009 36 Ϯ 12 conditioned medium harvested from COS-7 cells transfected H2227 31 Ϯ 6 with wild-type SEMA3B. In contrast, conditioned medium from NHBE 11 Ϯ 10 mutant SEMA3B-transfected COS-7 cells did not suppress growth. The difference between normal and cancer cells could *The experiments were performed as described in the legend for Fig. 5B with exist because of the difference in expression for receptors for cell counts performed at day 4. The values are the mean Ϯ SD of three separate experiments with triplicate wells for cell counting in each experi- semaphorins, other components of the SEMA3B signaling path- ment. Vector control-transfected COS-7 cell condition medium was applied way, or response to other products secreted by COS-7 cells after to the replicate cell cultures, and cell numbers at day 4 were set as showing SEMA3B transfection. We need to consider whether secreted 0% growth inhibition. NHBE, normal human bronchial͞tracheal epithelial SEMA3B acts directly to inhibit tumor growth or indirectly by cell cultures (Clonetics). causing COS-7 cells to secrete other factors that inhibit growth.

13958 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.231490898 Tomizawa et al. Downloaded by guest on September 29, 2021 In preliminary experiments, anti-NP1 antibodies did not block One model to consider is that the loss of SEMA3F function plays SEMA3B-transfected COS-7 cell conditioned medium growth a role in loss of cell adhesion and altered motility response to suppression (data not shown). In addition, NSCLC NCI-H23 was tumor-produced VEGF, whereas SEMA3B induces apoptosis resistant to overexpression of SEMA3B but sensitive to possibly also related to competition with tumor-produced SEMA3B-transfected COS-7 conditioned medium. These find- VEGF, which in this case would be acting as a tumor-survival or ings would seem to support an indirect mechanism. Bachelder et growth factor. al. found that vascular endothelial growth factor (VEGF) was an Several other reports have noted correlations between sema- autocrine survival factor for NP expressing breast cancer cells phorins and growth control or cancer. In Caenorhabditis elegans, (27), whereas SEMA3A competed with VEGF165 for binding to Sema2a as a null mutant led to errant epidermal cell migrations NP1 receptors and inhibited vascular endothelial cell motility, and affected epidermal enclosure of the embryo (30). In contrast suggesting a role in inhibiting angiogenesis (4). In fact, the to the tumor-suppressing function of SEMA3B, SEMA3C and different effects of SEMA3B on normal human bronchial͞ SEMA3E are overexpressed in metastatic human and mouse tracheal epithelial cells versus cancer cells may occur because tumors, and SEMA3C led to cancer therapy drug resistance normal cells do not depend on survival factors such as VEGF, (31–33). Thus, abnormalities of semaphorin family genes may whereas lung cancer cells may require these factor(s). NP1 was play a cooperative role in carcinogenesis analogous to the role expressed in all the lung cancers, and overexpression of NP1 these genes play in the developing nervous system. itself may contribute to oncogenesis by enhancing angiogenesis, In summary, we have provided evidence that SEMA3B is a whereas a soluble NP1 bound VEGF and inhibited tumorigen- potent lung cancer growth suppressor, acting through apoptosis esis (28, 29). Thus, one model to consider is that SEMA3B acts pathways, that is epigenetically inactivated in human lung cancer. to antagonize VEGF by acting through the NP1 receptors. The signaling pathway(s) mediating lung cancer cell growth Substantial data indicate that the complex of NP1 and Plexin 1 suppression and apoptosis after the reexpression of SEMA3B is is the physiologic signal transducer for SEMA3A (1–3). There- unclear, and indirect mechanisms must be considered. Further fore, plexins also need to be studied to understand the growth studies will be necessary to elucidate the mechanism of suppres- suppression and apoptosis induced in lung cancer by SEMA3B. sive effect of SEMA3B in human tumors and to determine whether the neighboring homologue SEMA3F can function as a SEMA3F is implicated also as a TSG by functional studies in tumor suppressor in vivo. mouse A9 cells and loss of expression or mislocalization in lung cancers, the latter correlating with high levels of lung tumor We thank A. Kolodkin for advice and neuropilin antibodies. This work VEGF staining (9–11, ††). Thus, although we did not find an in is supported by National Cancer Institute grants CA71618 and Lung vitro growth-suppressing effect of SEMA3F, it is conceivable Cancer SPORE P50 CA70907 and the G. Harold and Leila Y. Mathers that both semaphorins play a role in tumor suppression in vivo. Charitable Foundation. M.I.L. was funded by National Cancer Institute Contract N01-CO-56000. We are grateful for encouragement by Pro- fessor Masatomo Mori, First Department of Internal Medicine, Gunma ††Nalor, S., Davalos, A., Hense, C. & Xiang, R. (1998) Am. J. Hum. Genet. 63, abstr. 431, 80. University, School of Medicine, Japan.

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