Human Semaphorin 3B (SEMA3B) Located at Chromosome 3P21.3 Suppresses Tumor Formation in an Adenocarcinoma Cell Line1

[CANCER RESEARCH 62, 542–546, January 15, 2002] Human Semaphorin 3B (SEMA3B) Located at Chromosome 3p21.3 Suppresses Tumor Formation in an Adenocarcinoma Cell Line1

Christin Tse, Ruinua H. Xiang, Todd Bracht, and Susan L. Naylor2 Sagres Discovery, Davis, California 95616 [C. T.], and Department of Cellular and Structural Biology, The University of Texas Health Science Center, San Antonio, Texas 78229-3900 [R. H. X., T. B., S. L. N.]

ABSTRACT role in axonal guidance and can be classified as either membrane bound (classes 1, 4, 5, and 6) or secreted (classes 2 and 3) (Refs. The short arm of chromosome 3 has been shown to exhibit high loss of 21–23). All semaphorins contain ϳ500 amino acid NH -terminal heterozygosity in several types of cancer including ovarian, kidney, lung, 2 sema domains, an immunoglobulin-like domain (with the exception of and testicular cancers. In particular, overlapping homozygous deletions in lung cancers have been identified in region 3p21.3. Semaphorin 3B, a gene class 1 semaphorins), and a basic COOH-terminal domain (24). The that resides within this region, has been proposed to be involved in role of the secreted class 3 semaphorins in axonal guidance has been tumorigenesis. To address this hypothesis, we have examined the effects of clearly demonstrated; however, their role(s) in nonneuronal tissue semaphorin 3B on HEY cells, an ovarian cancer cell line. HEY cells remains to be elucidated (25). The receptors for the class 3 semaphor- expressing semaphorin 3B exhibited a diminished tumorigenicity in ins are the neuropilin receptors (21, 26, 27). In neuronal tissue, BALB/c nu/nu mice. Semaphorin 3B also severely reduced the anchorage signaling through the neuropilin receptor has been shown to involve a independence of HEY cells. These results demonstrate a role for sema- heteromeric complex with the plexins (25, 28). The intracellular phorin 3B in tumor suppression. signal transduction pathway is less clear but has been shown to involve the Rho family GTPases (29). Both semaphorin 3B and 3F reside in 3p21.3 (Fig. 1) and exhibit INTRODUCTION overlapping yet distinct tissue expression patterns (20). Recently, Tumorigenesis results from the abnormal regulation of genes in- immunohistochemical studies found SEMA3F localization and ex- volved in cellular homeostasis. One mechanism involves the alteration pression to be altered in lung tumors compared with normal lung in gene expression or mutation of tumor suppressor genes, which are tissue (19). These results suggest a role of SEMA3F in tumorigenesis. recessive genes where mutations result in a loss of function, e.g., However, The potential role of SEMA3B in tumorigenesis is less clear. unrestricted cell cycle progression (1). There are several regions SEMA3B has been found at reduced levels or not expressed in both within the human genome that when altered, lead to uncontrolled small cell and non-small cell lung carcinomas, suggesting a role in proliferation. One such “hot spot” lies within the short arm of chro- tumorigenesis (6). Furthermore, mutations resulting in amino acid mosome 3 (3p). Three tumor suppressors localized to this region changes were found; however, the ramifications of these mutations include the von Hippel-Lindau gene, the MLH1 repair gene, and the remain to be elucidated (6, 20). On the basis of these observations, we FHIT gene (2–4). LOH3 studies of 3p have identified deletions propose that SEMA3B plays a suppressive role in tumorigenesis. To associated with various cancers (5–7). In particular, the 3p21.3 region address this hypothesis, HEY cells, a tumorigenic adenocarcinoma exhibits a high LOH in both lung and ovarian carcinomas, suggesting cell line, were stably transfected with SEMA3B and examined for the existence of a putative tumor suppressor gene within this region. tumor suppression abilities. SEMA3B transfectants exhibited de- In small cell lung cancer, Ͼ90% of the tumors exhibit LOH at this site creased tumor formation. Thus, in addition to its role in axonal (7, 8). Furthermore, LOH rates Ͼ60% are observed in other cancers, development, we have identified a tumor suppressive role of e.g., breast, gastric, ovarian, and testicular cancers and renal cell SEMA3B. carcinoma (9–14). Ovarian cancer is one of the leading causes of death in women. Given the high rate of LOH in 3p, several studies have been conducted MATERIALS AND METHODS to identify any ovarian cancer tumor suppressor genes within this Material. The human ovarian cell line, HEY, was used as the tumor cell region. Monochromosomal transfer studies of 3p by Rimessi et al. model and was a gift of Dr. Ron Buick (University of Toronto, Toronto, (15) have identified three ovarian cancer suppression regions. One Ontario, Canada). Briefly, HEY cells were derived from a human ovarian region, OCSR-C (8), overlaps with 3p21.3, a region implicated in lung cancer xenograft (HX-62; Ref. 30). ␣-MEM and the Thermoscript RT-PCR kit cancer (6, 16–18). Interestingly, two class III semaphorin genes were obtained from Life Technologies, Inc. Fetal bovine serum was obtained (SEMA3B and SEMA3F) reside in this region and have been proposed from Hyclone. The mammalian expression vector, pTracer-SV40, and the to play a role in tumorigenesis (6, 18–20). In normal ovarian tissue, selection agent, zeocin, were obtained from Invitrogen. Total ovary RNA was SEMA3B is the predominant semaphorin expressed (20). Semaphor- obtained from Clontech. All other chemicals were of reagent grade. ins are a family of signaling molecules initially identified to play a Quantitative Real-Time RT-PCR. SEMA3B gene expression was quantitated using Taqman chemistry on an ABI 7700 SDS machine (Perkin-Elmer) as described by manufacturer. The primers and probes used for detection of Received 7/2/01; accepted 11/13/01. SEMA3B were: 5Ј-GCGACACCCACTTCGATCA-3Ј/5Ј-CGTGGAGAA- The costs of publication of this article were defrayed in part by the payment of page Ј Ј charges. This article must therefore be hereby marked advertisement in accordance with GACGGCATAGAG-3 , and the probe was 5 -6-carboxyfluorescein-CTC- 18 U.S.C. Section 1734 solely to indicate this fact. CAGGATGTGTTTCTGTTGTCCTCGC-6-carboxytetramethylrhodamine-3Ј. 1 This research was supported by National Cancer Institute Grants CA56266 (to Briefly, reverse transcription was performed at 48°C for 30 min using the S. L. N.) and CA084643 (to C. T.). Multiscribe reverse transcriptase (Perkin-Elmer). Samples were then denatured 2 To whom requests for reprints should be addressed, at Department of Cellular and Structural Biology, University of Texas Health Sciences Center, MSC 7762, 7703 Floyd at 95° for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Curl Drive, San Antonio, TX 78229-3900. Phone: (210) 567-3842; Fax: (210) 567-6781; Results were analyzed on the SDS software version 1.7. E-mail: [email protected]. SEMA3B cDNA Isolation and Cloning into Expression Vector. The 3 The abbreviations used are: LOH, loss of heterozygosity; RT-PCR, reverse transcrip- full-length cDNA of SEMA3B was obtained by RT-PCR using human ovary tion-PCR; FBS, fetal bovine serum; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H- tetrazolum-5-carboxanilide inner salt; Np, neuropilin; VEGF, vascular endothelial growth total RNA. The Thermoscript RT-PCR kit was used for this procedure. Briefly, factor. cDNA corresponding to the total mRNA was reverse transcribed using an 542

titated using a NucleoTech Imaging Workstation equipped with a colony counting program. Cell Proliferation and Cytotoxicity Assays. The Cell Proliferation Kit II (XTT; Roche) was used to assess both cell proliferation and cytotoxicity. This assay is based on the cleavage of XTT by metabolic active cells, resulting in an orange formazan dye. The amount of orange formazan dye produced is quantitated using a spectrophotometric plate reader to measure the absorbance at 450 nm. Both assays were carried out essentially as described by manufacturer. Briefly, the cell proliferation assay involved plating the cells at a low density (4 ϫ 103 cells/well) in a 96-well plate. Cells were plated in either ␣-MEM containing 0.5% FBS or 10% FBS (100 ␮l final volume) and incu- ␮ bated at 37°C and 5% CO2. Twenty-four h later, 50 l of XTT labeling mixture were added per well and incubated for an additional6hat37°C and 5% CO2. For the cytotoxicity assay, cells were plated at high density (5 ϫ 104 cells/well) in a 96-well plate. Cells were plated in either ␣-MEM containing either 0.5% FBS ϩ 0.25 ␮M Taxol or 1.0 ␮M Adriamycin or serum-free (100-␮l final ␮ volume) and incubated at 37°C and 5% CO2. Twenty-four h later, 50 lof XTT labeling mixture were added per well and incubated for an additional 6 h

at 37°C and 5% CO2. The absorbance was then measured on a Molecular Device ELISA plate reader.

RESULTS The Semaphorin 3B Gene Suppresses Tumor Formation in Athymic Nude Mice. SEMA3B is highly expressed in ovarian tissue; however, its expression level in HEY cells, an ovarian adenocarcinoma, is unknown. To evaluate the expression level, quantitative Fig. 1. A, schematic diagram of chromosome 3. Both SEMA3B and SEMA3F reside in RT-PCR was performed. SEMA3B expression in total human ovarian 3p21.3, a region of high LOH in many tumor types. B, quantitative RT-PCR analysis of SEMA3B expression in normal total human ovary RNA and HEY cell lines. SEMA3B RNA (Ambion) was determined to be 0.918 arbitrary units (Fig. 1B). expression was normalized across samples by dividing the absolute values by the glyc- In contrast, the SEMA3B expression in HEY cells was 0.038 arbitrary eraldehyde-2-phosphate dehydrogenase expression values from each sample. Bars, SD. units, ϳ25-fold less than that observed in the total human ovarian RNA. The substantial decrease in the expression of SEMA3B in the oligo-dT primer as directed by the manufacturer. The SEMA3B cDNA was ovarian adenocarcinoma cell line is analogous to the observations in amplified by PCR using the following forward/reverse primers: 5Ј-GAAC- lung carcinomas and suggests a role for SEMA3B in ovarian tumori- CCTGAGCACCCTGAG-3Ј/5Ј-CTGTCGTCTCCTGCTTGGTT-3Ј. 2.5 ␮lof genesis. To examine whether SEMA3B has a role in tumorigenesis, the cDNA synthesis reaction were added to a 50-␮l final reaction containing 1 HEY cells were stably transfected with a SEMA3B cDNA clone. The ␮M of each primer using Platinum Taq high fidelity DNA polymerase. The SEMA3B cDNA was isolated by amplification from total human thermocycle conditions were as follows: 95°C for 2.5 min; 95°C for 45 s, 60°C placenta RNA (Clontech) and cloned into the pTracer-SV40 mamma- for 1.5 min, and 72°C for 3 min for 35 cycles; and 72°C for 10 min. The lian expression vector (Invitrogen). Stable transfectants expressing product (2318 bp corresponding to the entire coding region of SEMA3B) was SEMA3B from the introduced cDNA were identified by RT-PCR cloned into the pCRII-TOPO vector (TOPO-TA Cloning kit; Invitrogen). The amplification product was verified by DNA sequence analysis (UTHSCSA using a forward primer specific to the vector backbone and a reverse Sequencing Core). The SEMA3B cDNA was excised from the pCRII-TOPO primer specific for SEMA3B (Fig. 2A). Use of the vector-specific vector by digestion with EcoRI (one site flanking the multiple cloning site) and forward primer allowed us to identify only transfectants that were subcloned into the pTracer-SV40 mammalian expression vector (Invitrogen). expressing SEMA3B from the pTracer expression vector. To assess the Transfection and Isolation of Stable Clones. pTracer-SV40-SEMA3B tumor suppression properties of SEMA3B, five stable HEY clones (SEMA3B) constructs were stably integrated into HEY cells using Lipo- were injected into five male athymic nude mice, and tumor growth fectamine reagent (Life Technologies, Inc.) as described by manufacturer. The was monitored in intervals of 3–5 days for 38 days. Mice receiving cells were diluted 1:10 48-h after transfection into ␣-MEM containing 75- injections of two distinct sets of control HEY cells containing the ␮ g/ml zeocin. Single clones were isolated after 10 days, and a PCR screen was pTracer-SV40 vector alone exhibited exponential tumor growth after used to identify clones containing SEMA3B. RNA was then isolated from the 21 days (Fig. 2B) with an average volume of 933 mm3 on day 38. In remaining clones to verify SEMA3B expression via RT-PCR. Stables were maintained in 75-␮g/ml zeocin to ensure retention of the SEMA3B construct. contrast, the SEMA3B transfectants exhibited a substantial decrease in In Vivo Assay for Tumorigenesis. Single SEMA3B transfectants were tumorigenicity with an average volume ranging from 8.0 to 740.0 3 injected s.c. into the shoulders of five BALB/c nu/nu mice (5–6 weeks of age) mm on day 38. The wet mass of tumors produced from the SEMA3B at 2.0 ϫ 106 cells/mouse in a volume of 0.2 ml. Control HEY cells were transfectants was substantially smaller than controls, consistent with also injected at 2.0 ϫ 106 cells/mouse. Tumor volume (4/3 ϫ the results with the tumor sizes (Fig. 2C). Interestingly, the ␲ ϫ width ϫ width ϫ length) was assessed at 2–3-day intervals. Tumors were SEMA3B-10 clone produced a tumor that more closely resembled the excised at day 38, wet weight was determined, and tumors were placed into control than the other SEMA3B transfectants. If SEMA3B functions as culture for 3 days prior to DNA and RNA extraction. a tumor suppressor, no tumors should form. One explanation for In Vitro Assay for Anchorage Dependence. Sixty-mm soft agar plates tumor formation in the SEMA3B transfectants is whether SEMA3B consisted of ␣-MEM containing 10% FBS in a 0.4% agar medium. Cells (103) expression is lost during the course of the experiment. To determine cells of either control HEY or SEMA3B transfectants were resuspended in ␣-MEM containing 10% FBS in a 0.4% agar medium prewarmed to 37°C, and whether this occurred, tumors formed from the SEMA3B transfectants 2.5 ml were added to each plate. Agar plates containing cells were carefully were analyzed using RT-PCR. RT-PCR was performed on RNA placed into the incubator and were supplemented with medium once a week. isolated from the tumor explants using a vector-specific forward The number of cell colonies formed was determined by staining the agar plates primer and a SEMA3B-specific reverse primer as discussed above. As with p-iodonitrotetrazolium violet (Sigma Chemical Co.). Colonies were quan- expected, RNA isolated from the control tumors explants did not yield 543

a RT-PCR product because they did not contain the pTracer-SEMA3B construct (Fig. 2D, Lanes 1 and 2). In comparison, RNA isolated from the tumors formed from the SEMA3B transfectants also did not result in a RT-PCR product, indicating the loss of SEMA3B expression from the pTracer-SEMA3B construct (Fig. 2D, Lanes 3–10). In vitro, cancer cells lose contact inhibition and acquire anchorage independence. To examine the anchorage independence, both controls and SEMA3B transfectants were grown on soft agar (Fig. 3). Over a 3-week period, control HEY cells formed several hundred foci (Fig. 3A). In contrast, the number of foci formed from the SEMA3B transfectants was reduced up to 10-fold (Fig. 3B). SEMA3B Alters the Proliferation Rate of HEY Cells. One mechanism by which SEMA3B may reduce tumor formation is by affecting cell proliferation. To examine this possibility, we performed an XTT metabolic assay. This assay involves the reduction of the tetrazolium salt (XTT) by viable cells. The assay principle is based on the fact that proliferating cells are more metabolically active than resting cells. Both control and SEMA3B transfectants were plated at the same density and grown for 24 h in ␣-MEM containing either 0.5 or 10% FBS (Fig. 4). The metabolic activity of the control HEY cells grown in medium containing 10% FBS is Ͼ2-fold higher than those grown in 0.5% FBS. In contrast, the SEMA3B transfectants only exhibited a 1.2–1.6-fold change. Importantly, no cell death was observed in the cells grown in medium containing 0.5% FBS, as determined by trypan blue exclusion (data not shown). Because no cell death was observed, we consider the metabolic activity to reflect proliferative properties of the cell population. Thus, the cell proliferation rate of SEMA3B transfectants is reduced by ϳ36% compared with controls (Fig. 4B). SEMA3B Increases the Susceptibility of HEY Cells to Cytotoxic Environments. To further characterize the effects of SEMA3B, we performed the XTT assay under various cytotoxic conditions. The degree of cytotoxicity can be determined using this assay because cytotoxic factors reduce the rate of tetrazolium salt cleavage as a

Fig. 2. In vivo analysis of tumorigenesis in athymic nude mice. A, HEY cells containing the stable integration of the SEMA3B cDNA were assessed by RT-PCR of RNA from single clones after several rounds of expansion. Transcripts expressed from pTracer-SV40 containing SEMA3B was identified using a forward primer that recognized the T7 promoter (5Ј-TAATACGACTCACTATAGGG-3Ј) and a unique SEMA3B reverse primer (5Ј-TGATGTTGTCCAGGTTGAGG-3Ј). Five clones expressing SEMA3B were identified. B, each clone was injected into the right shoulder of five BALB/c nu/nu mice, and the size was monitored. As controls, two individual sets of HEY cells containing the Fig. 3. In vitro analysis of anchorage dependence. Cells (4 ϫ 103) of either HEY cells pTracer-SV40 vector alone (Control) were included. One clone representative of both containing pTracer-SV40 vector alone or pTracer-SV40 containing the SEMA3B cDNA control clones is illustrated in the figure. C, the wet weight of the tumors was determined were plated on a soft agar medium as described in “Materials and Methods.” Controls by excision on day 38. Bars, SD. D, RT-PCR of RNA isolated from explants of tumors containing vector alone and each SEMA3B transfectant clone were repeated in triplicate. formed. After excision, tumors were placed back into culture medium for 3 days prior to A, representative pictures of both the control and S3F transfectant plate. B, quantitation of RNA isolation. The presence of SEMA3B expression from the pTracer-SV40 construct the number of colonies formed. Results are plotted as the total number of colonies formed was assessed using the same T7/SEMA3B primers in A. versus sample. Bars, SD. 544

that corroborate this result: (a) 3p21.3 exhibits Ͼ60% LOH in ovarian carcinomas and Ͼ90% in small cell lung carcinomas, suggesting that a tumor suppressor(s) resides within this region; (b) SEMA3B expression appears to be absent or altered in both small cell and non-small cell lung carcinomas (5, 6, 20); and (c) we found that SEMA3B expression in an ovarian carcinoma cell line is ϳ25-fold less than the expression in total RNA from human ovarian tissue. Moreover, mutations in SEMA3B have been identified in tumor cell lines (6, 20). In this study, we demonstrate that expression of SEMA3B decreases both tumorigenicity and cell proliferation rates. Furthermore, the charac- teristic anchorage independence of cancer cells is severely diminished by SEMA3B expression. Suppression of Tumorigenesis by SEMA3B May Occur through Multiple Mechanisms. Our data indicate that there is an intrinsic change in the cells that have been transfected with semaphorin 3B. Perhaps the most significant difference is that transfected cells no longer exhibit anchorage-independent growth. This coupled with the

Fig. 4. Effects of SEMA3B on cell proliferation. The rate of cell proliferation was assessed by the XTT proliferation assay as described in “Materials and Methods.” This assay is based on the quantitation of metabolically active or viable cells. Briefly, this assay measures metabolic activity that is directly proportional to the number of viable cells. If more viable cells are present, then the metabolic activity signal will be greater and vice versa. A, measurement of the proliferation rate over a 24-h period. Control HEY cells as well as five SEMA3B transfectants assayed in the presence of 10 or 0.5% medium. f, controls; o, all of the SEMA3B transfectants. Bars, SD. B, assessment of the growth rate of the SEMA3B transfectants relative to the controls over a 24-h period. f, control cells; Ⅺ, all of the SEMA3B transfectants. result of decreasing the metabolic activity or viability of the cells (increased cell death; Refs. 31, 32). It is important to note that this measure of cytotoxicity or cell death is inclusive of cells that undergo both apoptosis and necrosis. Control and SEMA3B transfectants were grown for 24–48 h in the absence or presence of Taxol, a genotoxic agent that interferes with microtubule remodeling, leading to program cell death. As shown in Fig. 5A, control HEY cells containing only the pTracer-vector exhibit a ϳ22% decrease in metabolic activity after a 24-h exposure to Taxol. The metabolic activity decreases by ϳ40% after 48 h. In contrast, a ϳ35–59% decrease in metabolic activity is observed in the SEMA3B transfectants after 24–48 h, respectively. In the presence of Adriamycin, another genotoxic agent that inhibits topoisomerase activity, both controls and SEMA3B transfectants exhibited a similar behavior (Fig. 5B). This is analogous to previous observations that HEY cells exhibit resistance to cisplatin, another DNA interacting agent (30). Serum starvation also induces cell death but in a more obscure pathway than Taxol and Adriamycin. Under serum starvation, control HEY cells did not display a significant decrease in metabolic activity until 48 h (40%; Fig. 5C). In contrast, the metabolic activity of the SEMA3B transfectants decreased by 40–80% over 24–48 h, respectively. In sum, the presence of SEMA3B increased the cytotoxic effects of Taxol and serum starvation but not Adriamycin on HEY cells.

Fig. 5. SEMA3B alters the response of HEY cells to various cytotoxic environments. DISCUSSION The XTT cytotoxicity assay was used to evaluate the effects of Taxol, Adriamycin, and serum starvation on SEMA3B transfectants. All cells were normalized to cells in ␣-MEM Cancer progression is a multistep process that requires the mutation containing 0.5% FBS. A, control and SEMA3B transfectant cells were exposed to Taxol for and/or alteration of expression of multiple genes. Here we demon- 24 and 48 h as described in “Materials and Methods.” f, controls; p, clones. Bars, SD. B, control and SEMA3B transfectant cells were exposed to Adriamycin for 24 and 48 h. strate that SEMA3B, a gene that resides in 3p21.3, suppresses tumor f, controls; p, clones. Bars, SD. C, control and SEMA3B transfectant cells were serum formation in an ovarian carcinoma. There are several lines of evidence starved for 24 and 48 h. f, controls; p, clones. Bars, SD. 545

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Christin Tse, Ruinua H. Xiang, Todd Bracht, et al.

Cancer Res 2002;62:542-546.

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