Identification of a Putative Tumor Suppressor Gene Rap1gap in Pancreatic Cancer

Research Article

Identification of a Putative Tumor Suppressor Gene Rap1GAP in Pancreatic Cancer

Lizhi Zhang,1 Li Chenwei,1 Redah Mahmood,1 Kenneth van Golen,2 Joel Greenson,3 Gangyong Li,1 Nisha J. D’Silva,4 Xiangquan Li,2 Charles F. Burant,2,5 Craig D. Logsdon,6,7 and Diane M. Simeone1,5

Departments of 1Surgery, 2Internal Medicine, 3Pathology, 4Oral Medicine, and 5Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan; Departments of 6Cancer Biology and 7Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas

Abstract association with a new GTP molecule, which activates Rap1. GAPs stimulate the low intrinsic GTPase activity of Rap1 and induce GTP Human chromosome 1p35-p36 has long been suspected to harbor a tumor suppressor gene in pancreatic cancer and hydrolysis, thus inactivating Rap1 in cells. other tumors. We found that expression of rap1GAP, a gene Rap1GAP is a member of a family of related GAP proteins, each located in this chromosomal region, is significantly down- with a unique expression profile and cellular distribution (6). Little regulated in pancreatic cancer. Only a small percentage of is known about the role of rap1GAP in cancer, although alterations preneoplastic pancreatic intraductal neoplasia lesions lost in other rap1GAP family members play a role in cancer develop- rap1GAP expression, whereas loss of rap1GAP expression ment and/or progression. SPA-1 is a rap1GAP expressed in peri- occurred in 60% of invasive pancreatic cancers, suggesting pheral T cells and hematopoietic progenitors in bone marrow. that rap1GAP contributes to pancreatic cancer progression. Mice deficient in SPA-1 developed a spectrum of myeloid disorders In vitro and in vivo studies showed that loss of rap1GAP that resemble chronic myeloid leukemia (7). Another member of promotes pancreatic cancer growth, survival, and invasion, the rap1GAP family, E6TP1, is targeted for ubiquitin-mediated and may function through modulation of integrin activity. degradation by the viral E6 oncoprotein and its virally mediated Furthermore, we showed a high frequency of loss of down-regulation may contribute to cervical cancer and other cancers associated with chronic human papillomaviruses infection heterozygosity of rap1GAP in pancreatic cancer. Collectively, our data identify rap1GAP as a putative tumor suppressor (8). Tuberin, the product of the tuberous sclerosis type 2 (TSC2) gene, is a rap1GAP homologue and functions as a GAP for the Ras- gene in pancreatic cancer. (Cancer Res 2006; 66(2): 898-906) like protein Rheb (9). Mutations in the GAP domain of tuberin cause tuberous sclerosis (10) and somatic inactivation of TSC2 Introduction predisposes to formation of sporadic gliomas (11). Pancreatic cancer is a formidable disease with a 5-year survival In this study, we examined the role of rap1GAP in pancreatic rate <5%. A major hallmark of pancreatic cancer is its extensive tumor growth and progression. We verified that rap1GAP is down- local tumor invasion and early systemic dissemination. These regulated in pancreatic cancer and provide data to support its features likely contribute to the fact that the vast majority of critical role in pancreatic cancer progression. Furthermore, we also patients present with advanced disease and cannot be offered show that a significant number of pancreatic cancer patients have curative treatment. The molecular basis for these characteristics loss of heterozygosity (LOH) of the rap1GAP gene located at of pancreatic cancer is incompletely understood. A better chromosome 1p36.1-p35, indicating that rap1GAP may be a novel understanding of the genes involved in tumor growth and invasion tumor suppressor gene in pancreatic cancer. may allow development of novel treatment strategies to tackle this rapidly progressive disease. Materials and Methods We recently found that Rap1 GTPase-activating protein 1 (rap1GAP) was significantly down-regulated in pancreatic cancer Pancreatic tissue and pancreatic cancer cell lines. Paraffin-embedded (1). Rap1GAP, a 663-amino-acid protein with a molecular weight of human pancreatic tissues came from the University of Michigan Medical Center and conformed to the policies and practices of the University of 73 kDa, was the first identified member of a family of proteins with Michigan Institutional Review Board. The pancreatic cancer cell lines MIA GAP function specific for Rap1 (2). Rap1 belongs to the Ras family PaCa-2, Panc-1, BxPC-3, CAPAN-2, HPAF-11, HPAC, AsPc-1, Su 8686, and of small GTP-binding proteins and is implicated in a host of CFPAC were obtained from the American Type Culture Collection cellular processes, including proliferation, adhesion, and morpho- (Manassas, VA). The immortalized pancreatic ductal cell line HPDE was genesis (3–5). Activation of Rap1, like other Ras family proteins, kindly provided by M.S. Tsao (University of Toronto, Toronto, ON, Canada). occurs by cycling between GTP-bound and GDP-bound forms. The Immunohistochemical analysis. A pancreatic cancer tissue microarray kinetics of Rap1 GTP hydrolysis and GDP dissociation are regulated containing five samples of normal pancreas and 47 samples of pancreatic by two protein classes: guanine-nucleotide exchange factors (GEF) adenocarcinoma was used for immunohistochemical analysis of rap1GAP and GAPs. GEFs promote the release of bound GDP and the expression. Immunohistochemistry was done using a standard biotin-avidin complex technique (Vector Labs, Burlingame, CA) and a rabbit polyclonal antibody against rap1GAP (Santa Cruz, Santa Cruz, CA) at a dilution of 1:1,000. Twenty-five paraffin-embedded human pancreatic tissue samples Requests for reprints: Diane M. Simeone, University of Michigan Medical Center, containing pancreatic intraductal neoplasia (PanIN) lesions of varying TC 2922D, Box 0331, 1500 East Medical Center Drive, Ann Arbor, MI 48109. Phone: 734- stages were also stained with an anti-rap1GAP antibody. Rap1GAP 615-1600; Fax: 734-936-5830; E-mail: [email protected]. I2006 American Association for Cancer Research. expression in PanIn lesions was evaluated by an experienced pancreatic doi:10.1158/0008-5472.CAN-05-3025 pathologist and graded as negative, weak positive or positive.

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Creation of stable cell lines. Cell lines with stable expression of rap1GAP cytometry using a single laser emitting excitation light at 488 nm was done were generated by transfecting Panc1 and MiaPaCa-2 cells with a pcDNA3.1 to detect Annexin V–positive cells. vector containing Flag-rap1GAP. Control cell lines were generated by trans- Cell motility assays. Motility was assessed using colloidal gold random fecting Panc-1 and MiaPaCa-2 cells with an empty pcDNA3.1 vector. motility assays as described (14). Motility was induced by stimulating the Selection for neomycin resistance was initiated 48 hours after transfection cells with 10% FBS. Phagokinetic tracks were visualized by microscopy and by adding 500 Ag/mL of G418 (Life Technologies) to the culture medium. were digitally recorded. The areas of the phagokinetic tracks were measured The selection medium was changed every 3 days for 5 weeks. Clones of G418- using ImageJ version 1.30 software and expressed as pixels squared. resistant cells were isolated and expanded for further characterization. Immunofluorescent staining of cytoskeleton and focal adhesion Reverse transcription-PCR. Reverse transcription-PCR (RT-PCR) was components. Cells were serum starved for 24 hours and then were done using total RNA prepared from human samples of normal pancreas, stimulated with 10% FBS for 45 minutes. Immunofluorescent staining for chronic pancreatitis and pancreatic cancer. Reverse transcription was phalloidin and vinculin was done to detect focal adhesion and F-actin conducted for 45 minutes at 45jC from 500 ng of purified RNA in 25 ALof assembly using a FAK100 kit from Chemicon International, Inc. (Temecula, Reserve Transcription system reaction mixture using avian myeloblastosis CA). Briefly, cells were fixed with 4% paraformaldehyde for 15 minutes at virus reverse transcripase (Promega, Madison, WI). Reverse transcription room temperature and then were permeabilized with 0.1% Triton X-100 for was followed by 25 cycles of PCR (30 seconds denaturation at 94jC, 30 5 minutes. After blocking with 1% bovine serum albumin for 30 minutes, the seconds annealing at 58jC, and 30 seconds extension at 72jC). The primers cells were incubated with a monoclonal antivinculin antibody for 1 hour at designed for rap1GAP were as follows: forward 5V-GGAGCTTCGCGCG- room temperature. After washing, the slides were incubated with FITC- CCCAACAACC-3V and reverse, 5V-CTCCGTCCACACCCTCCGTCTCCT-3V. conjugated secondary antibody and tetramethyl rhodamine isothiocyanate Primers designed for ribosomal protein S6 (RPS6) were as described (12). (TRITC)–conjugated phalloidin antibody for 30 minutes. Nuclei counter- Amplified products were separated on 1.5% agarose gels and visualized staining was done by incubating cells with 4V,6-diamidino-2-phenylindole by ethidium bromide. Images of the RT-PCR ethidium bromide–stained (DAPI) for 5 minutes. The slides were mounted using a Vectashield agarose gels were acquired with a Gel Doc EQ system and quantified using mounting solution (Vector Labs). Fluorescent images were visualized and Multi-Analyst Version1.1 software (Bio-Rad Laboratories, Hercules, CA). recorded with an Olympus FV-500 confocal microscope. Rap1GAP mRNA expression was normalized to RPS6 expression levels from In vitro cell invasion assays. Matrigel invasion assays were done as the same samples. previously described using precoated Transwell filters with 8 Am pores (14). Immunoblot analysis. Immunoblot analysis was done as previously Orthotopic pancreatic cancer model. Male CB17 severe combined described (1) using rabbit polyclonal anti-phospho-focal adhesion kinase immunodeficient (SCID) mice (Charles River, Wilmington, MA) were (Upstate, Lake Placid, NY), rabbit polyclonal anti-total focal adhesion kinase housed under pathogen-free conditions in accordance with University of and anti-Rap1, goat polyclonal anti-rap1GAP (Santa Cruz), and mouse anti- Michigan Animal Care and Use Committee guidelines. Ten-week-old mice Flag antibodies (Sigma, St. Louis, MO). Images were visualized using the were anesthetized with an i.p. injection of xylazine (9 mg/kg) and ketamine ECL Detection System (Amersham, Arlington Heights, IN). Film images (100 mg/kg). A median laparotomy was done and an aliquot of 5 Â 105 were scanned with an Agfa Arcus II (Bayer Corp., Ridgefield Park, NJ) to MiaPaCa-2 cells (empty vector or rap1GAP stably transfected) in a volume create a digital image. of 30 AL was injected into the pancreatic tail using a 30-gauge needle Rap1 activity assays. Rap1 activity assays were done according to (six per group). To prevent leak from the injection site, the needle was instructions of the manufacturer (Upstate). Briefly, whole cell lysates were slowly withdrawn after a 1-minute delay and slight pressure was applied prepared by incubating the cells in ice-cold lysis buffer [50 mmol/L Tris-HCl with a sterile cotton swab. The mice were sacrificed 7 weeks later and

(pH 7.4), 0.5 mol/L NaCl, 1% NP40, 2.5 mmol/L MgCl2,10Ag/mL aprotinin, autopsies were done. Primary tumor size was measured and tumor volume 10 Ag/mL leupeptin, and 10% glycerol]. The cells were sonicated for was calculated using the formula for hemi-ellipsoids: V = length (mm) Â 5 seconds and the lysates centrifuged at 14,000 Â g for 5 minutes at 4jC. width (mm) Â height (mm) Â 0.5236 (15). The extent of local invasion and The supernatant was removed and assayed for protein concentration using distant metastasis was also recorded. Primary and metastatic lesions were the Bio-Rad protein assay. Thirty microliters of Ral GDS-RBD agarose were excised, fixed in 10% formalin, and embedded in paraffin. Tissue sections added to each tube containing 0.5 mL cell extract and then incubated were obtained and stained with H&E. for 45 minutes at 4jC. Pellet beads were collected by centrifugation LOH analysis of rap1GAP. DNA was extracted from nine micro- (10 seconds, 14,000 Â g) and washed thrice with lysis buffer. The beads dissected samples of human pancreatic cancer, nine pancreatic cancer cells were resuspended in 40 ALof2Â Laemmli buffer and 2 AL of 1 mol/L DTT lines, and the immortalized human pancreatic ductal cell line C6H11. was added, followed by boiling for 5 minutes. Western blotting was done A rap1GAP–specific marker, rap1GA1 3+4 (GDB:1006983), was selected from to detect Rap1. the Genome Database (www.gdb.org). This marker is contained within Cell proliferation assays. Cell proliferation was measured using a intron 9 of the rap1GAP gene at chromosome 1p36.1-p35. Primer sequences CellTiter 96 AQueous nonradioactive cell proliferation assay (Promega) as to amplify this region were designed from the Genome Database and PCR we have previously described (13). primers were purchased from Invitrogen (Carlsbad, CA). Forty nanograms Soft-agar colony formation assays. Assays of colony formation in soft of DNA were used as a template in each sample. PCR was done in a 25 AL agar were done by preparing 1 mL underlayers consisting of 0.6% agar reaction volume containing 0.2 Amol/L PCR primers, 1Â PCR Master Mixer medium in 35 mm dishes by combining equal volumes of 1.2% Noble agar (Promega), 1Â PCR buffer, 200 Amol/L deoxynucleotide triphosphate, (Difco, Detroit, MI) with 2Â DMEM and 20% fetal bovine serum (FBS). Cells 1.5 mmol/L MgCl2, and 0.6 units of Taq DNA Polymerase. DNA was were trypsinized, centrifuged, and resuspended in 0.3% agar medium amplified for 30 cycles at 94jC for 30 seconds, 56jC for 30 seconds, and followed by plating onto the previously prepared underlayers. The cells were 72jC for 30 seconds, followed by a 10-minute extension at 72jC. PCR kept wet by adding a small amount of DMEM with 10% FBS every other day. products were evaluated using a DNA 500 Labchip kit and analyzed using

The cells were incubated at 37jC in a humidified 5% CO2 atmosphere for an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). We used 4 weeks and were then stained with methylene blue. Stained colonies were the formula of Cawkwell et al. (16) with a change in allelic ratios of 0% photographed and counted. to 50% scored as LOH. This criterion permits LOH detection in tumor Detection of apoptosis by flow cytometry. To detect apoptotic cells, an samples with up to 50% stromal contamination, as is seen with pancreatic ApoAlert Annexin V-FITC Apoptosis kit was used (BD Biosciences, Palo adenocarcinoma. Alto, CA). Cells were induced to undergo apoptosis with serum starvation A and treatment with 5-fluorouracil (5-FU, 300 g/mL) or etoposide Results (200 Ag/mL) for 16 hours. Cells were collected and rinsed once with binding buffer. Resuspended cells were stained with Annexin V-FITC/ Rap1GAP1 is down-regulated in pancreatic cancer. Because propidium iodide according to the instructions of the manufacturer. Flow significant evidence has emerged that dysregulation of Rap1 www.aacrjournals.org 899 Cancer Res 2006; 66: (2). January 15, 2006

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research activation is responsible for the development of malignancy in a such as K-ras mutation and inactivation of p16 and p53. To variety of cell types (17), we determined if alterations in the Rap1 examine if loss of rap1GAP expression is associated with pancreatic signaling pathway might contribute to pancreatic tumor develop- tumorigenesis, we measured rap1GAP expression in 25 human ment and/or progression by examining expression levels of a pancreatic tissue samples (19 cancers and 6 chronic pancreatitis) variety of components of the Rap1 signaling pathway. According to containing PanIN lesions. Only a small proportion of PanIN lesions our gene profiling data (1), expression levels of Rap1a and Rap1h, lost rap1GAP expression, with 2 of 12 (17%) PanIN1, 2 of 13 (15%) both isoforms of Rap1, were not altered in pancreatic cancer. The PanIN2, and 2 of 11 (18%) PanIN3 lesions showing loss of rap1GAP expression levels of Rap1 GEFs, which can activate Rap1, including expression, with no evidence of progressive loss of rap1GAP C3G and Epacs, were also unchanged in pancreatic cancer. Analysis expression with PanIn progression. This contrasts to the observed of rap1GAP family members revealed that rap1GAP levels were 60% loss of rap1GAP expression in invasive pancreatic cancers significantly down-regulated in pancreatic cancer, whereas other (Fig. 1C). These results suggest that inactivation of rap1GAP is rap1GAP family members, including TSC2 and SPA-1, were not not critical in the early stages of pancreatic cancer development significantly changed. but rather occurs at a later stage of tumor progression. Rap1GAP was significantly down-regulated in pancreatic cancer Overexpression of rap1GAP blocks Rap1 activation in and pancreatic cancer cell lines, compared with both normal and pancreatic cancer cells. To examine the functional role of chronic pancreatitis samples (Fig. 1A). Rap1GAP expression was rap1GAP in pancreatic cancer, we increased rap1GAP expression lower in chronic pancreatitis than normal pancreas, likely because in pancreatic cancer cells by stably transfecting the human the large number of stroma cells in chronic pancreatitis do not pancreatic cancer Panc1 and MiaPaCa-2 cells with a Flag-tagged express rap1GAP by immunohistochemistry (data not shown). rap1GAP vector or empty expression vector as a control. Results We used RT-PCR to independently examine mRNA levels for obtained from the two different cell lines were similar and, rap1GAP in five samples each of normal pancreas and pancreatic therefore, experiments described in this report are from the adenocarcinoma and four samples of chronic pancreatitis. RT-PCR MiaPaca-2 cell line. Two independent clonal cell lines of Flag- showed expression of rap1GAP in normal pancreas and chronic rap1GAP-transfected MiaPaCa-2 cells (designated R1 and R2) pancreatitis but little or no expression of rap1GAP in pancreatic showed increased rap1GAP expression compared with the cancer samples (Fig. 1B). RPS6 served as an internal control. corresponding wild-type and control vector-transfected cells Using a pancreatic cancer tissue microarray, we next evaluated (Fig. 2A). High basal Rap1 activity was detected in nontransfected the expression of rap1GAP protein in samples of normal pancreas and control empty vector-transfected cancer cells. However, in (n = 5) and pancreatic adenocarcinoma (n = 47). The expression rap1GAP-overexpressing cells (R1 and R2), Rap1 activity was level of rap1GAP in individual samples was scored as negative, completely abolished (Fig. 2B). Thus, introduction of rap1GAP weak positive, and positive by a blinded pancreatic pathologist. into pancreatic cancer cells results in inhibition of Rap1 binding Rap1GAP protein expression was observed primarily in the activity. cytoplasm (Fig. 1E). In normal tissue, strong staining of rap1GAP Overexpression of rap1GAP inhibits cell proliferation and was observed in ductal and islet cells with weak staining in acinar survival. Rap1 has been shown to be important in regulation of cells. In rap1GAP-expressing pancreatic cancers, staining was seen cell proliferation; however, its effects are cell type specific. For only in the neoplastic epithelium. Most pancreatic cancer samples example, overexpression of Rap1 in Swiss 3T3 cells induces DNA were graded as negative (28 of 47, 60%), whereas 40% were weakly synthesis and decreases doubling time (3, 19), but introduction of positive. These results are summarized in Fig. 1C and confirm constitutively activated Rap1 in astrocytes (20) and in keratinocytes a significant down-regulation of rap1GAP levels in pancreatic (21) inhibits cell proliferation. To examine whether increasing adenocarcinoma. rap1GAP expression influences the growth properties of pancreatic To determine if the expression of rap1GAP correlated with the cancer cells, we examined the effect of rap1GAP on in vitro differentiation status of pancreatic adenocarcinoma, cancer proliferation rates and the ability of cells to form colonies in samples on the tissue microarray were graded as well, moderately, soft agar. Rap1GAP-transfected R1 and R2 cell lines showed a and poorly differentiated. The level of rap1GAP expression was significant decrease in proliferation (Fig. 2C). The effect of rap1GAP then analyzed in a blinded manner and correlated with tumor was even more pronounced in inhibiting anchorage-independent differentiation status. We found a direct correlation between loss growth, measured by colony formation (Fig. 2D and E). of rap1GAP expression with more poorly differentiated tumors. Resistance to apoptosis is a key factor in the survival of Over 65% (9 of 14) of poorly differentiated cancers lost rap1GAP malignant cells. To determine the role of rap1GAP in pancreatic expression compared with 25% (2 of 8) of well-differentiated cancer cell apoptosis, we examined the effect of rap1GAP on basal cancers (Fig. 1D). We did not observe a correlation between apoptotic rates and in response to the apoptosis-inducing rap1GAP expression levels and patient survival (data not shown); chemotherapeutic drugs 5-FU and etoposide using Annexin V however, it is likely that a larger study group would be needed for staining. The basal apoptotic rate of rap1GAP-overexpressing adequate statistical power to determine the effect of rap1GAP pancreatic cancer cells increased 2-fold compared with control expression on this end point. vector-transfected cells (data not shown). Treatment with 5-FU or Rap1GAP expression in PanIN lesions. A progression model of etoposide for 16 hours resulted in an apoptosis rate of 4% to 5% in pancreatic cancer has become widely accepted in which normal control MiaPaCa-2 cells; however, rap1GAP-overexpressing cells duct epithelium progresses to infiltrating cancer through a series of showed much higher rates of apoptosis in response to 5-FU and morphologically defined pancreatic cancer precursors named etoposide (28% and 13%, respectively; Fig. 2F). These results PanINs (18). PanIN1 lesions show hyperplasia without dysplasia, suggest that loss of rap1GAP expression in human pancreatic PanIN2 lesions show dysplasia, and PanIN3 lesions show severe adenocarcinoma may render cells resistant to apoptosis and, nuclear atypia characteristic of carcinoma in situ. This progression therefore, play a role not only in tumor progression but is associated with the accumulation of specific genetic alterations, chemotherapeutic resistance.

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Rap1GAP inhibits cell motility and invasion ability in vitro. invasion in parental MiaPaCa-2 cells and rap1GAP transfectants Increasing cell motility and invasion are two key components of using colloidal gold random motility and in vitro invasion assays. tumor progression. Several studies have documented that Rap1 As seen in Fig. 3A, R1 and R2 cells showed a significant decrease functions in integrin-mediated morphologic changes, cell adhesion, in random motility, measured by white cell movement tracks, and cell motility (22). To examine whether rap1GAP expression is compared with control pancreatic cancer cells, which showed associated with cancer cell invasiveness, we measured motility and wider and longer white tracks. Quantitation of track area revealed a

Figure 1. Down-regulation of rap1GAP in pancreatic cancer correlates with pancreatic cancer progression status. A, cDNA microarray analysis (1) was done using Affymetrix chips containing 6,800 genes. Microdissected samples from pancreatic adenocarcinoma (10), pancreatic cancer cell lines (7), chronic pancreatitis (CP, 5), and normal pancreas (5) were analyzed. mRNA expression levels of rap1GAP were expressed as Affymetrix units. *, P < 0.05. B, validation of microarray results of rap1GAP mRNA levels using RT-PCR analysis. Total RNA was collected from pancreatic adenocarcinoma (5), chronic pancreatitis (4), and normal pancreas (5), and 100 ng was subjected to RT-PCR for rap1GAP mRNA. RPS6 mRNA was used as an internal control for variance in loading samples. Representative of a single experiment. Experiments were repeated twice with essentially the same results. C, summarized results from analysis of the level of rap1GAP expression in individual samples in the pancreatic cancer tissue array containing five samples of normal pancreas and 47 samples of pancreatic adenocarcinoma. Columns, percentage of total sample type. D, samples from the tissue array were graded as well (8), moderate (25), and poorly differentiated (14) and the extent of rap1GAP expression was quantitated as negative (white columns), weakly positive (gray columns), or positive (black columns). Columns, percentage of total sample type. E, rap1GAP immunostaining of two samples of pancreatic adenocarcinoma. High-power photomicrograph of rap1GAP immunostaining demonstrating strong staining of benign pancreatic ducts on the bottom and negative staining of a well-differentiated pancreatic adenocarcinoma on the top (left). High-power photomicrograph of rap1GAP immunostaining showing weak staining of pancreatic acinar cells on the right and negative staining of poorly differentiated pancreatic adenocarcinoma cells on the left (right). www.aacrjournals.org 901 Cancer Res 2006; 66: (2). January 15, 2006

Figure 2. Inhibition of cancer cell proliferation and survival by rap1GAP. A, a representative immunoblot demonstrating levels of rap1GAP expression in MiaPaCa-2 cells stably expressing rap1GAP and their wild-type and vector control transfectants (top). The same membrane was stripped and reblotted with anti-Flag antibody showing overexpression of the transfected Flag-tagged rap1GAP gene (bottom). B, Rap1 activation is inhibited in rap1GAP stably transfected cancer cells. Rap1 activity assay was done in MiaPaCa-2 cells. Thirty microliters of Ral GDS-RBD agarose were used in 0.5 mL cell extract to pull down the activated form of Rap1 (GTP-Rap1). Western blotting was done to detect the GTP-Rap1 (top). The same amount cell extract (30 Ag) was also used to perform a regular Western blotting with anti-Rap1 antibody as an internal control for loading (bottom). Similar results were also observed in Panc-1 cells. Representative of a single experiment. Experiments were repeated twice with essentially the same results. C, the R1 and R2 rap1GAP-transfected MiaPaCa-2 cell lines and vector-transfected control cells were plated in 96-well plates at a density of 500 cells per well in 150 AL DMEM with 5% FBS for 5 days. At each day, 15 AL/well of combined 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate solution was added and the absorbance was determined by using an ELISA plate reader. Points, average absorbance of three wells in one experiment. The experiment was repeated twice with similar results. D and E, colony formation in soft agar. Five thousand parent cells or their transfectants per well were plated in 35 mm dishes containing 0.6% agar medium (underlay) and 0.3% agar medium (uplayer). The cells were incubated at 37jC in a humidified 5% CO2 atmosphere for 4 weeks and were then stained with methylene blue. Stained colonies were photographed and counted. A representative stained well for control and rap1GAP-transfected cells is shown in Fig. 4B. The summary results from three separate experiments are presented in Fig. 4C.*,P < 0.01. F, rap1GAP promotes cell apoptosis. Apoptosis was induced by treating rap1GAP-transfected MiaPaCa-2 cells or controls with either 5-FU (300 Ag/mL) or etoposide (200 Ag/mL) for 16 hours. Cells were collected and stained with Annexin V-FITC/propidium iodide. Flow cytometry was done to detect Annexin V–positive cells. Experiments were repeated thrice. *, P < 0.01.

68% (R1) and 60% (R2) decrease in cell motility compared with Rap1GAP may function through integrins. Rap1 has been control cells (Fig. 3B). In vitro invasive assays showed that the reported to affect cell function through effects on the Ras/ invasion ability of R1 and R2 cells was decreased 60% compared extracellular signal-regulated kinase (ERK) signaling pathway and with wild-type and control vector-transfected MiaPaCa-2 cells activation of integrins. We observed no difference in phospho-ERK (Fig. 3C). These results show that rap1GAP expression is inversely or total ERK levels between control pancreatic cancer cells and associated with motility and invasiveness of pancreatic cancer their rap1GAP-transfected counterparts (data not shown). We cells in vitro. next determined the effect of rap1GAP on two integrin-dependent

Figure 3. Effect of rap1GAP on cell motility and invasion. A and B, random cell motility assays were done using a colloidal gold random motility assay. A, representative pictures for control and the rap1GAP-transfected cell lines R1 and R2. B, summary results from three separate experiments. C, in vitro invasion assays were done by determining the percentage of rap1GAP-transfected and control-transfected cells that penetrated through Matrigel-coated Transwell chambers. Experiments were repeated thrice. *, P < 0.05.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Rap1GAP in Pancreatic Cancer events, focal adhesion formation, and F-actin formation. Control done. All six animals injected with control MiaPaCa-2 cells MiaPaCa-2 cells stimulated with serum (Fig. 4A, left) manifested developed large pancreatic tumors with extensive invasion to local morphologic changes suggestive of integrin activation, such as structures, including the portal vein, stomach, and spleen (Fig. 5A spreading and nuclear enlargement. F-actin formation, assessed by and B). Control mice also had extensive liver and peritoneal TRITC-conjugated phalloidin staining, was observed at the leading metastasis (Fig. 5C). However, the mice injected with rap1GAP edge of spreading cells. Vinculin, a component of focal adhesion stably transfected MiaPaCa-2 cells had significantly smaller protein complexes, translocated from the cytoplasm to the cell primary tumors (76.4 F 25.2 mm3 of R1 versus 320.4 F 98.7 mm3 membrane at the leading edge following serum exposure (Fig. 4A, of control; Fig. 5A and B), with two of six animals demonstrating arrows). In contrast, MiaPaCa-2 cells overexpressing rap1GAP did no gross tumor. Only one of six mice in the rap1GAP animal group not show morphologic changes associated with integrin activation had liver and peritoneal metastases, which were much smaller and after 45 minutes of exposure to serum (Fig. 4A, right). fewer in number than those seen with the control animals (Fig. 5C). The effect of rap1GAP on integrin activity was further examined These results show that overexpression of rap1GAP suppresses by testing the ability of rap1GAP to inhibit focal adhesion kinase primary pancreatic cancer growth and metastasis in an orthotopic phosphorylation. Control MiaPaCa-2 cells showed focal adhesion pancreatic cancer model. kinase activation as early as 5 minutes after 2.5% serum treatment, High frequency of LOH of rap1GAP gene at chromosome with peak stimulation at 20 to 40 minutes, with return toward 1p36.1-p35 in pancreatic cancer. Like other cancers, pancreatic control values by 60 minutes (Fig. 4B, top). Focal adhesion kinase adenocarcinoma is thought to arise from a multistep process of activation was not detected in cells overexpressing rap1GAP accumulation of genetic alterations, including activating mutations (Fig. 4B, bottom). These data further support the role of rap1GAP in of specific oncogenes, such as K-ras, and loss of function of specific modulating focal adhesion formation and integrin activation in tumor suppressor genes, such as p16, p53, and DPC4 (23). To deter- pancreatic cancer cells. mine the mechanism of rap1GAP expression in pancreatic cancer, Rap1GAP inhibits tumor formation and progression in vivo. we explored whether the epigenetic mechanism of hypermethyla- To determine whether rap1GAP can suppress tumorigenesis and tion at CpG dinucleotide sites in or near key cis-acting trans- metastasis in vivo, we used an orthotopic SCID mouse model. criptional regulatory elements was the cause of inactivation of Mice were injected with 0.5 Â 106 vector-transfected or rap1GAP- rap1GAP, particularly because a CpG-rich island is located 2.5 kb expressing MiaPaCa2 cells into the pancreatic tail. Animals were upstream of the rap1GAP transcription initiation site. Treatment of sacrificed 7 weeks after injection and pathologic examination was four pancreatic cancer cell lines with absent rap1GAP expression

Figure 4. Rap1GAP may function by regulating integrin activity. A, confocal fluorescence microscopy of focal adhesion and F-actin formation in MiaPaCa-2 cells. Focal adhesion formation was detected using antivinculin monoclonal antibody and a FITC-conjugated secondary antibody (green), and F-actin was detected using TRITC-conjugated phalloidin (red). Nuclei were counterstained using DAPI (purple). B, Western blot analysis of the effect of rap1GAP expression on phosphorylation of focal adhesion kinase (p-FAK) following serum exposure for indicated times. The same membrane was stripped and reblotted with anti–total focal adhesion kinase antibody to show equal loading. The experiment was repeated thrice with similar results.

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(MiaPaCa-2, Panc1, BxPC3, and CAPAN) with the DNA methyl- Rap1GAP expression is infrequently lost in PanIN lesions, transferase inhibitor 5-aza-2V-deoxycytidine did not induce suggesting that loss of rap1GAP expression comes at a later stage rap1GAP expression (data not shown). This suggested that in pancreatic cancer progression. Furthermore, using both in vitro hypermethylation may not cause rap1GAP down-regulation in and in vivo models, we have shown that increased rap1GAP pancreatic cancer. expression is associated with the decreased growth, motility, and LOH at loci on chromosome 1p is frequent in pancreatic invasive capability of pancreatic cancer cells, presumably through adenocarcinoma (23, 24) but the gene(s) targeted for inactivation modulating activation of integrins. Finally, we show that there is a by 1p LOH are not known. Rap1GAP is located at human high frequency of LOH of rap1GAP gene at human chromosome chromosome 1 p36.1-p35, a region characterized as a peak of 1p36.1-p35, suggesting that rap1GAP may function as an important LOH frequency in pancreatic cancer. To determine if a mutational tumor suppressor gene in pancreatic cancer. mechanism was responsible for rap1GAP inactivation in pancreatic Rap1 was first identified as a protein that could revert the cancer, we examined the LOH status of rap1GAP in pancreatic morphologic phenotype of Ras-transformed cells, suggesting that cancer. We chose a marker within intron 9 of the rap1GAP gene Rap1 either mediates growth-inhibitory signals or interferes with (Rap1GA1 3+4, GDB:1006983) from the Genome Database and Ras-effector signaling (26). However, later studies suggested that designed PCR primers to amplify this region. DNA was isolated Rap1 delivers an oncogenic signal, making it unclear whether Rap1 from nine primary pancreatic adenocarcinomas using PCR, and the plays a ‘‘good role’’ or is a ‘‘bad actor’’ in cancer. PCR products were analyzed using DNA LabChips. Three of the Alterations in other molecules that regulate Rap1 signaling nine primary tumors analyzed (33.3%) showed LOH, evidenced by have been found to play a role in oncogenesis in a cell type– losing one PCR product from one of two alleles (Fig. 6A and B). specific fashion, including E6TP1, SPA-1, and tuberin (7, 8, 11). One of nine cancers showed homozygous deletion with this Mutations in RapGEFs have also been identified in human tumors marker. Furthermore, six of nine pancreatic cancer cell lines and cancer cell lines. For example, myeloid leukemias in BXH-2 (MiaPaCa-2, Panc1, BxPC3, CAPAN, HPAF-11, and HPAC) also mice contain a proviral insertion of the CalDAG-GEF1 gene and showed LOH with this marker, whereas AsPc1, Su8686, CFPAC, and the leukemia cells exhibited constitutive Rap1 activation (27). the immortalized pancreatic ductal cell line HPDE did not have These studies suggest that increased Rap1 activity correlates with LOH. Lack of rap1GAP expression in pancreatic cancer cell lines oncogenesis. correlated with LOH status (Fig. 6C). The percentage of LOH we Very little is known about the role of rap1GAP in cancer observed was similar to a previous report of LOH frequency (41%) development. Rap1GAP is expressed in a tissue-specific manner at chromosome 1p36.1-p35 in pancreatic cancer (25). These results (28) with low levels of expression in proliferating cells and an suggest that rap1GAP is a putative tumor suppressor gene increase in expression levels with differentiation (6). A recent important in the progression of pancreatic cancer. report by Tsygankova et al. (29) showed that rap1GAP levels are dynamically regulated in thyroid-stimulating hormone–dependent thyroid cells and suggest that rap1GAP may regulate thyroid cell Discussion proliferation and differentiation. In a separate report, cDNA In the current study, we have identified a putative tumor microarrays were used to examine follicle stimulating hormone– suppressor function for rap1GAP, a protein that regulates the Rap1 induced gene expression in ovarian cancer cells compared with signaling cascade, in pancreatic cancer. Specifically, we have shown immortalized normal human ovarian cells (30). Rap1GAP was one that rap1GAP expression is down-regulated in primary pancreatic of nine genes induced by follicle stimulating hormone in normal cancers and pancreatic cancer cell lines compared with normal ovarian cells but not in cancer cells, suggesting that rap1GAP pancreas and chronic pancreatitis and that loss of rap1GAP might possess tumor suppressor activity. In this study, we have expression correlates with poor differentiation status of tumors. shown that rap1GAP is down-regulated in pancreatic cancer and

Figure 5. The effect of rap1GAP expression on tumor growth and metastasis in vivo. A, mean primary tumor size in animals injected with 0.5 Â 106 rap1GAP-transfected or vector-control transfected MiaPaCa-2 cells into the tail of the pancreas of SCID mice (six mice per group). B, a representative example of a resected primary tumor and liver from a SCID mouse injected with wild-type MiaPaCa-2 cells compared with a resected primary tumor from a mouse injected with rap1GAP-expressing cells. Note the large primary tumor with extensive desmoplastic reaction and liver metastases in the control animal, whereas the rap1GAP-overexpressing tumor is much smaller, with no evidence of local invasion and or liver metastases. C, incidence of grossly evident primary tumor, liver, peritoneal, and lymph node metastases (Mets), and evidence of local invasion in control (gray columns) and rap1GAP-expressing tumors (black columns).

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found to be important in the progression and spread of cancer. It has been firmly established that overexpression of active Rap1 activates integrins and that inhibition of Rap1 signaling inhibits integrin function. In the current study, we assessed if the effects of rap1GAP on pancreatic cancer cell function might be mediated through integrin-dependent events (focal adhesion formation and focal adhesion kinase activation). We found that overexpression of rap1GAP in pancreatic cancer cells blocked focal adhesion formation and focal adhesion kinase activation, effects similar to those seen by inhibition of Rap1 signaling in other cell types, supporting the concept that rap1GAP may be functioning solely by altering Rap1 activity levels. Although not addressed in this study, other studies suggest that rap1GAP may have actions distinct from its effects on Rap1. For example, in thyroid cells, whereas overexpression of rap1GAP and the dominant-negative Rap1A17N both impaired Rap1 activity, they had opposite effects on growth rate and differentiated gene expression (33). In addition, in a yeast two-hybrid screen, rap1GAP interacted with the Gz member of the Gi family of trimeric G proteins independent of the ability of rap1GAP to interact with Rap1 (34), raising the possibility that different multiprotein complexes containing rap1GAP may exist and elicit distinct cellular activities. To address mechanisms underlying loss of rap1GAP expression in pancreatic cancer, we examined both epigenetic and mutational mechanisms of rap1GAP gene inactivation. Our data indicate a high frequency of LOH of rap1GAP, with loss of expression of rap1GAP in pancreatic cancer likely due loss of the second allele of the gene. The rap1GAP gene is located on chromosome 1p, which has been long suspected, on the basis of LOH and other data, to harbor a tumor suppressor gene important in pancreatic cancer and other tumors. The chromosomal region in which rap1GAP is located, 1p36-p35, has been identified to site of high frequency not only in pancreatic adenocarcinoma (24, 25) but also in other Figure 6. LOH of rap1GAP gene in pancreatic cancer. A and B, representative pictures show LOH with PCR products from cancer and normal tissue tumors, including neuroblastoma (35, 36), breast cancer (37), analyzed using an Agilent 2100 Bioanalyzer. A larger allele (Nl) and a smaller hepatocellular carcinoma (38), hepatoblastoma (39), and endocrine allele (Ns) are shown as two peaks in (A). PCR products on a pseudogel are neoplasia (40). However, a specific candidate tumor suppressor depicted on the right, demonstrating loss of the larger allele (Tl), whereas a smaller allele (Ts) is still present in the tumor sample. C, immunohistochemical gene within this region has not been identified. Further studies will analysis of rap1GAP staining in rap1GAP LOH negative and positive cells. Lack be needed to assess the LOH status of rap1GAP in other tumors of rap1GAP expression correlates with LOH status. A, 293 cells; B, Su8686 with a high frequency of LOH at chromosome 1p36-35. Because of cells; C, BxPC-3 cells; D, Panc-1 cells. Red, rap1GAP; green, actin. the length and complexity of rap1GAP1 gene (27 exons spanning over 100 kb), significant work will be required to analyze the that rap1GAP likely serves a tumor suppressor function based on mutational status of rap1GAP in pancreatic cancer. our findings that restoration of rap1GAP activity inhibits tumor growth, invasion, and apoptosis in both in vitro and in vivo studies. An important question is whether the effects of rap1GAP are Acknowledgments mediated solely through alterations in Rap1 activity or if rap1GAP Received 8/24/2005; revised 9/26/2005; accepted 10/26/2005. elicits signals distinct from negative regulation of Rap1 activity. Grant support: NIH grant DK061507 (D.M. Simeone) and Michigan Life Sciences Corridor grant MEDC03-622 (D.M. Simeone and C.D. Logsdon). One of the most consistent findings of Rap1 function is its The costs of publication of this article were defrayed in part by the payment of page involvement in integrin-mediated cell adhesion and motility charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (31, 32). Integrins are heterodimeric intracellular molecules that We thank Tom Giordano (Department of Pathology, University of Michigan, Ann possess a unique ability to regulate cell adhesion and have been Arbor, MI) for providing the pancreatic cancer tissue array slides.

References 2. Rubinfeld B, Munemitsu S, Clark R, et al. Molecular a direct target of cAMP in regulated exocytosis. Nat Cell cloning of a GTPase activating protein specific for the Biol 2000;2:805–11. 1. Logsdon CD, Simeone DM, Binkley C, et al. Molecular Krev-1 protein p21rap1. Cell 1991;65:1033–42. 5. Asha H, de Ruiter ND, Wang MG, Hariharan IK. The profiling of pancreatic adenocarcinoma and chronic 3. Altschuler DL, Ribeiro-Neto F. Mitogenic and onco- Rap1 GTPase functions as a regulator of morphogenesis pancreatitis identifies multiple genes differentially genic properties of the small G protein Rap1b. Proc Natl in vivo. EMBO J 1999;18:605–15. regulated in pancreatic cancer. Cancer Res 2003;63: Acad Sci U S A 1998;95:7475–9. 6. Kurachi H, Wada Y, Tsukamoto N, et al. Human SPA-1 2649–57. 4. Ozaki N, Shibasaki T, Kashima Y, et al. cAMP-GEFII is gene product selectively expressed in lymphoid tissues www.aacrjournals.org 905 Cancer Res 2006; 66: (2). January 15, 2006

is a specific GTPase-activating protein for Rap1 and 18. Hruban RH, Goggins M, Parsons J, Kern SE. 30. Ho S-M, Lau K-M, Mok SC, Syed V. Profiling follicle Rap2. Segregate expression profiles from a rap1GAP Progression model for pancreatic cancer. Clin Cancer stimulating hormone-induced gene expression changes gene product. J Biol Chem 1997;272:28081–8. Res 2000;6:2969–72. in normal and malignant human ovarian surface 7. Ishida D, Kometani K, Yang H, et al. Myeloproliferative 19. Yoshida Y, Kawata M, Miura Y, et al. Microinjection of epithelial cells. Oncogene 2003;22:4243–56. stem cell disorders by deregulated Rap1 activation in smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA 31. Reedquist KA, Ross E, Koop EA, et al. The small SPA-1-deficient mice. Cancer Cell 2003;4:55–65. synthesis and morphological changes. Mol Cell Biol GTPase, Rap1, mediates CD31-induced integrin adhe- 8. Singh L, Gao Q, Kumar A, et al. The high-risk human 1992;12:3407–14. sion. J Cell Biol 2000;148:1151–8. papillomavirus type 16 E6 counters the GAP function of 20. Apicelli AJ, Uhlmann EJ, Baldwin RL, et al. Role of the 32. Caron E. Cellular functions of the Rap1 GTP-binding E6TP1 toward small Rap G proteins. J Virol 2003;77: Rap1 GTPase in astrocyte growth regulation. Glia protein: a pattern emerges. J Cell Sci 2003;116:435–40. 1614–20. 2003;42:225–34. 33. Tsygankova OM, Saavedra A, Rebhun JF, Quilliam LA, 9. Zhang Y, Gao X, Saucedo LJ, Ru B, Edgar BA, Pan D. 21. D’Silva NJ, Mitra RS, Zhang Z, et al. Rap1, a small Meinkoth JL. Coordinated regulation of Rap1 and Rheb is a direct target of the tuberous sclerosis tumour GTP-binding protein is upregulated during arrest of thyroid differentiation by cyclic AMP and protein kinase suppressor proteins. Nat Cell Biol 2003;5:578–81. proliferation in human keratinocytes. J Cell Physiol A. Mol Cell Biol 2001;21:1921–9. 10. Manning BD, Cantley LC. Rheb fills a GAP between 2003;196:532–40. 34. Meng J, Glick JL, Polakis P, Casey PJ. Functional TSC and TOR. Trends Biochem Sci 2003;28:573–6. 22. Bos JL, de Bruyn K, Enserink J, et al. The role of Rap1 interaction between Gaz and Rap1GAP suggests a 11. Gutmann DH, Saporito-Irwin S, DeClue JE, Wienecke in integrin-mediated cell adhesion. Biochem Soc Trans novel form of cellular cross-talk. J Biol Chem 1999; R, Guha A. Alterations in the rap1 signaling pathway are 2003;31:83–6. 274:36663–9. common in human gliomas. Oncogene 1997;15:1611–6. 23. Bardeesy N, DePinho RA. Pancreatic cancer biology 35. Fong CT, Dracopoli NC, White PS, et al Loss of 12. Binkley CE, Zhang L, Greenson JK, et al. The and genetics. Nat Rev Cancer 2002;2:897–909. heterozygosity for the short arm of chromosome 1 in molecular basis of pancreatic fibrosis: common stromal 24. Hilgers W, Tang DJ, Sugar AY, Shekher MC, Hruban human neuroblastomas: correlation with N-myc gene expression in chronic pancreatitis and pancreatic RH, Kern SE. High-resolution deletion mapping of amplification. Proc Natl Acad Sci U S A 1989;86: adenocarcinoma. Pancreas 2004;29:254–63. chromosome arm 1p in pancreatic cancer identifies a 3753–7. 13. Zhang L, Duan CJ, Binkley C, et al. Simeone DM. A major consensus region at 1p35. Genes Chromosomes 36. Martinsson T, Sjoberg RM, Hedborg F, Kogner P. transforming growth factor h-induced Smad3/Smad4 Cancer 1999;24:351–5. Deletion of chromosome 1p loci and microsatellite complex directly activates protein kinase A. Mol Cell 25. Hahn SA, Seymour AB, Hoque AT, et al. Allelotype of instability in neuroblastomas analyzed with short- Biol 2004;24:2169–80. pancreatic adenocarcinoma using xenograft enrich- tandem repeat polymorphisms. Cancer Res 1995;55: 14. van Golen KL, Wu ZF, Qiao XT, Bao LW, Merajver SD. ment. Cancer Res 1995;55:4670–5. 5681–6. RhoC GTPase, a novel transforming oncogene for 26. Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y, Noda 37. Nagai H, Negrini M, Carter SL, et al. Detection and human mammary epithelial cells that partially recapit- M. A ras-related gene with transformation suppressor cloning of a common region of loss of heterozygosity at ulates the inflammatory breast cancer phenotype. activity. Cell 1989;56:77–84. chromosome 1p in breast cancer. Cancer Res 1995;55: Cancer Res 2000;60:5832–8. 27. Dupuy AJ, Morgan K, von Lintig FC, et al. Activation 1752–7. 15. Maeda H, Segawa T, Kamoto T, et al. Rapid detection of the Rap1 guanine nucleotide exchange gene, CalDAG- 38. Yeh SH, Chen PJ, Chen HL, Lai MY, Wang CC, Chen of candidate metastatic foci in the orthotopic inocula- GEF I, in BXH-2 murine myeloid leukemia. J Biol Chem DS. Frequent genetic alterations at the distal region of tion model of androgen-sensitive prostate cancer cells 2001;276:11804–11. chromosome 1p in human hepatocellular carcinomas. introduced with green fluorescent protein. Prostate 28. Polakis PG, Rubinfeld B, Evans T, McCormick F. Cancer Res 1994;54:4188–92. 2000;45:335–40. Purification of a plasma membrane-associated GTPase- 39. Kraus JA, Albrecht S, Wiestler OD, von Schweinitz D, 16. Cawkwell L, Lewis FA, Quirke P. Frequency of allele activating protein specific for rap1/Krev-1 from HL60 Pietsch T. Loss of heterozygosity on chromosome 1 in loss of DCC, p53, RBI, WT1, NF1, NM23 and APC/MCC cells. Proc Natl Acad Sci U S A 1991;88:239–43. human hepatoblastoma. Int J Cancer 1996;67:467–71. in colorectal cancer assayed by fluorescent multiplex 29. Tsygankova OM, Feshchenko E, Klein PS, Meinkoth 40. Williamson C, Pannett AA, Pang JT, et al. Local- polymerase chain reaction. Br J Cancer 1994;70:813–8. JL. Thyroid-stimulating hormone/cAMP and glycogen isation of a gene causing endocrine neoplasia to a 4 17. Hattori M, Minato N. Rap1 GTPase: functions, synthase kinase 3h elicit opposing effects on Rap1GAP cM region on chromosome 1p35-p36. J Med Genet 1997; regulation, and malignancy. J Biochem 2003;134:479–84. stability. J Biol Chem 2004;279:5501–7. 34:617–9.

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Lizhi Zhang, Li Chenwei, Redah Mahmood, et al.

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