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ARTICLES |
Analysis of Integrin 
7 Mutations in Prostate Cancer, Liver Cancer, Glioblastoma Multiforme, and Leiomyosarcoma
Affiliations of authors: Departments of Pathology (BR, YPY, CW, KC, UNR, GKM, JHL), Biostatistics (GCT), and Urology (JN), University of Pittsburgh, Pittsburgh, PA
Correspondence to: Jian-Hua Luo, MD, PhD, 3550 Terrace St, Scaife Hall A-725, Pittsburgh, PA 15261 (e-mail: luoj{at}msx.upmc.edu).
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ABSTRACT |
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 Top Abstract Context and CaveatsPatients, materials, and methodsResultsDiscussionReferencesNotes |
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Background: Integrins are the major adhesive molecules in mammalian cells. Each integrin subtype plays a unique role in cell differentiation and embryo development. However, integrin involvement in carcinogenesis has not been well defined.
Methods: We identified mutations in integrin 
7 by sequencing genomic DNAs and cDNAs from 122 specimens, including 62 primary human tumor samples, four cell lines, and 56 matched normal tissues. We evaluated the tumor suppressor activity of integrin 7 with colony formation, soft agar colony growth, and cell migration assays by forcing its expression in PC-3 and Du145 prostate cancer cells and SK-UT-1 leiomyosarcoma cells. PC-3 and Du145 xenograft tumors with increased levels of integrin 7 in severe combined immune deficient mice were used to assess the effect of integrin 7 on tumor growth and metastasis. Immunostaining was used to localize and to measure the level of integrin 7 in 701 and 141 specimens of prostate and smooth muscle, respectively. A meta-analysis of integrin 7 mRNA microarray data from four studies was performed. Kaplan–Meier analyses were used to assess survival. All statistical tests were two-sided.
Results: Integrin 7 mutations that generate truncations were found in specimens of 16 of 28 prostate cancers (57%, 95% confidence interval [CI] = 37% to 76%), five of 24 hepatocellular carcinomas (21%, 95% CI = 7% to 42%), five of six glioblastomas multiforme (83%, 95% CI = 36% to 99%), and one of four leiomyosarcomas (25%, 95% CI = 0.6% to 81%). Integrin 7 mutations were associated with increased recurrence of human prostate cancer (nine recurrences among 13 patients with integrin 7 mutations versus one among eight without such mutations; odds ratio [OR] = 14, 95% CI = 1.15 to 782, P = .024) and hepatocellular carcinoma (five recurrences among eight patients with integrin 7 mutations versus one among 16 without such mutations, OR = 21, 95% CI = 1.6 to 1245; P = .007). Forced expression of normal integrin 7 in prostate cancer and leiomyosarcoma cell lines suppressed tumor growth and metastasis both in vitro and in vivo. Focal or no integrin 7 expression in human prostate cancer and soft tissue leiomyosarcoma was associated with a reduction of metastasis-free survival (for example, for prostate cancer with focal or no expression, 5-year metastasis-free survival was 32%, 95% CI = 24.4% to 40.3%, and for prostate cancer with at least weak expression, it was 85%, 95% CI = 79% to 91%; P<.001). Microarray analysis indicated that cyclin D kinase inhibitor 3 and GTPase-activating protein may be possible targets for integrin 7–mediated tumor suppressor activity and inhibition of cell motility.
Conclusion: Integrin 7 appears to be a tumor suppressor that operates by suppressing tumor growth and retarding migration.
Prior knowledge
Integrin Study design
Molecular, cellular, human tumor specimen, and human xenograft tumor studies of integrin Contribution
Integrin Implications
Impairing the function of integrin Limitations
The signaling pathway used by integrin
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As a major class of adhesive molecules in mammalian cells, the integrins are involved in many cellular processes, including development, immune responses, leukocyte traffic, and hemostasis (1). Integrin knockout mice have distinctive developmental defects, including kidney tubule defects, severe skin blistering, chylothorax, and muscular dystrophy (2–7). The integrin superfamily contains 24 members, each of which mediates a unique function in mammals. For example, integrins 3, 6, or 7 combine with a 
1 subunit to form receptors for laminin; and the combination of a 1 subunit with 1, 2, 10, or 11 forms a receptor for collagen; heterodimers between 2 and L, M, X, or D form leukocyte-specific receptors; and heterodimers between V and several subunits form the RGD tripeptide receptor. Regulation of integrin expression is critical for certain aspects of tissue differentiation and regeneration [e.g., keratinocyte differentiation, hair follicle formation, and skeletal muscle development (8–10)], and abnormal integrin expression is associated with several human diseases [e.g., muscular dystrophy, Glanzmann thrombasthenia, and congenital cardiac myopathy (10–12)]. Integrin 7 is thought to be involved in smooth and skeletal muscle development (10,13). Very little is known about the role of integrin 7 in other tissues and organs.
Integrin 7 forms a heterodimer with integrin 1 in the plasma membrane and is responsible for communication between extracellular matrix and muscle cells (14). There are two distinct isoforms of integrin 7 that are generated by two mutually exclusive alternative splicings. Whether integrin 7 has a role in the development of cancer is largely unknown. However, the expression of integrin 7 has been shown to be altered in some malignances [e.g., human leiomyosarcoma and prostate cancer (15–18)]. To investigate the role of integrin 7 in human malignancies, we examined whether this gene is mutated in specimens of various human cancers, the level of integrin 7 expression is associated with clinical relapse of human malignancies, and integrin 7 has tumor suppressor activity.
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Patients, Materials, and Methods |
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Cells, Culture Conditions, and Antibodies
All cell lines, including PC-3 (prostate cancer), Du145 (prostate cancer), LNCaP (prostate cancer), SK-UT-1 (leiomyosarcoma), H1299 (lung cancer), and H358 (lung cancer), were purchased from American Type Cell Culture (Manassas, VA). PC-3 cells were cultured with F12K medium supplemented with 10% fetal bovine serum (InVitrogen, Carlsbad, CA). Du145 and SK-UT-1 cells were cultured with modified Eagle medium supplemented with 10% fetal bovine serum (InVitrogen). LNCaP, H358, and H1299 cells were cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum (InVitrogen). The 1573 cells, a renal cell carcinoma cell line, were cultured with modified Eagle medium supplemented with 10% fetal bovine serum (InVitrogen). SW-33, SW39, SW40, SW61, SW94, and SW95 (glioblastoma multiformes) were obtained from University of Pittsburgh Hillman Cancer Center and cultured in modified Eagle medium supplemented with 10% fetal bovine serum (InVitrogen).
Rabbit anti–integrin 7 serum (polyclonal) was raised through immunization of a rabbit with the synthetic peptide GTILRNNWGSPRREGPDAH. Mouse anti–integrin 7 monoclonal antibody was purchased from Novus Biologicals Inc (Littleton, CO). Mouse anti–cyclin D kinase inhibitor 3 (CDKN3) monoclonal antibody was purchased from Abnova Corp (Taipei, Taiwan). Goat anti–GTPase-activating protein (RACGAP1) antibody (polyclonal) was purchased from Abcam (Cambridge, MA). Goat anti–integrin 1 (polyclonal) and mouse anti–-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).
Human Cancer Patients and Specimens
The prostate cancer specimens that were analyzed had been archived as frozen or formalin-fixed paraffin-embedded specimens of tissues from radical prostatectomies from 1985 through 2000. Specimens were selected largely on the basis of their availability or whether sufficient amounts of tumor tissues were present. The ages of patients at the time of surgery ranged from 45 through 79 years. In total, 435 samples were collected. Two hundred ninety-four of the 435 corresponding patients were followed clinically for at least 5 years. Hepatocellular carcinoma specimens were analyzed that had been archived as frozen specimens of liver tissue resections from 1997 through 2002. In total, 24 specimens were collected, and the corresponding patients were followed clinically for at least 5 years. Soft tissue leimyosarcomas were analyzed that had been archived as frozen or formalin-fixed paraffin-embedded specimens of tumor tissue resections from 1970 through 2000. One hundred eleven samples were collected. Sixty-four of the 111 corresponding patients were followed clinically for at least 5 years. Six glioblastoma multiforme specimens were analyzed that had been archived as frozen specimens of tissue resections from 1998 through 2002. Four separate study protocols, all of which included informed consent exemptions, were approved by institutional review board.
Tissue Sample Preparation
Pure tumor specimens were obtained by dissecting freshly resected tissues, typically within 30 minutes of removal from patients. These tissues were frozen at –80 °C and were selected on the basis of tissue availability. Tissues were retrieved and microdissected immediately before the extraction of DNA or total RNA. Tumor cells were microdissected from frozen sections on slides by board-certified pathologists (J.-H. Luo and G. K. Michalopoulos). For matched normal samples, different tissue lineages from the tumor or blood cells (e.g., fat, blood vessels, and seminal vesicles) were obtained. Protocols for tissue banking (which was used for pathology), deidentification, and processing (for molecular analyses) were approved by the institutional review board. The study protocols were exempted from informed consent.
Sequencing Integrin 7 Genomic DNA and cDNA
Genomic DNA and total RNA were extracted from various tissues (i.e., prostate, liver, leiomyosarcoma, and glioblastoma multiforme) by use of a QiAmp blood kit and a RNeasy kit from Qiagen (Valencia, CA), respectively, according to the manufacturer's instructions. Five micrograms of total RNA was used for first-strand cDNA synthesis with d(T)24 primer and Superscript II reverse transcriptase (200 U; GIBCO-BRL, Rockville, MD). Second-strand cDNA synthesis was carried out at 16 °C by adding Escherichia coli DNA ligase (10 U), E. coli DNA polymerase I (40 U), and RNAse H (2 U) to the reaction mixture. T4 DNA polymerase (10 U in 20 µL) was added to blunt the ends of newly synthesized cDNA, and the cDNA was purified by phenol–chloroform extraction and ethanol precipitation. Purified genomic DNA or cDNA from various tissues served as templates for polymerase chain reactions (PCRs) that used a total of 31 sets of primers (Supplementary Table 1, available online) corresponding to the 27 exons of integrin 7. Each PCR product was gel purified by use of the Geneclean purification kit (Qbiogene, Irvine, CA) and then sequenced by use of the corresponding primers as described below. For cDNA sequencing, purified total RNA from various tissues was reverse transcribed with random hexamers (17) for double-stranded cDNA synthesis. PCR mixtures contained the cDNA templates and six sets of primers (Supplementary Table 1, available online) distributed along the entire integrin 7–coding region. Automated sequencing of all PCR products used 500 ng of DNA and the BigDye terminator 1.1 cycle sequencing kit (ABI, Foster City, CA), as described by the manufacturer. The fluorescence-labeled PCR products were separated by electrophoresis in 6% polyacrylamide gels and analyzed with an ABI Prism 377 DNA sequencer. When a mutation was identified in a genomic sample, cDNAs were prepared from the corresponding tissue, and the entire integrin 7 coding region was sequenced as described above. Mutations in alleles were determined by clonal sequencing of PCR products (cDNA or genome DNA) by use of primers encompassing the region of both mutations. Loss of heterogeneity was determined by comparing single-nucleotide polymorphisms in the introns or exons of integrin 7 between matched normal and tumor samples. For reverse transcription–PCR (RT–PCR) analysis of integrin 7 expression, the cDNAs from 16 cell lines (including PC3, Du145, LNCaP, H23, H522, H358, H1299, SK-UT-1, Hep3G, 1573, SW-33, SW39, SW40, SW61, SW94, and SW95) were synthesized as described above. cDNAs from 20 organs (including bone marrow, cerebellum, fetal brain, fetal liver, heart, kidney, lung, placenta, prostate, salivary gland, skeletal muscle, spleen, testis, thyroid, trachea, uterus, colon, small intestine, spinal cord, and stomach) were obtained from Clontech (Mountain View, CA). PCRs were performed with primers specific for integrin 7 (Supplementary Table 1, available online).
Peptide Antibodies Against Integrin 7
The peptide GTILRNNWGSPRREGPDAH, which corresponds to amino acids 1097–1115 of human integrin 7, was chemically synthesized and purified by high-pressure liquid chromatography at the University of Pittsburgh biotechnology support center. Rabbit antiserum against this peptide was raised by Cocalico Biologicals, Inc (Reamstown, PA). Antibodies against integrin 7 were purified by use of the synthetic peptide and a Carboxylink kit from Pierce (Rockford, IL). The specificity of rabbit preimmune serum and anti–integrin 7 antiserum was tested on immunoblots of PC-3 and 1573 cell protein extracts. Integrin 7 bands were specifically detected in extracts from both 1573 and PC-3 cells with anti–integrin 7 antiserum and with a monoclonal antibody against integrin 7 (a positive control), but no visible integrin 7 band was detected with either preimmune serum or antiserum depleted of integrin 7 peptide antibodies (see Supplementary Fig. 1, A; available online).
Immunohistochemistry and Tissue Array Analysis
For tissue microarray analysis, 701 formalin-fixed and paraffin-embedded prostate tissue specimens (407 from prostate cancer tissue and 294 from normal prostate tissue as described above) were arrayed onto six slides, with one or two samples from each specimen (19). Patients in this group ranged in age from 45 to 79 years, and complete 5-year follow-up data was available for 266 patients with prostate cancer (University of Pittsburgh Medical Center tissue collection archive, 1985 through 2000). Tissue array slides and thin sections of paraffin-embedded tissues were used to study soft tissue leiomyosarcoma specimens (34 normal tissue samples and 107 leiomyosarcomas, including samples from 60 patients with >5 years of follow-up). These specimens were arrayed onto three slides, with two samples from each specimen. Immunohistochemistry was performed as described previously (20) with purified integrin 7 peptide antiserum (1:1000 dilution). The peptide antibody was omitted in negative controls. The sections were then incubated with horseradish peroxidase–conjugated anti-rabbit IgG for 30 minutes at room temperature. Slides were exposed to a 3,3'-diaminobenzidine solution to visualize immunostaining. Integrin 7 immunostaining was graded on a scale of 0–3 as follows: 0 = no expression; 0.5 = focal positive; 1 = weak; 2 = moderate; 3 = strong. A threshold of score of 0.5 was used to determine the likelihood of tumor relapse, and it was chosen so that two groups had balanced sample sizes. Moving the threshold to 0 or 1 resulted in a similar conclusion (data not shown).
To construct the inducible integrin 7 expression vector pCDNA4-ITGA7, full-length integrin 7 cDNA was ligated at the NotI and KpnI sites of pcDNA4/TO/MYC/HIS-B (Invitrogen, CA). This plasmid was then cotransfected into PC-3 cells with pCDNA6/TR, which encodes the tetracycline repressor. Transfected cells were selected by use of zeomycin (pCDNA4/TO/MYC/HIS-B–transfected cells) and blasticidin S (pCDNA6/TR-transfected cells) (Invitrogen). Selected clonal cell lines, including two that were designated PITT1 and PITT2, were tested for doxycycline inducibility (1 µg/mL) by western blot analysis with antibodies specific for integrin 7 or -actin (the loading control) and by immunofluorescence analysis (Supplementary Fig. 1, B; available online). Coimmunoprecipitations using anti–integrin 7 antibodies indicated that integrin 7 and integrin 1 formed a protein complex because immunoprecipitates contained integrin 1 protein (Supplementary Fig. 1, C; available online).
Immunofluorescence Localization of Integrin 7
PITT1 cells, in which integrin 7 expression can be induced by treatment with tetracycline at 1 µg/mL, were used for these experiments. Cells were grown on covered slides in the presence of tetracycline, fixed with 3% paraformaldehyde, and then blocked with normal donkey serum for 30 minutes at 4 °C. Anti-ITGA7 serum or preimmune rabbit serum (as the control) was added, and slides were incubated for 1 hour at 4 °C. After three washes with phosphate-buffered saline (PBS), rhodamine-conjugated donkey anti-rabbit secondary antibodies were added and incubated for 1 hour at 4 °C. After three washes with PBS, immunofluorescence staining was visualized under an Olympus fluorescence inverted microscope IX (B & B Microscopes, Ltd, Pittsburg, PA) (Supplementary Fig. 1, B; available online).
Immunoblot Analysis of Integrin 7, Cyclin D Kinase Inhibitor 3, Rac GTPase-Activating Protein 1, and -Actin
Integrin 7 expression was examined in PC3, DU145, 1573, SK-UT-1, H1299, and H358 cells. First, cells were washed with PBS and lysed by RIPA buffer (50 mM Tris–HCl at pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, aprotinin at 1 µg/mL, leupeptin at 1 µg/mL, pepstatin at 1 µg/mL, and 1 mM Na3VO4). The lysates were sonicated and centrifuged at 12000g at 4 °C for 30 minutes to remove the insoluble materials. The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) in 8.5% polyacrylamide gels, and bands were blotted onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% powdered skim milk in Tris–Tween 20 buffer (0.1 M Tris–HCl and 0.1% Tween-20 [pH 7.4]) for 1 hour at room temperature, followed by a 2-hour incubation with primary anti-ITGA7 antibodies (1:1000 dilution), anti-CDKN3 antibodies (1:1000 dilution; Abnova Corp), or anti-RACGAP1 antibodies (1:500 dilution; Abcam). The membrane was then washed three times with Tris–Tween 20 buffer and incubated with a horseradish peroxidase–conjugated secondary antibody specific for rabbit (anti-ITGA7, 1:1000 dilution), mouse (anti-CDKN3, 1:1000 dilution), or goat (anti-RACGAP1, 1:1000 dilution) for 1 hour at room temperature. The protein expression was detected with the ECL system (Amersham, Life Science, Piscataway, NJ) according to the manufacturer's protocols.
Colony Formation, Soft Agar Anchorage–Independent, and Wound-Healing Assays
Colony formation and soft agar anchorage–independent assays were similar to those previously described (20). PC-3, Du145, and SK-UT-1 cells that were transfected with pCMVscript or pCMV-integrin 7 and H1299 and H358 cells that were transfected with pENTR-siITGA7 were used. For colony formation assay, 5000 cells were cultured in 60-mm dishes. Triplicate experiments were performed for each cell clones. Medium was changed every 4 days. On the 10th day, the plates were stained with 1% crystal violet, and colonies with diameter of more than 2 mm were counted.
For the soft agar colony formation assay, the same cell lines were used. In brief, 5000 cells were cultured on a plate containing 2% base agar and 0.43% top agar in the medium described above and incubated at 37 °C for 21 days. Plates were stained with 0.005% crystal violet for 1 hour. Colonies were counted by use of a dissecting microscope.
For the wound-healing assay (21), Du145, PC-3, or SK-UT-1 cells were cultured in six-well culture plates in the medium described above. After cells reached confluence, a plastic pipette tip was drawn across the center of the well to produce a clean crevice that was 1 mm wide. Microscopic images of the "wounds" were taken in five different areas for each experiment (at an original magnification of x10 with an Olympus inverted system microscope IX). After culturing for 24 hours at 37 °C in F12K medium (PC-3 cells) or modified Eagle medium (Du145 and SK-UT-1 cells) containing 10% fetal bovine serum, images of original locations were taken again, and recovered areas (i.e., the bare area into which cells migrated) were measured as a percentage of the original wound.
Construction of Integrin 7–Expressing Cell Lines and Small Interfering RNA Vectors
Integrin 7 cDNA was generated from total RNA from normal donor prostate tissue by extended long PCR (20) with primers specific for the 5' and 3' ends of integrin 7. The 3.7-kilobase PCR product was ligated into a TA cloning vector (Invitrogen) and from there cloned into a pCMVscript vector (Clontech) with HindIII and XhoI (New England Biolab, Ipswich, MA). The final pCMV-ITGA7 construct was sequenced by the automatic sequencing method, as described above, to confirm that no mutations had been introduced. This construct was transfected into Du145, PC-3, or SK-UT-1 cells. Colonies containing pCMV-ITGA7 were selected for with medium that included G418 (400 µg/mL).
To construct the small interfering RNA (siRNA) vectors for CDKN3, RACGAP1, integrin 7, and a scrambled control sequence, oligonucleotides corresponding to the following regions of CDKN3 mRNA (5'-CACCGGAGCTTACAACCTGCCTTAAATTGATATCCGTTTAAGGCAGGTTGTAAGCTC-3'/5'-AAAAGAGCTTACAACCTGCCTTAAACGGATATCAATTTAAGGCAGGTTGTAAGCTCC-3'), RACGAP1 (5'-CACCGTTTGCACTTTGGATGCTGAAATTGATATCCGTTTCAGCATCCAAAGTGCAAA-3'/5'-AAAATTTGCACTTTGGATGCTGAAACGGATATCAATTTCAGCATCCAAAGTGCAAAC-3'), integrin 7 (5'-CACCGACTCCCAACCACTGGTTCTCCTTGCCGAAGCAAGGAGAACCAGTGGTTGGGAGT-3'/5'-AAAAACTCCCAACCACTGGTTCTCCTTGCTTCGGCAAGGAGAACCAGTGGTTGGGAGTC-3'), or scrambled siRNA (5'-CACCGTAATGTATTGGAACGCATATTTTGATATCCGAATATGCGTTCCAATACATTA-3'/5'-AAAATAATGTATTGGAACGCATATTCGGATATCAAAATATGCGTTCCAATACATTA-3') were annealed and ligated into a pENTR/U6 vector. The ligated products were transfected into E. coli and plated on kanamycin plates (50 µg/mL). Six colonies per transfection were picked and sequenced for the presence of inserts. The selected clones, which suppress the expression of integrin 7 (ITGA7), CDKN3, or RACGAP1, respectively, were then transfected into cultured cells to generate pENTR-siITGA7–transfected H1299 or H358 cells or pENTR-siCDKN3– and pENTR-siRACGAP1–transfected PITT1 and PITT2 cells.
Coimmunoprecipitation
We investigated the possibility that overexpression of integrin 7 could abrogate formation of the 71 heterodimer by use of protein extracts that were obtained as described above. Protein extracts that were prepared from PITT1 cells that had been induced with tetracycline to express integrin 7 were incubated with anti-ITGA7 antibody for 16 hours and then with protein G-Sepharose beads for 3 hours to immunoprecipitate integrin 7 complex. The complex was washed five times with RIPA buffer, and the bound proteins were eluted from the beads with SDS–PAGE sample buffer. The precipitated complexes were separated by SDS–PAGE, electroblotted to a polyvinylidene difluoride membrane, and immunoblotted with anti-integrin 1 antibodies (1:500 dilution, Santa Cruz Biotechnology, Inc; Supplementary Fig. 1, C; available online). The membrane was then washed three times with Tris–Tween 20 buffer and incubated with a horseradish peroxidase–conjugated secondary antibody specific for goat antibodies (1:1000 dilution) for 1 hour at room temperature. The coimmunoprecipitated integrin 1 was detected with the ECL system (Amersham Life Science), according to the manufacturer's protocols.
Tumor Growth and Spontaneous Metastasis
Approximately 1 x 107 viable PC-3 and Du145 cells, suspended in 0.2 mL of Hanks’ balanced salt solution (Krackeler Scientific, Inc, Albany, NY) were subcutaneously implanted in the abdominal flanks of 48 severe combined immune deficient (SCID) mice to generate one tumor per mouse. Mice were observed daily, and their body weight, tumor size, and lymph-node enlargement were recorded weekly. Tumor and lymph node size were measured on the diameter. After 6 weeks or when mice became moribund, which ever occurred first, mice were killed, and necropsies were performed. Serial sections of formalin-fixed, paraffin-embedded lung, brain, liver, kidney, vertebra, and lymph node specimens were collected, stained with hematoxylin and eosin, and examined microscopically. All animal procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
mRNA Microarray Analysis
Total RNA was extracted from uninduced and induced PITT1 cells and purified with Qiagen RNeasy kit (Qiagen). Five micrograms of total RNA was used for first-strand cDNA synthesis with T7-d(T)24 primer [GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24] and Superscript II reverse transcriptase (200 U; GIBCO-BRL). Second-strand cDNA synthesis was carried out at 16 °C by adding E. coli DNA ligase (10 U), E. coli DNA polymerase I (40 U), and RNAse H (2 U) to the reaction mixture. T4 DNA polymerase (10 U in 20 µL) was added to blunt the ends of newly synthesized cDNA, and the cDNA was purified by phenol–chloroform extraction and ethanol precipitation. Purified cDNAs were then incubated at 37 °C for 4 hours in an in vitro transcription reaction mixture containing 10 mM ATP, 10 mM biotin–CTP, 10 mM GTP, and 10 mM biotin–UTP to produce biotin-labeled complementary RNA (cRNA) by use of the MEGAscript system (Ambion, Inc, Austin, TX). cRNA (15–20 µg) was fragmented by incubating in a buffer containing 200 mM Tris–acetate (pH 8.1), 500 mM potassium acetate, and 150 mM magnesium acetate at 95 °C for 35 minutes. The fragmented RNA was then hybridized to a preequilibrated Affymetrix chip (u133 2.0) at 45 °C for 14–16 hours. After the hybridization buffer was removed, the chips were washed in a fluidic station with a low stringency buffer (6x SSPE [5.25% NaCl, 0.83% sodium phosphate, and 0.22% EDTA], 0.01% Tween-20, and 0.005% antifoam) for 10 cycles (two automated mixes per cycle) and in a stringent buffer (100 mM morpholinoethanesulfonic acid, 0.1 M NaCl, and 0.01% Tween-20) for four cycles (15 automated mixes per cycle), and stained with streptoavidin-conjugated phycoerythrin to identify hybridized biotin–labeled cRNA. This procedure was followed by an incubation with biotinylated mouse anti-avidin antibody and restaining with streptoavidin-conjugated phycoerythrin to amplify the signal for hybridized biotin-labeled cRNA. The chips were scanned in a HP ChipScanner (Affymetrix Inc, Santa Clara, CA) to detect hybridization signals. Hybridization data were normalized to an average target intensity of 500 per chip, and then analysis of induced versus uninduced PITT1 cells at baseline was performed with the program GCOS version 1.0.
A PubMed search was conducted to identify articles containing Affymetrix datasets on human leiomyosarcoma or prostate cancer by use of the following search terms: "Affymetrix," "primary prostate cancer," and "primary leiomyosarcoma." Seven relevant articles about prostate cancer and one for human soft tissue leiomyosarcoma were found. Four sets of data from these eight articles were selected because of their availability. Among them, three were from University of Pittsburgh and one was from Memorial Sloan-Kettering Cancer Institute. For meta-analysis, Affymetrix CEL files of all samples from the four articles (15–17,19) were reanalyzed with GCOS version 1.0 and normalized to an average target intensity of 500 for each sample. The results were exported to Microsoft Excel for statistical analysis. Fold changes of integrin 7 in tumor samples were calculated as average intensity of tumor samples over average of the normal controls in the same set of data. Two-sided Student's t tests were performed to obtain P values. Confidence intervals (CIs) were calculated as described below. The number of samples analyzed from each article was as follows: 156 samples from Yu et al. (17), 30 from Luo et al. (16), 26 from LaTulippe et al. (15), and 29 from Ren et al. (19).
Statistical Methods
Confidence intervals for individual proportions were calculated by use of the exact binomial test (function "binom.test" in R package of statistical computer programs), and those for a numerical distribution were calculated with conventional independent and normal assumption (i.e., mean ± [1.96 x SD/n1/2], where SD = standard deviation and n = sample size) (18). Comparison of two proportions was inferred by Fisher's exact test because of relatively small sample size (function "fisher.test" in R package) (18). The odds ratio (OR) estimates and the confidence intervals were inferred by conditional maximum likelihood estimate rather than conventional sample odds ratio. Survival was analyzed by the Kaplan–Meier method, and survival curves were compared by use of the log-rank test (22). All statistical tests were two-sided.
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Mutations of Integrin
7 in Human Malignancies
To investigate whether qualitative alterations in the integrin 7 gene occur in human cancers, we sequenced integrin 7 genomic DNA and cDNA from 66 human cancer specimens (including 28 prostate cancers, 24 hepatocellular carcinomas, six glioblastoma multiformes, and four leiomyosarcomas specimens) and cancer cell lines (including PC3, Du145, and LNCaP cells derived from prostate cancers and SK-UT-1 cells derived from a leiomyosarcoma). In addition, integrin 7 genomic DNA and cDNA from 56 specimens from matched nontumor tissues were sequenced. Two types of alterations in the integrin 7 sequence were associated with human malignancies: changes in the amino acid sequence caused by missense mutations and protein truncations caused by nonsense, deletion, or insertion mutations (Supplementary Fig. 2, A and B; available online).
Integrin 7 contains only 50 amino acid residues in its carboxyl-terminal cytoplasmic domain; truncations in this domain should adversely affect in its signal transduction ability and other functions. Because truncation mutations have the strongest impact on the structure of the protein, we focused our analysis on such mutations. Truncation mutations of integrin 7 occurred at high frequency in samples from human malignancies (Table 1). In the prostate cancer specimens, the rate of integrin 7 mutations was 57% (95% CI = 37% to 76%; i.e., 16 mutations in 28 specimens). In glioblastoma multiforme specimens, the rate reached 83% (95% CI = 36% to 99%; i.e., five mutations in six specimens); in leiomyosarcoma specimens, the rate was 25% (95% CI = 0.6% to 81%; i.e., one mutation in four specimens); and in hepatocellular carcinoma specimens, the rate was 21% (95% CI = 7% to 42%; i.e., five in 24 specimens). All of these mutations had major structural consequences (Supplementary Fig. 2, A; available online), including protein truncation because of a premature stop codon, a frameshift because of deletions or insertions of nucleotides, or loss of the translational start site.
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The integrin
7 mutations were spread across the coding region, but a hot spot (n = 9 specimens) was identified in codon 921, in which a glutamine codon was mutated to a stop codon. Fourteen samples contained both truncation and missense mutations, 10 of which were identified as mutations in separate alleles. Prostate cancers with integrin 7 mutations, compared with those without such a mutation, were generally less differentiated (Fisher's exact test, P = .009), had a more advanced stage (P = .005), and were more likely to be associated with relapse (nine recurrences among 13 patients with integrin 7 mutations versus one among eight without such mutations; odds ratio = 14, 95% CI = 1.15 to 782, P = .024). However, the only association for hepatocellular carcinomas with integrin 7 mutations was with shorter relapse-free survival than tumors without such mutations (five recurrences among eight patients with integrin 7 mutations versus one among 16 without such mutations, OR = 21, 95% CI = 1.6 to 1245; P = .007) (Table 2).
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PC-3 cells contain a frameshift mutation at codon 759 in one integrin
7 allele, and Du145 cells contain a two–amino acid deletion mutation in integrin 7. SK-UT-1 cells have a premature stop codon at position 350 in one integrin 7 allele, so that integrin 7 protein is expressed only from the remaining nonmutated allele. Cell lines H1299 and H358 express normal wild-type levels of integrin 7 and lack integrin 7 mutations. To examine the effect of alterations in the level of integrin 7 mutations on tumorigenesis (as assessed by colony formation and growth in soft agar), we increased the level of integrin 7 in the deficient cell lines (i.e., PC3, Du145, and SK-UT-1) to normal wild-type levels by use of an integrin 7 expression vector (pCMV-integrin 7 vector) or decreased its level by 70% by use of siRNA against integrin 7. For the cells with deficient levels of integrin 7, we selected PC-3 and Du145 cells, which were derived from human metastatic prostate cancers, and SK-UT-1 cells, which were derived from a human leiomyosarcoma (Supplementary Fig. 2, A; available online). For cell lines with normal levels of integrin 7 expression, we selected H1299 and H358 cells, which were derived from human lung cancers. We first increased integrin 7 expression to normal wild-type levels in PC-3, Du145, and SK-UT-1 cells by transfecting them with an integrin 7 expression vector (pCMV-integrin 7 vector) and then compared the ability of these cells to form colonies and grow on soft agar with that of corresponding pCMVscript-transfected control cells (Fig. 1, A). In the colony formation assay, the rate of colony formation was reduced by 7.1-fold (95% CI = 4.91-fold to 9.38-fold) in integrin 7–transfected PC-3 cells as compared with pCMVscript-transfected control PC-3 colonies, by 6-fold (95% CI = 3.87-fold to 8.13-fold) in integrin 7–transfected Du145 cells as compared with pCMVscript-transfected control Du145 colonies, and by 5.9-fold (95% CI = 5.59-fold to 6.28-fold) in integrin 7–transfected SK-UT-1 cells as compared with pCMVscript-transfected control SK-UT-1 colonies. In the soft agar growth assay, pCMVscript-transfected control cells formed large colonies with up to 100 cells on soft agar, but integrin 7–transfected cells with higher (normal) levels of integrin 7 expression formed fewer and smaller colonies (Fig. 1, B). Specifically, for PC-3 cells, there was a 3.8-fold (95% CI = 3.15-fold to 4.39-fold) reduction in colony formation; for Du145 cells, there was a 3.2-fold (95% CI = 2.83-fold to 3.62-fold) reduction; and for SK-UT-1 cells, there was a 2.6-fold (95% CI = 2.25-fold to 2.80-fold) reduction. In a second set of experiments, we decreased the level of integrin 7 in H1299 and H358 cells by transfecting cells with integrin 7–specific siRNAs or scrambled siRNAs expressing vectors and then investigated the colony formation ability and growth on soft agar of these cells. Both integrin 7–specific siRNA-expressing cell lines formed more colonies and grew better on soft agar than their corresponding scramble control cell lines (Fig. 1).
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To investigate the role of integrin
7 in metastasis, we examined the relationship between the level of integrin 7 expression and cell migration by use of wound-healing assays with PC-3, Du145, SK-UT-1, H1299, and H358 cells. When the expression of integrin 7 was increased in PC-3, Du145, and SK-UT-1 cells with low integrin 7 expression by transfecting cells with integrin 7 expression vectors, the rate of migration, compared with that in corresponding pCMVscript-transfected cells, was reduced by 5.4-fold (95% CI = 4.68-fold to 6.19-fold), 4.3-fold (95% CI = 3.86-fold to 4.64-fold), and 11.7-fold (95% CI = 5.59-fold to 17.85-fold), respectively (Fig. 1, C). When integrin 7 expression in H1299 and H358 cells, which express a normal level of integrin 7 and have low motility, was decreased by transfection with an integrin 7–specific siRNA increased the rate of migration by 2-fold (95% CI = 1.57-fold to 2.41-fold) compared with that of corresponding scrambled siRNA-transfected control cells. Thus, the level of integrin 7 expression appears to be inversely associated with tumor cell migration.
To investigate the tumor suppressor activity of integrin 7, we generated xenograft tumors in SCID mice implanted with vector-transfected PC-3 and Du145 prostate cancer cells and corresponding cells transfected with integrin 7 expression constructs and then compared the volume of tumors as a function of integrin 7 expression. Six weeks after implantation, tumors from integrin 7–transfected Du145 cells had an average volume of 0.8 cm3, and tumors from vector-transfected Du145 cells had an average volume of 2.2 cm3 (difference = 1.4 cm3, 95% CI of difference = 0.9 to 2.1, P = .001) (Fig. 1, D). Similarly, 6 weeks after implantation, the volume of tumors from integrin 7–transfected PC-3 cells was 0.7 cm3 and that from vector-transfected PC-3 cells was 2.9 cm3 (difference = 2.2, 95% CI of difference = 1.5 to 2.9, P<.001) (Fig. 1, D). No visible metastases were identified in mice with integrin 7–transfected Du145 or PC-3 tumors. However, metastasis was observed in three (25%) of the 12 mice with vector-transfected Du145 tumors and in four (33%) of the 12 mice with vector-transfected PC-3 tumors (Fig. 1, E). Furthermore, the 6-week survival of mice bearing integrin 7–transfected Du145 tumors (83%, 95% CI = 62% to 100%) or PC-3 tumors (92%, 95% CI = 76% to 100%) was higher than that of mice bearing tumors from the corresponding vector-transfected cells tumors (25%, 95% CI = 0.5% to 49.5%, and 42%, 95% CI = 13.8% to 69.5%) (Fig. 1, F). Thus, increased integrin 7 was associated with decreased tumor growth and metastasis in vivo.
To determine whether the expression of integrin 7 alters the global gene expression profile, we transfected PC-3 and SK-UT-1 cells with a tetracycline-inducible integrin 7 expression vector (pCDNA4-ITGA7) and used microarray analysis to compare gene expression in these cells in the presence of tetracycline with that in uninduced cells. Within 24 hours of integrin 7 induction, we found a 6.1-fold (95% CI = 5.5-fold to 6.8-fold) increase in the expression of CDKN3 mRNA in induced PC-3 cells transfected with pCDNA4-ITGA7 compared with uninduced cells (that is, 8593 arbitrary units in induced cells and 1408 arbitrary units in uninduced cells) and a 5.8-fold (95% CI = 5.23-fold to 6.41-fold) increase in the expression of CDKN3 protein (with CDKN3/-actin ratio increasing from 0.043 in noninduced cells to 0.249 in induced cells) (Fig. 2, A). We found a 5-fold (95% CI = 4.26-fold to 5.70-fold) increase in CDKN3 protein expression in SK-UT-1 cells transfected with integrin 7 compared with the same cells transfected with vector control (with the CDKN3/-actin ratio increasing from 0.042 in uninduced cells to 0.214 in induced cells). Within 24 hours of integrin 7 induction, we also found a 3-fold (95% CI = 3.35-fold to 3.65-fold) increase of RACGAP1 mRNA in induced PC-3 cells transfected with pCDNA4-ITGA7 compared with uninduced cells (from 715 units in uninduced cells to 2146 units in induced cells). Within 24 hours of integrin 7 induction, RACGAP1 protein expression was increased 2.8-fold (95% CI = 2.60-fold to 3.00-fold) in pCDNA4-ITGA7–transfected PC-3 cells, compared with uninduced cells (with RACGAP1/-actin ratio increasing from 0.049 in uninduced cells to 0.139 in induced cells), and 3.3-fold (95% CI = 2.73-fold to 3.86-fold) in SK-UT-1 cells (with RACGAP1/-actin ratio increasing from 0.044 in uninduced cells to 0.148 in induced cells). Thus, integrin 7 expression may lead to the activation of several genes, including CDKN3 and RACGAP1.
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To evaluate the importance of CDKN3 and RACGAP1 in integrin
7–mediated tumor suppressor and motility inhibition activities, we used RNA interference for CDKN3 and RACGAP1, PC-3 cells that were transfected with a tetracycline-inducible integrin 7 expression vector (pCDNA4-ITGA7), and SK-UT-1 cells that were transfected with pCMV-ITGA7 or pCMVscript. We evaluated tumor suppressor activity with the colony formation assay and motility with a wound-healing assay. Inhibition of CDKN3 expression by 80% in PC3 cells transfected with pCDNA4-ITGA7 and induced with tetracycline reduced integrin 7–mediated soft agar colony growth inhibition by 85% (95% CI = 83.9% to 87.6%), and inhibition of RACGAP1 reduced it by 32% (95% CI = 26.4% to 37.5%) (Fig. 2, B–D). When the expression of both CDKN3 and RACGAP1 was inhibited with corresponding siRNAs, integrin 7 tumor suppressor activity was virtually abolished (i.e., reduced by 99%, 95% CI = 99% to 100%). Thus, the combination of CDKN3 and RACGAP1 may mediate integrin 7 tumor suppression, although CDKN3 appears to be the dominant target. Similar results were also found with the leiomyosarcoma cell line SK-UT-1. In contrast, inhibition of RACGAP1 with a RACGAP1 siRNA reversed the inhibition of motility by integrin 7 by 70%, whereas CDKN3 alone was virtually ineffective in motility inhibition (5%) (Fig. 2, D). The combination of CDKN3 and RACGAP1 siRNAs in PITT1 and PITT2 cells did not result in additional reversal of inhibition of motility, indicating that RACGAP1 is the main target for motility inhibition induced by integrin 7.
Meta-analysis of microarray data on integrin 7 expression found low integrin 7 expression (2.7-fold decreased to 4.5-fold decreased expression) in human prostate cancer specimens from Memorial Sloan-Kettering Cancer Institute and University of Pittsburgh that did not metastasize but even lower expression (4.4-fold decreased to 6.1-fold decreased expression) in those that metastasized, when compared with normal prostate (4325 units on average) (15–17) (Table 3). Soft tissue leiomyosarcomas that did not metastasize and normal smooth muscle tissue had approximately the same level of integrin 7 expression, but integrin 7 expression in highly aggressive soft tissue leiomyosarcomas was decreased by 41.1-fold (95% CI = 37.4-fold to 44.8-fold), compared with normal smooth muscle (23). RT–PCR analyses of 20 human organs and 16 cell lines derived from tumors of prostate, brain, liver, smooth muscle, lung, and kidney detected expression of integrin 7 mRNAs in all tissues and cell lines examined (data not shown).
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We used immunostaining of tissues to determine whether integrin
7 protein expression was decreased in prostate cancer and leiomyosarcoma, compared with normal tissues. Immunostaining of prostate tissues with rabbit anti–integrin 7 antiserum showed that normal prostate gland tissue had a moderate level of integrin 7 expression, with an average score of 1.82 (95% CI = 1.74 to 1.89). The acinar cells were more intensely stained than the basal cells. In contrast, many prostate cancer tissues had no integrin 7 or only focal positive staining for integrin 7, with an average score of 0.740 (95% CI = 0.699 to 0.789, P<.001) (Fig. 3, A, and Table 4). A further decrease in the level of integrin 7 expression was observed in metastasizing prostate cancer tumors, with an average score of 0.414 (95% CI = 0.348 to 0.480) (Table 4). Strong integrin 7 expression was identified in smooth muscle tissue surrounding small vessels. Soft tissue leiomyosarcoma tissue from patients with a relatively mild clinical course (i.e., tumor-free survival of patients was >5 years) had slightly lower integrin 7 expression (average score = 1.125, 95% CI = 0.876 to 1.373) than normal smooth muscle (1.43, 95% CI = 1.239 to 1.614), and aggressive soft tissue leiomyosarcoma tissue (from patients with a relapse within 5 years) had much lower integrin 7 expression (0.625, 95% CI = 0.456 to 0.794) (Table 4). Among patients contributing prostate cancer or leiomyosarcoma samples (Fig. 3, B), statistically significant decreases in 5-year metastasis-free survival were associated with little or no expression of integrin 7 in tumors, compared with at least weak expression of integrin 7 in tumors (for example, among patients with prostate cancer, 5-year metastasis-free survival rate associated with tumors with focal or no integrin 7 expression was 32%, 95% CI = 24.4% to 40.3%, and that associated with higher integrin 7 expression was 85%, 95% CI = 79.0% to 91.0%; P<.001). These results support a role of integrin 7 in cancer metastasis and indicate that integrin 7 may have a role in cancer behavior.
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Discussion |
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TopAbstractContext and CaveatsPatients, materials, and methodsResultsDiscussionReferencesNotes |
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We report, to our knowledge for the first time, that mutations in the integrin
7 gene appear to be widespread and frequent in human malignancies. We also found by use of RT–PCR that integrin 7 mRNA was readily detected in tissues of 20 normal organs (data not shown). Moreover, we detected integrin 7 mRNA in all 16 cell lines examined that were derived from tumors of prostate gland, lung, brain, smooth muscle, liver, and kidney (data not shown). The presence of mutations in cDNA and genomic DNA from tumor samples and the absence of similar mutations in the matched normal samples largely eliminated the possibility of pseudogene mutations because pseudogene should be present in both normal and tumor samples. Thus, the ubiquitous expression of integrin 7 in human organs and widespread mutations of this gene in human cancers raise the possibility that integrin 7 may have a role in the development of many human malignancies.
To our knowledge, this is also the first report that integrin 7 appears to function as a tumor suppressor in human malignancies. Several lines of evidences support a tumor suppressor role of integrin 7 in mammalian cells. First, in three different tumor cell culture systems, a normal level of integrin 7 expression suppressed tumor growth, and lower levels of integrin 7 expression promoted tumor growth. In addition, mice bearing xenograft tumors from either of two highly aggressive prostate cancer cell lines had reduced tumor volume, fewer metastases, and fewer deaths if the expression of integrin 7 in the cells from which the tumors were derived was increased by transfection with integrin 7 constructs, compared with those in mice bearing xenografts from cell lines transfected with control vector. Second, decreased integrin 7 expression was detected in human prostate tumor tissue samples and in highly aggressive soft tissue leiomyosarcoma samples by two comprehensive protein expression analyses that used data from immunostaining assays. These findings were further supported by findings from several independent microarray datasets in which prostate cancer and soft tissue leiomyosarcoma specimens expressed lower levels of integrin 7 mRNA than corresponding normal tissue specimens. Third, integrin 7 expression appeared to activate the expression of CDKN3 and RACGAP1. CDKN3 has been shown to dephosphorylate tyrosine residues of several CDKs (including CDK2, CDK3, and CDC2) and inhibit cell cycle progression in yeast and mammalian cells (24,25). RACGAP1 has been shown to suppress growth and induce differentiation in hematopoietic cells (26). Thus, by activating CDKN3 and RACGAP1, integrin 7 appears to prevent cell cycle progression and suppress tumor growth. Consistent with these findings, the expression of integrin 7 was strongest in the terminally differentiated prostate acinar cells of the prostate gland but was weakest in basal or stem cell layers of both organs, indicating that integrin 7 may prevent the overgrowth of highly differentiated tissues. This cell growth inhibitory activity of integrin 7 may be mediated by activating the expression of CDKN3 and RACGAP1. Our analyses also indicated that integrin 7 inhibits cell motility and reduces metastases. Inhibition of both growth and motility may mean that integrin 7 is in a position to counteract proliferation and invasion of malignant cells.
Our study has several limitations. Limitations involving the interpretation of data from cells with forced expression of integrin 7 include the artificial cultural system, variations in clonal selection, and the lack of an antitumor immune system in the mice used in our experiments. However, when integrin 7 expression in nonmutant cell lines was reduced by use of siRNA against integrin 7, the tumorigenicity of these cell lines increased, which is consistent with our hypothesis that removal of integrin 7 enhances tumorigenesis. Another limitation is that the signaling pathway used by integrin 7 to activate the transcription of CDKN3 and RACGAP1 mRNAs has not been identified. Micorarray analysis indicates that PC-3 cells express other integrin and types in addition to integrin 7 and integrin 1 (data not shown), but induction of integrin 7 expression did not appear to alter the expression of other integrin molecules. Consequently, to form a heterodimer with 1 subunit, integrin 7 may have to displace mutated integrin 7 (or another integrin ) subunit from the complex, which could alter the homeostasis of integrin signaling and thus alter cell growth.
The function of integrin 7 in prostate gland and smooth muscle appears to be related to the adhesion of cells to the basement membrane and prevention of the random migration of these cells to other organs. Another important function of integrin 7 appears to be its role in limiting cell proliferation because expression of integrin 7 induced the expression of proteins that inhibit cell cycle progression and cell growth. When the level of integrin 7 protein was decreased or the protein was mutated, cells appeared to lose inhibitory signals for both cell migration and proliferation. This loss may lead to unchecked tumor cell proliferation and a higher incidence of metastases. Thus, impairing the function of integrin 7 may be an efficient mechanism of carcinogenesis.
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NOTES |
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TopAbstractContext and CaveatsPatients, materials, and methodsResultsDiscussionReferencesNotes |
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This work was supported by grants from National Cancer Institute to G. K. Michalopoulos (1UO1CA88110-01), to C. Wu (RO1 GM65188), and to J.-H. Luo (RO1 CA098249); the development fund from Department of Urology; and the John Rangos Foundation for Enhancement of Research in Pathology.
The authors had full responsibility for the design of the study, the collection of the data, the analysis and interpretation of the data, the decision to submit the manuscript for publication, and the writing of the manuscript.
Funding to pay the Open Access publication charges for this article was provided by a grant (R01 CA098249) from the National Cancer Institute.
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Manuscript received November 9, 2006; revised March 23, 2007; accepted April 20, 2007.
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