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• W1966451921 abstract "The HIV Tat-interacting protein (TIP30), also called CC3 or HTIP2, is encoded by Tip30, a putative tumor-suppressor gene located on human chromosome 11p15.1. In this study, we investigated the role of TIP30 in the progression and metastasis of lung cancer. TIP30 expression was analyzed in 206 paired lung cancers and adjacent non-tumor tissues, as well as in 70 matched lymph node metastases using a high-density tissue microarray. Results were compared with the clinicopathologic features of the patients from whom the tissues were taken. Low TIP30 expression levels were found in all 9 cases of small cell lung cancer and in 36.5% (72/197) of non-small cell lung cancer, which were correlated with lymph node metastasis in non-small cell lung cancer and with poor differentiation and advanced stage of tumor cells in squamous cell carcinoma. The immunostaining scores were significantly lower in the metastatic lesions than in the primary lesions. Down-regulation of TIP30 by a short hairpin RNA enhanced cell survival, migration, and invasion through Matrigel in vitro, and promoted lung metastasis and vascularization in nude mice. Further studies revealed that the down-regulation of TIP30 enhanced the expression of osteopontin, as well as matrix metalloproteinase-2 and vascular endothelial growth factor. Our results suggest that the down-regulation of TIP30 promotes metastatic progression of lung cancer, hence it could serve as a potential target for the development of lung cancer therapies. The HIV Tat-interacting protein (TIP30), also called CC3 or HTIP2, is encoded by Tip30, a putative tumor-suppressor gene located on human chromosome 11p15.1. In this study, we investigated the role of TIP30 in the progression and metastasis of lung cancer. TIP30 expression was analyzed in 206 paired lung cancers and adjacent non-tumor tissues, as well as in 70 matched lymph node metastases using a high-density tissue microarray. Results were compared with the clinicopathologic features of the patients from whom the tissues were taken. Low TIP30 expression levels were found in all 9 cases of small cell lung cancer and in 36.5% (72/197) of non-small cell lung cancer, which were correlated with lymph node metastasis in non-small cell lung cancer and with poor differentiation and advanced stage of tumor cells in squamous cell carcinoma. The immunostaining scores were significantly lower in the metastatic lesions than in the primary lesions. Down-regulation of TIP30 by a short hairpin RNA enhanced cell survival, migration, and invasion through Matrigel in vitro, and promoted lung metastasis and vascularization in nude mice. Further studies revealed that the down-regulation of TIP30 enhanced the expression of osteopontin, as well as matrix metalloproteinase-2 and vascular endothelial growth factor. Our results suggest that the down-regulation of TIP30 promotes metastatic progression of lung cancer, hence it could serve as a potential target for the development of lung cancer therapies. Lung cancer is one of the leading causes of death worldwide.1Carney DN Lung cancer–time to move on from chemotherapy.N Engl J Med. 2002; 346: 126-128Crossref PubMed Scopus (252) Google Scholar, 2Houwen L State of the art: lung cancer in China.Ann Thorac Cardiovasc Surg. 2003; 9: 147-148PubMed Google Scholar In China, about 300,000 new cases of lung cancer, causing more than 250,000 deaths, occur annually.2Houwen L State of the art: lung cancer in China.Ann Thorac Cardiovasc Surg. 2003; 9: 147-148PubMed Google Scholar The prognosis for lung cancer remains poor despite of the improvements made over the years to its management and treatment. The overall survival of patients with lung cancer remains at a dismal 15% because of metastasis and the development of resistance to chemotherapy.1Carney DN Lung cancer–time to move on from chemotherapy.N Engl J Med. 2002; 346: 126-128Crossref PubMed Scopus (252) Google Scholar Therefore, it is important to better understand the molecular mechanisms of lung cancer progression and find more effective targets for improving the outcomes for patients with lung cancer. The 30-kDa HIV Tat-interacting protein (TIP30), also called CC3 or HTIP2, is a tumor suppressor with pro-apoptotic and anti-metastasis properties.3Shtivelman E A link between metastasis and resistance to apoptosis of variant small cell lung carcinoma.Oncogene. 1997; 14: 2167-2173Crossref PubMed Scopus (106) Google Scholar, 4Baker ME TIP30, a cofactor for HIV-1 Tat-activated transcription, is homologous to short-chain dehydrogenases/reductases.Curr Biol. 1999; 9: R471Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 5Xiao H Tao Y Greenblatt J Roeder RG A cofactor, TIP30, specifically enhances HIV-1 Tat-activated transcription.Proc Natl Acad Sci USA. 1998; 95: 2146-2151Crossref PubMed Scopus (70) Google Scholar The Tip30 gene is located on human chromosome 11p15.1. It was originally identified by a differential display analysis of mRNA from the highly metastatic human variant small cell lung cancer (SCLC) versus less metastatic classic SCLC cell line.3Shtivelman E A link between metastasis and resistance to apoptosis of variant small cell lung carcinoma.Oncogene. 1997; 14: 2167-2173Crossref PubMed Scopus (106) Google Scholar The expression of TIP30 is mainly located in cytoplasm.6Ito M Jiang C Krumm K Zhang X Pecha J Zhao J Guo Y Roeder RG Xiao H TIP30 deficiency increases susceptibility to tumorigenesis.Cancer Res. 2003; 63: 8763-8767PubMed Google Scholar Decreased TIP30 expression has been detected in some tumor cells, such as melanoma, breast cancer, neuroblastoma, glioblastoma, colon cancer, and hepatocellular carcinoma.3Shtivelman E A link between metastasis and resistance to apoptosis of variant small cell lung carcinoma.Oncogene. 1997; 14: 2167-2173Crossref PubMed Scopus (106) Google Scholar, 6Ito M Jiang C Krumm K Zhang X Pecha J Zhao J Guo Y Roeder RG Xiao H TIP30 deficiency increases susceptibility to tumorigenesis.Cancer Res. 2003; 63: 8763-8767PubMed Google Scholar, 7Hewitt RE Brown KE Corcoran M Stetler-Stevenson WG Increased expression of tissue inhibitor of metalloproteinases type 1 (TIMP-1) in a more tumourigenic colon cancer cell line.J Pathol. 2000; 192: 455-459Crossref PubMed Scopus (47) Google Scholar, 8Liu Y Thor A Shtivelman E Cao Y Tu G Heath TD Debs RJ Systemic gene delivery expands the repertoire of effective antiangiogenic agents.J Biol Chem. 1999; 274: 13338-13344Crossref PubMed Scopus (92) Google Scholar, 9NicAmhlaoibh R Shtivelman E Metastasis suppressor CC3 inhibits angiogenic properties of tumor cells in vitro.Oncogene. 2001; 20: 270-275Crossref PubMed Scopus (49) Google Scholar About 24% of various types of cancer cells had Tip30 missense mutation in exon 3 by comparing the Tip30 cDNA sequences in National Center for Biotechnology Information databases.6Ito M Jiang C Krumm K Zhang X Pecha J Zhao J Guo Y Roeder RG Xiao H TIP30 deficiency increases susceptibility to tumorigenesis.Cancer Res. 2003; 63: 8763-8767PubMed Google Scholar G134V mutation derived from liver cancer significantly shortened the half-life of TIP30 protein. Besides gene mutations, TIP30 expression was recently found frequently down-regulated by promoter hypermethylation in liver cancer.10Lu B Ma Y Wu G Tong X Guo H Liang A Cong W Liu C Wang H Wu M Zhao J Guo Y Methylation of Tip30 promoter is associated with poor prognosis in human hepatocellular carcinoma.Clin Cancer Res. 2008; 14: 7405-7412Crossref PubMed Scopus (43) Google Scholar TIP30 was considered to have tumor suppressor activity by inhibiting tumor growth,11Zhao J Zhang X Shi M Xu H Jin J Ni H Yang S Dai J Wu M Guo Y TIP30 inhibits growth of HCC cell lines and inhibits HCC xenografts in mice in combination with 5-FU.Hepatology. 2006; 44: 205-215Crossref PubMed Scopus (42) Google Scholar invasion,3Shtivelman E A link between metastasis and resistance to apoptosis of variant small cell lung carcinoma.Oncogene. 1997; 14: 2167-2173Crossref PubMed Scopus (106) Google Scholar, 11Zhao J Zhang X Shi M Xu H Jin J Ni H Yang S Dai J Wu M Guo Y TIP30 inhibits growth of HCC cell lines and inhibits HCC xenografts in mice in combination with 5-FU.Hepatology. 2006; 44: 205-215Crossref PubMed Scopus (42) Google Scholar and angiogenesis,9NicAmhlaoibh R Shtivelman E Metastasis suppressor CC3 inhibits angiogenic properties of tumor cells in vitro.Oncogene. 2001; 20: 270-275Crossref PubMed Scopus (49) Google Scholar and by inducing apoptosis.3Shtivelman E A link between metastasis and resistance to apoptosis of variant small cell lung carcinoma.Oncogene. 1997; 14: 2167-2173Crossref PubMed Scopus (106) Google Scholar, 11Zhao J Zhang X Shi M Xu H Jin J Ni H Yang S Dai J Wu M Guo Y TIP30 inhibits growth of HCC cell lines and inhibits HCC xenografts in mice in combination with 5-FU.Hepatology. 2006; 44: 205-215Crossref PubMed Scopus (42) Google Scholar, 12Xiao H Palhan V Yang Y Roeder RG TIP30 has an intrinsic kinase activity required for up-regulation of a subset of apoptotic genes.EMBO J. 2000; 19: 956-963Crossref PubMed Scopus (90) Google Scholar Depletion of Tip30 predisposed mammary epithelial cells to neoplastic transformation,13Pecha J Ankrapp D Jiang C Tang W Hoshino I Bruck K Wagner KU Xiao H Deletion of Tip30 leads to rapid immortalization of murine mammary epithelial cells and ductal hyperplasia in the mammary gland.Oncogene. 2007; 26: 7423-7431Crossref PubMed Scopus (22) Google Scholar and Tip30-deficient mice spontaneously developed various tumors at high incidence as compared with wild-type mice.6Ito M Jiang C Krumm K Zhang X Pecha J Zhao J Guo Y Roeder RG Xiao H TIP30 deficiency increases susceptibility to tumorigenesis.Cancer Res. 2003; 63: 8763-8767PubMed Google Scholar G134V and R106H mutations identified in liver cancer not only abrogated the tumor suppressor potential but also gained oncogenic activities and promoted cell growth and invasion, and inhibited cisplatin-induced apoptosis through up-regulation the expression of N-cadherin and c-MYC, and down-regulation the expression of p53 and E-cadherin.6Ito M Jiang C Krumm K Zhang X Pecha J Zhao J Guo Y Roeder RG Xiao H TIP30 deficiency increases susceptibility to tumorigenesis.Cancer Res. 2003; 63: 8763-8767PubMed Google Scholar, 14Jiang C Pecha J Hoshino I Ankrapp D Xiao H TIP30 mutant derived from hepatocellular carcinoma specimens promotes growth of HepG2 cells through up-regulation of N-cadherin.Cancer Res. 2007; 67: 3574-3582Crossref PubMed Scopus (30) Google Scholar It has been proved that TIP30 acts as a transcription cofactor and regulates expressions of genes involved in apoptosis, cell growth, and metastasis. TIP30 was shown to interact with human immunodeficiency virus-1 Tat and enhance Tat-activated transcription.4Baker ME TIP30, a cofactor for HIV-1 Tat-activated transcription, is homologous to short-chain dehydrogenases/reductases.Curr Biol. 1999; 9: R471Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar TIP30 was also found to interact with estrogen receptor α-interacting coactivator and negatively regulate estrogen receptor α-mediated c-myc expression.15Jiang C Ito M Piening V Bruck K Roeder RG Xiao H TIP30 interacts with an estrogen receptor alpha-interacting coactivator CIA and regulates c-myc transcription.J Biol Chem. 2004; 279: 27781-27789Crossref PubMed Scopus (61) Google Scholar Recently TIP30 was found to interact with Ets-1 and inhibit osteopontin (OPN) transcription.16Zhao J Lu B Xu H Tong X Wu G Zhang X Liang A Cong W Dai J Wang H Wu M Guo Y Thirty-kilodalton Tat-interacting protein suppresses tumor metastasis by inhibition of osteopontin transcription in human hepatocellular carcinoma.Hepatology. 2008; 48: 265-275Crossref PubMed Scopus (44) Google Scholar Recent studies have linked TIP30 to metastatic progression of several different cancer types. Introduction of TIP30 into v-SCLC cells suppressed metastasis in SCID-hu-L mice.3Shtivelman E A link between metastasis and resistance to apoptosis of variant small cell lung carcinoma.Oncogene. 1997; 14: 2167-2173Crossref PubMed Scopus (106) Google Scholar The expression of TIP30 was inversely associated with axillary lymph node metastasis and vascular invasion in breast cancer.17Zhao J Ni H Ma Y Dong L Dai J Zhao F Yan X Lu B Xu H Guo Y TIP30/CC3 expression in breast carcinoma: relation to metastasis, clinicopathologic parameters, and P53 expression.Hum Pathol. 2007; 38: 293-298Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar The conditioned media from CC3-expressing tumor cells greatly inhibited the proliferation and migration of endothelial cells in vitro.9NicAmhlaoibh R Shtivelman E Metastasis suppressor CC3 inhibits angiogenic properties of tumor cells in vitro.Oncogene. 2001; 20: 270-275Crossref PubMed Scopus (49) Google Scholar Our recent studies showed that TIP30 inhibited tumor metastasis through suppressing the expression of OPN in human hepatocellular carcinoma.16Zhao J Lu B Xu H Tong X Wu G Zhang X Liang A Cong W Dai J Wang H Wu M Guo Y Thirty-kilodalton Tat-interacting protein suppresses tumor metastasis by inhibition of osteopontin transcription in human hepatocellular carcinoma.Hepatology. 2008; 48: 265-275Crossref PubMed Scopus (44) Google Scholar The role of TIP30 in the development and progression of lung cancer has not been fully characterized. In the present study, TIP30 expression was examined by immunohistochemistry in 206 lung carcinoma tissues, including 197 non-small cell lung cancers (NSCLC) and 9 SCLCs, and adjacent non-tumor tissues, as well as 70 matched lymph node metastases using a high-density tissue microarray. The correlations of TIP30 expression with tumor stage, histological grade, and lymph node metastasis were evaluated. The effects of TIP30 on tumor cell survival, invasion, and angiogenesis were assessed both in vitro and in nude mice. Tissue samples were obtained from 206 patients with lung cancer without chemotherapy in Changhai Hospital, Shanghai, P. R. China, from January 2001 to June 2006. The use of all of the human samples and the experimental procedures for this study were reviewed and approved by the university and hospital ethics committees. The specimens consisted of 197 tumors of NSCLC and 9 SCLC, as well as corresponding adjacent non-tumor lung tissues taken from the same patients; 70 (62 NSCLC and 8 SCLC) matched lymph nodes with metastatic tumors were also collected. Tissue microarrays were constructed as previously described.18Kononen J Bubendorf L Kallioniemi A Barlund M Schraml P Leighton S Torhorst J Mihatsch MJ Sauter G Kallioniemi OP Tissue microarrays for high-throughput molecular profiling of tumor specimens.Nat Med. 1998; 4: 844-847Crossref PubMed Scopus (3558) Google Scholar Briefly, tissues were stained with H&E to identify viable and representative areas of the specimen. From the defined areas, core biopsies were taken with a Tissue Arrayer (Beecher Instruments, Silver Spring, MD). Duplicate 2-mm tissue cores were used to construct the tissue microarrays. Four tissue microarray blocks were constructed. Array blocks were sectioned to produce serial 4-μm sections, and the first section was stained with H&E to assess adequacy. Bacterially produced glutathione S-transferase fusion proteins were used in the production of anti-human TIP30 antibody. The fusion proteins were injected subcutaneously into rabbits to get antiserum and the antibody purification was performed by using Protein A Sepharose CL-4B (Amersham Pharmacia Biotech, Piscataway, NJ), according to manufacturer's instructions. The specificity of the polyclonal antibody was demonstrated by Western blot and immunohistochemical analysis. The expressions of TIP30 protein in the specimens were detected by immunohistochemistry assay with a polyclonal antibody against human TIP30 as described previously.17Zhao J Ni H Ma Y Dong L Dai J Zhao F Yan X Lu B Xu H Guo Y TIP30/CC3 expression in breast carcinoma: relation to metastasis, clinicopathologic parameters, and P53 expression.Hum Pathol. 2007; 38: 293-298Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar For antibody control, one set of samples was incubated with non-immune rabbit IgG (1:150) instead of primary antibody. Evaluation of TIP30 staining was independently performed by two experienced pathologists. The intensity of TIP30 immunostaining was semiquantitatively estimated according to the signal intensity and distribution. Briefly, a mean percentage of positive tumor cells was determined in at least five areas ×400 magnification and assigned to one of the five following categories: 0, <5%; 1, 5% to 25%; 2, 25% to 50%; 3, 50% to 75%, and 4, >75%. The intensity of immunostaining was scored as follows: 1, weak; 2, moderate and 3, intense. For tumors that showed heterogeneous staining, the predominant pattern was taken into account for scoring. The percentage of positive tumor cells and the staining intensity were multiplied to produce a weighted score for each case. Tissues with immunohistochemical scoring ≤4 were considered as having low expression, and scores of 5 to 12 were considered high expression. Human lung cancer cells (A549, NCI-H460, SK-MES-1, LTEP-a-2, H1299) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified incubator at 37°C in 5% CO2 atmosphere. Infection of lentivirus encoding Tip30 cDNA and short hairpin (sh)RNA targeting Tip30 was performed as reported previously.16Zhao J Lu B Xu H Tong X Wu G Zhang X Liang A Cong W Dai J Wang H Wu M Guo Y Thirty-kilodalton Tat-interacting protein suppresses tumor metastasis by inhibition of osteopontin transcription in human hepatocellular carcinoma.Hepatology. 2008; 48: 265-275Crossref PubMed Scopus (44) Google Scholar Briefly, the double-strand oligo DNAs for Tip30 were as follows: top strand, 5′-CACCGATGGAACTGCTGGAGAACAATCAAGAGTTGTTCTCCAGCAGTTCCATC-3′, and bottom strand, 5′-AAAAGATGGAACTGCTGGAGAACAACTCTTGATTGTTCTCCAGCAGTTCCATC-3′). For negative control, we used scramble shRNA, top strand, 5′-CACCGAATTCTCCGAACGTGTCACGTTCAAGAGACGTGACACGTTCGGAGAATT-3′ and bottom strand, 5′-AAAAAATTCTCCGAACGTGTCACGTCTCTTGAACGTGACACGTTCGGAGAATTC-3′. pENTR-U6-shRNA plasmid was recombined with destination vectors pLenti6/BLOCKiT-DEST vector to generate the shRNA constructs. For construction of lentiviral vector expressing human Tip30 gene, Tip30 cDNA was amplified and subcloned to pLenti6/V5-TOPO vectors (Invitrogen, Carlsbad, CA). For virus production, HEK-293T cells were co-transfected with the resulting vector described above and ViraPower Packaging Mix (Invitrogen) using Lipofectamine 2000 according to the manufacturer's guidelines. Infectious lentiviruses were harvested and concentrated and the infectious titer was determined by counting the blue-stained colonies after crystal violet staining in 293 cells. Lung cancer cells were infected with concentrated virus at a multiplicity of infection of 20 in the presence of 8 μg/ml polybrene (Sigma-Aldrich, St. Louis, MO). Supernatant was removed after 24 hours and replaced with complete culture medium. Seventy-two hours after infection, the expressions of TIP30 were confirmed by Western blot. Expression of Tip30 mRNA was determined by quantitative reverse-transcription (qRT-PCR) using the LightCycler system (Roche, Mannheim, Germany) as described previously.17Zhao J Ni H Ma Y Dong L Dai J Zhao F Yan X Lu B Xu H Guo Y TIP30/CC3 expression in breast carcinoma: relation to metastasis, clinicopathologic parameters, and P53 expression.Hum Pathol. 2007; 38: 293-298Abstract Full Text Full Text PDF PubMed Scopus (45) Google ScholarActin was used as an endogenous control to normalize for differences in the amount of total RNA in each sample. The primers used for PCR were as follows: Tip30: sense 5′-TCACCTTCGACGAGGAAGCT-3′; antisense 5′-GCTCTGCAGACTTCAGACCA-3′; Actin: sense 5′-CGTGGACATCCGTAAAGACC-3′; antisense 5′-ACATCTGCTGGAAGGTGGAC-3′. For Western blot, the cells were lysed in RIPA buffer. Proteins at the same amount were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After probing with antibodies, the signals were visualized by Supersignal enhanced chemiluminescence reagent (Pierce, Rockford, IL). The antibodies used were anti-osteopontin (R&D Systems, Minneapolis, MN), anti-matrix metalloprotein (MMP)-2, and anti-vascular endothelial growth factor (VEGF) (Boster Biotechnology, Wuhan, P. R. China), and anti-glyceraldehyde-3-phosphate dehydrogenase (KangChen Bio-tech, Shanghai, P. R. China). Cell growth was measured by 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison, WI) in 96-well plates (1000 cells per well) following the instructions of the manufacturer. Each experiment was done in triplicate and repeated three times. For anchorage-independent growth assays, the cells in single-cell suspension were plated in 0.3% agarose over a 0.6% agarose bottom layer at a density of 200 cells per well in 24-well plates and incubated for 14 days and stained. Colonies with a diameter greater than 100 μm were counted. For wound-healing assays, the cells were first seeded in 6-well culture plates. A wound was made in the confluent monolayer with a plastic pipette tip and the migration of the cells at the wound front was photographed using an inverted microscope at indicated times after the scratch. Cell invasion assays were quantified in vitro using Transwell chambers with polycarbonate membrane filters (8 μm pore size; Corning, NY) coated with a Matrigel (Sigma) according to the manufacturer's instructions. In brief, the lower chamber was filled with 0.6 ml medium containing 20% fetal bovine serum, and 0.2 ml of medium that contained 3 × 105 cells under serum-starved conditions was plated in the upper chamber in triplicate wells and incubated at 37°C for 72 hours. Then cells attached to the upper side of the membrane were removed gently with a cotton swab and rinsed. The cells that migrated through the membrane and attached to the bottom of the membrane were fixed and stained with crystal violet. The number of cells invading through Matrigel was counted by randomly selecting five visual fields, and the extent of invasion was expressed as the average number of cells per microscopic field at a magnification of ×200. All experiments were performed for three times. Two independent investigators were blinded when reading the assays for wound-healing and Matrigel invasion. Nuclear morphology was assessed using Hoechst staining. Four days after lentivirus infection, cells were deprived of serum for 72 hours and fixed with 70% ethanol and labeled with Hoechst 33342 (Sigma) for 10 minutes. Apoptotic cells were distinguished by their characteristic patterns of nuclear condensation, cytoplasmic rounding, and membrane blebbing. The morphological aspect of nuclei was observed with Olympus IX71 fluorescence microscopy (Olympus, Shinjuku Monolith, Tokyo, Japan) by using UV light excitation. In situ apoptosis assay was performed with the Fluorescein FragEL DNA Fragmentation Detection Kit (Calbiochem, San Diego, CA). Formalin-fixed paraffin sections were deparaffinized and incubated with terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) reaction mixture. Apoptotic cells carrying DNA labeled with fluorescein isothiocyanate-dUTP were observed under fluorescence microscope. Female BALB/c nude mice and female severe combined immunodeficient/beige mice at 6 weeks old, were purchased from the Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, P. R. China). A549 cells were infected with lentiviral (LV)-shTip30 or LV-shNon, whereas H1299 cells were infected with LV-Tip30 or LV-green fluorescent protein (GFP). Viability of cells was determined by trypan blue exclusion staining 4 days after infection. For the tumorigenicity assay, viable cells (1 × 107) were injected subcutaneously into the right flank of each mouse (6 mice/group). The tumor volume was calculated using the equation V (mm3) = a × b2/2, where a is the largest dimension and b is the perpendicular diameter. For tumor metastasis analysis, female severe combined immunodeficient/beige mice (for H1299) and female nude mice (for A549) were inoculated with 1 × 106 viable cells in 200 μl of phosphate buffered saline via tail vein injection as described previously.19Lakka SS Rajagopal R Rajan MK Mohan PM Adachi Y Dinh DH Olivero WC Gujrati M Ali-Osman F Roth JA Yung WK Kyritsis AP Rao JS Adenovirus-mediated antisense urokinase-type plasminogen activator receptor gene transfer reduces tumor cell invasion and metastasis in non-small cell lung cancer cell lines.Clin Cancer Res. 2001; 7: 1087-1093PubMed Google Scholar Four weeks later, mice were sacrificed and examined for development of pulmonary metastasis under microscopy. Tumor microvessel density (MVD) was quantified using sections immunostained for CD31 (BioLegend, San Diego, CA) by two investigators independently and in a blinded manner according to the method described previously.20Weidner N Semple JP Welch WR Folkman J Tumor angiogenesis and metastasis–correlation in invasive breast carcinoma.N Engl J Med. 1991; 324: 1-8Crossref PubMed Scopus (5361) Google Scholar The areas with the greatest density of CD31-positive endothelial cells were designated “hot spots.” The whole section was scanned at low power (×40) to identify the best fields for counting. Counting was performed on five separate fields within a hot spot at ×200 magnification. Each stained endothelial cell or cell cluster was counted as one microvessel. If two or more CD31-positive foci appeared to belong to a single continuous vessel, this was counted as one microvessel. The mean vessel count from these fields was used for MVD scoring. Pearson χ2 tests were used to evaluate the relationship between the expression of TIP30 and clinicopathologic variables, as well as the TIP30 expression levels between primary and metastatic lesions. Comparisons between groups of related samples were assessed with the Wilcoxon paired-sample test. All other in vitro assay results represent the arithmetic mean ± SE of triplicate determinations of at least two independent experiments done under the same conditions. Student's t-test was used to determine the differences between groups and P < 0.05 was considered as statistically significant. All statistical tests were two-sided. Calculations were done with the Statistical Package for the Social Sciences version 13.0 (SPSS, Inc., Chicago, IL). The expressions of TIP30 were examined in 206 lung cancers and adjacent non-tumor tissues in a tissue microarray using anti-human TIP30 polyclonal antibody. In the non-tumor tissues, immunopositive staining of TIP30 was detected in the cytoplasm of alveolar epithelial cells (Figure 1A), as well as in ciliated and basal epithelial cells in bronchial mucosa (Figure 1B). Weak immunostaining pictures of TIP30 was found in 72 of 197 (36.5%) cases of NSCLC, with 33/90 (36.7%) in squamous cell carcinoma (SCC) (Figure 1C) and 39/107 (36.4%) in adenocarcinoma (AC) (Figure 1E). Low expression of TIP30 was found in all of the 9 cases of SCLC (Figure 1G). Statistical analysis indicated that the immunostaining scores in the adjacent non-tumor tissues were significantly higher than the scores in tumor tissues in lung cancer (P < 0.001). Lymph node metastases were found in 8 out of 9 cases of SCLC, in which TIP30 expressions were almost undetectable (Figure 1, H–J). The expression levels of TIP30 in the primary lesions were inversely associated with lymph node metastasis in SCC and AC (P = 0.033; P = 0.038) (Table 1). Moreover, when TIP30 expression in the lymph node metastatic lesions was compared with that in the paired primary lesions, in 62 cases of NSCLC, 43 cases showed equal TIP30 expression, 4 cases showed increased TIP30 expression, and 15 cases showed decreased TIP30 expression in metastatic lesions (Figure 1, D and F). The immunostaining scores in the metastatic lesions were significantly lower than the scores in the primary lesions (P = 0.001, Table 2). These data suggest that decreased expression of TIP30 may promote tumor metastasis in lung cancer patients.Table 1The Association between TIP30 Expression and Clinicopathological CharacteristicsAC (n = 107)SCC (n = 90)Clinicopathological characteristicsLow expressionHigh expressionP valueLow expressionHigh expressionP valueAge (years) <6021340.70214200.489 ≥6018341937Sex3968 Male19330.98530510.827 Female203536Tumor size(cm)3968 <316240.5559150.921 ≥323442442Lymph node involvement3968 −16420.038*P < 0.05;17420.033*P < 0.05; +23261615Stage3968 I–II21310.41120480.012*P < 0.05; III–IV1837139Differentiation Well differentiated390.1233180.005**P < 0.01. Moderately differentiated15362135 Poorly differentiated212394* P < 0.05;** P < 0.01. Open table in a new tab Table 2Comparative Analysis of TIP30 Expression in Primary and Lymph Node Metastatic NSCLC TissuesTIP30 expression in primary tumorTIP30 expression in metastatic lymph nodeP valueLow expressionHigh expressionLow expression1940.001**P < 0.01.High expression1524** P < 0.01. Open table in a new tab The correlation of TIP30 expression in the primary lesions and clinicopathologic features was further analyzed in NSCLC. There was no significant correlation between TIP30 expression and age, sex of patients, and tumor size, as determined by χ2 test (Table 1). However, TIP30 expression was inversely associated with the stage of the disease (P = 0.012) and positively associated with the degree of differentiation of the tumor (P = 0.005) in SCC, which indicates low expression of TIP30 may be associated with the progression of SCC" @default.
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• W1966451921 date "2009-05-01" @default.
• W1966451921 modified "2023-09-29" @default.
• W1966451921 title "Decreased TIP30 Expression Promotes Tumor Metastasis in Lung Cancer" @default.
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• W1966451921 doi "https://doi.org/10.2353/ajpath.2009.080846" @default.
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