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Published online before print April 6, 2009
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From the International Cancer Institute & Eastern Hospital of Hepatobiliary Surgery,* The Second Military Medical University, Shanghai; the Chinese National Engineering Center for Antibody Medicine and Shanghai Key Lab for Cell Engineering, Shanghai; and the Department of Pathology, Changhai Hospital, The Second Military Medical University, Shanghai, China
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 30-kDa HIV Tat-interacting protein (TIP30), also called CC3 or HTIP2, is a tumor suppressor with pro-apoptotic and anti-metastasis properties.3-5 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.3 The expression of TIP30 is mainly located in cytoplasm.6 Decreased TIP30 expression has been detected in some tumor cells, such as melanoma, breast cancer, neuroblastoma, glioblastoma, colon cancer, and hepatocellular carcinoma.3,6-9 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.6 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.10
TIP30 was considered to have tumor suppressor activity by inhibiting tumor growth,11
invasion,3,11
and angiogenesis,9
and by inducing apoptosis.3,11,12
Depletion of Tip30 predisposed mammary epithelial cells to neoplastic transformation,13
and Tip30-deficient mice spontaneously developed various tumors at high incidence as compared with wild-type mice.6
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.6,14
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.4
TIP30 was also found to interact with estrogen receptor 
-interacting coactivator and negatively regulate estrogen receptor -mediated c-myc expression.15
Recently TIP30 was found to interact with Ets-1 and inhibit osteopontin (OPN) transcription.16
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.3 The expression of TIP30 was inversely associated with axillary lymph node metastasis and vascular invasion in breast cancer.17 The conditioned media from CC3-expressing tumor cells greatly inhibited the proliferation and migration of endothelial cells in vitro.9 Our recent studies showed that TIP30 inhibited tumor metastasis through suppressing the expression of OPN in human hepatocellular carcinoma.16
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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.18 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.
Antibody Generation
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.
Immunohistochemical Staining
The expressions of TIP30 protein in the specimens were detected by immunohistochemistry assay with a polyclonal antibody against human TIP30 as described previously.17 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 x400 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.
Cell Culture and Lentiviral Infection
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.16 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.
Quantitative Reverse-Transcription PCR and Western Blot
Expression of Tip30 mRNA was determined by quantitative reverse-transcription (qRT-PCR) using the LightCycler system (Roche, Mannheim, Germany) as described previously.17 Actin 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 Assays
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.
Wound-Healing and Matrigel Invasion Assays
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 x 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 x200. All experiments were performed for three times. Two independent investigators were blinded when reading the assays for wound-healing and Matrigel invasion.
Detection of Apoptosis
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.
Animal Experiments
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 x 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 x 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 x 106 viable cells in 200 µl of phosphate buffered saline via tail vein injection as described previously.19 Four weeks later, mice were sacrificed and examined for development of pulmonary metastasis under microscopy.
Analysis of Tumor Vascular Density
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.20 The areas with the greatest density of CD31-positive endothelial cells were designated "hot spots." The whole section was scanned at low power (x40) to identify the best fields for counting. Counting was performed on five separate fields within a hot spot at x200 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.
Statistic Analysis
Pearson 
2 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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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. 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.
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2 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. Down-Regulation of TIP30 Promotes Lung Cancer Cell Survival
Previous data suggest that decreased expression of TIP30 may play an important role in the development of lung cancer. To more rigorously explore this possibility, we detected the levels of Tip30 mRNA and protein in five lung cancer cell lines (Figure 2A)
. The level of Tip30 mRNA and protein was relatively high in the AC cell line A549 and SCC cell line SK-MES-1, and decreased in the AC cell line H1299. We used lentivirus to knockdown the expression of TIP30 by shRNA targeting Tip30 (shTip30) in A549 and SK-MES-1 cells or elevate it by lentivirus encoding Tip30 cDNA (LV-Tip30) in H1299 cells (Figure 2B)
. LV-shTip30 significantly promoted cell growth and anchorage-independent growth in A549 and SK-MES-1 cells as compared with LV-shNon or mock treatment, while LV-Tip30 inhibited cell growth and anchorage-independent growth in H1299 cells (Figure 3A)
. The suppressive effect of TIP30 on lung cancer cell growth was confirmed by ex vivo assay. As shown in Figure 3B
, down-regulation of TIP30 in A549 cells dramatically promoted tumor development in nude mice compared with LV-shNon control group, whereas introduction of TIP30 into H1299 significantly inhibited tumor growth.
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. Apoptosis was further examined by in situ TUNEL assay on A549 and H1299 xenografted tumor tissues. The apoptotic nuclei, seen as green color under fluorescence microscopy, were greatly decreased in LV-shTip30 infected A549 xenografts than that in shNon controls. On the contrary, when elevating the level of TIP30 in H1299, the apoptotic cells were greatly increased (Figure 3D)
. Down-Regulation of TIP30 Enhances Tumor Metastasis and Angiogenesis in Lung Cancer
To confirm the inhibitory effects of TIP30 on tumor metastasis observed in lung cancer tissues, the metastatic potentials of lung cancer cells were examined in vitro and in nude mice. A549 and SK-MES-1 cells infected with LV-shTip30 migrated rapidly and filled in the wound faster than LV-shNon infected cells. In contrast, H1299 cells infected with LV-Tip30 filled in the wound slower than LV-GFP infected cells (Figure 4A)
. Consistent with the data of wound-healing assay, the invasive abilities through Matrigel were significantly increased in LV-shTip30 infected A549 and SK-MES-1 cells, relative to that in LV-shNon infected cells, and decreased in LV-Tip30 infected H1299 cells relative to that in LV-GFP infected cells (Figure 4B)
.
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). However, mice injected with H1299/LV-Tip30 cells exhibited a lower occurrence of lung metastases than those injected with H1299/LV-GFP cells (20 ± 13.28 vs. 59 ± 13.27 clusters per mice, P < 0.01, Figure 4Ca
). These data suggest that loss of TIP30 may promote the motile and invasive abilities of lung cancer cells.
Angiogenesis is a prerequisite for advanced tumor growth and is logically believed to be an important factor in tumor metastasis. We evaluated tumor angiogenesis by down-regulation and overexpression of TIP30 in A549 and H1299 respectively. Tumor-associated neovascularization, as indicated by MVD, was determined by immunohistochemistry using anti-CD31 antibodies. As shown in Figure 4Cb
, a significant increase in tumor MVD was observed by down-regulation of TIP30 in A549 xenografts as compared with control tumors. While in H1299 xenografts, a significant reduction of MVD was observed by introduction of TIP30. Thus, TIP30 negatively regulates tumor metastasis and angiogenesis.
We have previously reported that TIP30 inhibited tumor metastasis through inhibition of OPN expression, a key molecule involved in tumor metastasis in hepatocellular carcinoma.16
The expression of OPN on knock-down or introduction of TIP30 was examined in A549 and H1299, respectively. Consistent with the observation in hepatocellular carcinoma cells, TIP30 negatively regulated OPN expression in lung cancer cells as well (Figure 4D)
. Previous studies have demonstrated that OPN enhanced tumor metastasis through induction of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) expression.21,22
Concomitance with the expressions of OPN, the expressions of MMP-2 and VEGF were enhanced by down-regulation of TIP30 in A549 cells, and decreased by introduction of TIP30 in H1299 cells (Figure 4D)
. Thus, down-regulation of TIP30 might enhance tumor metastasis and angiogenesis through induction of OPN expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of TIP30 was found to be inversely associated with axillary lymph node metastasis and vascular invasion in breast cancer in our previous study.17 In this study, we found that low expression of TIP30 was associated with lymph node metastasis in lung cancer, and the expressions of TIP30 in the metastatic lesions were lower than that in the primary lesions. These data suggest that decreased expression of TIP30 may promote tumor metastasis in lung cancer patients. The inhibitory effects of TIP30 on lung cancer metastasis were further demonstrated in vitro and in nude mice. Inhibition of TIP30 expression significantly promoted tumor cell invasion through Matrigel and wound healing in vitro, and lung metastasis and angiogenesis in nude mice. Consistent with our previous observation in hepatocellular carcinoma,16 down-regulation of TIP30 might promote tumor metastasis through induction of OPN expression, which enhances tumor metastasis through induction of MMPs and VEGF expression.21,22 These data strongly suggest down-regulation of TIP30 promotes tumor metastasis and angiogenesis through induction of OPN expression in lung cancer.
However, it must be noted that there were certain patients with lymph node metastasis whose primary tumors were immunohistochemically TIP30 positive and in some cases TIP30 expression was increased relative to that in adjacent benign tissues. These differences with respect to TIP30 expression pointed to several possible explanations: the anti-TIP30 polyclonal antibody cannot discriminate between a normal and a mutant protein, or a gene mutation could give rise to an altered protein with diminished normal function. Therefore, mutation analyses in TIP30-increased samples are needed to further answer these questions.
In conclusion, TIP30 may function as a tumor suppressor gene and play important roles in suppressing the progression and metastasis of lung cancer. Given that introduction of TIP30 into tumor cells with decreased TIP30 expression promotes tumor apoptosis and inhibits tumor metastasis, TIP30 may be a potent target for the development of therapeutic strategies for patients with lung cancer.
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Acknowledgements |
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Footnotes |
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Supported by grants from National Nature Science Foundation of China, Ministry of Science and Technology of China (973 & 863 Projects), Shanghai Commission of Science and Technology, and Shanghai Municipal Education Commission, as well as a special grant from Shanghai Pudong Commission of Science & Technology. Jian Zhao is a recipient of Shuguang Scholar Award from Shanghai Commission of Education.
X.T., K.L., and Z.L. contributed equally to this work.
Accepted for publication January 23, 2009.
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