Japanese Journal of Clinical Oncology 32:85-89 (2002)
© 2002 Foundation for Promotion of Cancer Research
ßIGH3, a TGF-ß Inducible Gene, is Overexpressed in Lung Cancer
Department of Surgery II, Nagoya City University Medical School, Nagoya, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Background: Transforming growth factor-ß (TGF-ß) is capable of affecting the proliferation of many cell types. ßIGH3, TGF-ß inducible gene, was originally isolated from lung adenocarcinoma cell line (A549). We have hypothesized that ßIGH3 mRNA levels could be predictors of the development and invasion of lung cancer.
Methods: The study included 71 lung cancer cases. The ßIGH3 mRNA levels were quantified by real time reverse transcription polymerase chain reaction (RT-PCR) using LightCycler.
Results: The ßIGH3 mRNA levels were elevated in tumor tissues from lung cancer (1.058 ± 1.212) compared with non-malignant lung tissues (0.304 ± 0.159) (p = 0.0001). No significant difference in ßIGH3 mRNA levels was found among gender, age, pathological subtype and lymph node metastasis. The ßIGH3 mRNA levels were elevated in tumor tissues from T4 lung cancer (1.425 ± 1.470) compared with those from T1 lung cancer (0.702 ± 0.655) (p = 0.05) and T2 lung cancer (0.736 ± 0.734) (p = 0.044).
Conclusion: Using the LightCycler RT-PCR assay, the determination of ßIGH3 mRNA level might provide a potential marker for the aggressiveness of lung cancer. However, further studies and a longer follow-up are needed to confirm the impact of ßIGH3 on the biological behavior of the tumor.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Lung cancer is a major cause of death from malignant diseases, owing to its high incidence, malignant behavior and lack of major advances in treatment strategy (1). Lung cancer was the leading indication for respiratory surgery (42.2%) in 1998 in Japan (2). More than 15 000 patients underwent surgical operation at Japanese institutions in 1998 (2). The clinical behavior of lung cancer is largely associated with its stage. The cure of the disease by surgery is only achieved in cases representing an early stage of lung cancer (3).
The relationships between transforming growth factor-ß (TGF-ß) and cancer are varied and complex (4,5). During early stages of carcinogenesis, TGF-ß acts predominantly as a potent tumor suppressor and may mediate the actions of chemo-preventive agents. However, at some point during the development and progression of malignant neoplasms, bioactive TGF-ßs make their appearance in the tumor microenvironment and the tumor cells escape from TGF-ß-dependent growth arrest. In many cases, this resistance to TGF-ß is the consequence of loss or mutational inactivation of the genes that encode signaling intermediates (4).
To identify genes whose protein products may mediate cellular response to TGF-ß, a cDNA library was made by Skonier et al. from mRNA isolated from a human lung adenocarcinoma cell line (A549) that had been treated with TGF-ß. The library was screened by differential hybridization and a cDNA clone, ßIGH3, was isolated by Skonier et al. (6). ßIGH3 has an RGD sequence that can serve as a ligand recognition site for several integrins (6). ßIGH3 supported attachment and spreading of fibroblasts, suggesting that ßIGH3 might function as an extracellular attachment protein (7). Recently, cDNA microarray data showed that ßIGH3 gene was overexpressed in colorectal carcinoma compared with the normal mucosa (8). ßIGH3 gene was increased in most of the highly invasive breast cancer cell lines but not in the weakly invasive cell lines (9). However, to our knowledge, there has been no report of ßIGH3 mRNA expression in clinical samples from lung cancers. Hence the clinical significance of the ßIGH3 gene in lung cancer is not well known.
As many tumor markers have been searched for in lung carcinoma for screening and diagnostic purposes, we queried whether the ßIGH3 mRNA level could possibly serve as a marker to identify patients with lung cancer, especially in terms of invasive potential. In this work, we investigated ßIGH3 mRNA levels by real-time reverse transcription polymerase chain reaction (RT-PCR) assay using LightCycler (10). The findings were compared with the clinicopathological features of lung cancer.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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The study group consisted of 71 lung cancer cases who had undergone surgery at the Department of Surgery II, Nagoya City University Medical School, between 1997 and 1999. The lung cancers were classified according to the general rules for clinical and pathological recording of lung cancer (11). All tumor samples were immediately frozen and stored at –80°C until assayed.
The clinical and pathological characteristics of the 71 lung cancer patients are shown in Table 1; they include 29 cases at stage I, 15 at stage II, 24 at stage III and three at stage IV. The mean age was 63.2 years (range, 42–80 years). Among the lung cancer patients, 46 (64.8%) were diagnosed as adenocarcinoma and 17 (23.9%) as squamous cell carcinoma (Table 1).
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RT-PCR Assay for ßIGH3
Total RNA was extracted from lung cancer tissues and adjacent non-malignant lung tissues using an Isogen kit (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. Total RNA was also extracted from a lung cancer cell line, CCL185 (A549, adenocarcinoma cell line). This RNA was used as a positive control. RNA concentration was determined spectrophotometrically and adjusted to a concentration of 200 ng/ml. RNA (1 µg) was reverse transcribed by Superscript II enzyme (Gibco BRL, Gaithersburg, MD) with 0.5 mg of oligo-(dT)12–16 (Amersham Pharmacia Biotech, Piscataway, NJ). The reaction mixture was incubated at 42°C for 50 min followed by incubation at 72°C for 15 min. To ensure the fidelity of mRNA extraction and reverse transcription, all samples were subjected to PCR amplification with oligonucleotide primers specific for the constitutively expressed gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and normalized. PCRs were performed using a LightCycler-FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals, Mannheim, Germany). The primer sequences for ßIGH3 gene were forward primer 5-CCATGTCATCACCAATCGCTTTA-3 and reverse primer 5-GCATTCCTCCTGTAGTGCTT-3 and the size of the PCR product was 201 base pairs. The cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 60 cycles at 94°C for 15 s, 57°C for 5 s and 72°C for 9 s.
Statistical Methods
Statistical analyses were performed using the Mann–Whitney U-test for unpaired samples and Wilcoxon’s signed rank test for paired samples. Linear relationships between variables were determined by means of simple linear regression. Correlation coefficients were determined by rank correlation using Spearman’s test. Differences among the means of the stage and pathological subtypes in the patients were examined using the Kruskal–Wallis test and Fisher’s PLSD test. The overall survival of lung cancer patients was examined by the Kaplan–Meier method and differences were examined by the log-rank test. All analyses were carried out using the Stat-View software package (Abacus Concepts, Berkeley, CA) and were considered significant when the p-value was <0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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ßIGH3 RNA Expression in Lung Cancer
In the tissues from lung cancer, the mean value for the ßIGH3 mRNA level as standardized by the mRNA level of GAPDH (1.058 ± 1.212, mean ± standard deviation) was significantly higher than that in non-malignant lung tissue (0.304 ± 0.159, p = 0.0001) and variations in the level were not related to age. The mRNA levels of ßIGH3 of the lung cancer samples were stage I 0.794 ± 0.776, stage II 0.927 ± 1.263, stage III 1.088 ± 1.233 and stage IV 0.851 ± 0.310 (Table 1). The difference in ßIGH3/GAPDH mRNA levels between the pathological subtype of lung cancer was not significant (adenocarcinoma 0.817 ± 0.812, squamous cell carcinoma 0.923 ± 0.944, other carcinoma 1.543 ± 2.001). No significant difference in ßIGH3/GAPDH mRNA levels was found among gender and age. Patients’ groups were further stratified according to clinico-pathological factors. ßIGH3/GAPDH mRNA levels were significantly different among tumors with different T-factors (T1 0.702 ± 0.655, T2 0.736 ± 0.734, T3 1.097 ± 1.362, T4 1.425 ± 1.470) but not with N-factors (N0 1.141 ± 1.838, N1 1.373 ± 1.803, N2 1.846 ± 2.632). Thus there was a higher ßIGH3 expression level in tumors with T4 compared with tumors with T1 (p = 0.05) and T2 (p = 0.044) (Fig. 1).
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The overall survival of the 71 lung cancer patients was studied with reference to the ßIGH3/GAPDH mRNA levels. The difference in prognosis between the group with low ßIGH3/GAPDH mRNA levels (<1.05, 39/49, 79.59% were alive) and the group with elevated ßIGH3/GAPDH mRNA levels (>1.05, 20/22, 90.91% were alive) was not statistically significant (p = 0.2128), although the observation period was short.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Our findings indicate that ßIGH3 mRNA expression levels were significantly higher in lung cancer tissues than in non-malignant lung tissues. In addition, there was a significantly higher ßIGH3 expression levels in tumors with T4 lung cancers compared with those with T1 and T2 cancers.
A number of transgenic mouse studies have provided strong evidence for the idea that one of the physiological roles of the TGF-ß signaling pathway is to provide protection against malignant transformation (4). For example, transgenic mice that produce a constitutively active form of TGF-ß1 are resistant to DMBA-induced mammary tumor formation (12). Furthermore, when these mice were crossed with mice that develop mammary tumors at a high rate because they overexpress the epithelial mitogen TGF-
, the incidence of mammary tumors in the offspring was markedly reduced (12). Conversely, the absence of TGF-ß1 appears to promote tumor formation. Although targeted homozygous deletion of the TGF-ß1 gene results in mid-gestation loss due to defects in yolk sac vasculogenesis or death shortly postpartum due to a multisystemic inflammatory syndrome, primary skin keratinocytes isolated from TGF-ß1-null embryos have been shown to be highly susceptible to malignant conversion by the v-rasHa oncogene (13–15). Moreover, treatment of heterozygous (TGF-ß1–/+) animals with various tumor induction protocols also resulted in a much higher number of malignant lung and mammary tumors (16). Thus during the early stage of carcinogenesis, TGF-ß acts predominantly as a potent tumor suppressor.
However, at some point during the development and progression of malignant neoplasm, bioactive TGF-ßs make their appearance in the tumor microenvironment and the tumor cells escape from TGFß-dependent growth arrest. Many human carcinoma cell lines have been shown to secret bioactive TGF-ßs in vitro, although the molecular mechanisms remain to be elucidated (17).
Moreover, in many cases, TGF-ßs are also produced by human tumors in vivo (4). For example, plasma levels of TGF-ß are elevated in patients with lung cancer and correlated with disease status (18). In addition, a previous study has shown that TGF-ßs can be detected in tissue specimens from pulmonary adenocarcinoma (19). Using immunohistochemistry, tumor tissue from pulmonary adenocarcinoma appears to express a higher level of TGF-ß than the corresponding normal tissue and the overexpression of TGF-ß has a negative impact on prognosis (19). Another study has shown that the TGF-ß1 level correlates with angiogenesis, tumor progression and prognosis in patients with non-small cell lung carcinoma (20). These investigators concluded that enhanced production and activation of TGF-ß were likely to be implicated in the progression of carcinoma.
ßIGH3 gene, which is induced after long-term treatment by TGF-ß, was isolated from A549 lung adenocarcinoma cell line (6). In addition to A549 cells, ßIGH3 was induced by TGF-ß in H2981 (lung adenocarcinoma cell line) and HEPM cells, but not in 293S (embryo kidney cell line), MCF-7, MDA453 and MDA468 (breast carcinoma cell lines) cells, suggesting that this was not a universal result of TGF-ß treatment (6). DNA sequence analysis of ßIGH3 revealed an open reading frame encoding a 683-amino acid protein containing a secretory leader signal and an RGD sequence (6). ßIGH3 contains four internal repeats with limited homology with insect adhesion molecule, fasciclin-I (21), and also with periostin, that is also overexpressed in lung cancer (22). On the basis of the homology, ßIGH3 may encode a surface recognition protein (6). Also, the recombinant ßIGH3 supported attachment and spreading of dermal fibroblasts (7). Analysis of cDNA microarrays and RT-PCR assays showed that ßIGH3 gene was up-regulated in colorectal carcinoma compared with normal mucosa (8). Hence ßIGH3 represents a key molecule for adhesion and migration of malignant cells once cancer has developed (8). ßIGH3 gene was increased in most of the highly invasive breast cancer cell lines but not in the weakly invasive cell lines (9). We have also shown that ßIGH3 mRNA levels showed a positive correlation with T-status in lung cancers. These data suggested that ßIGH3 plays a role in the progression and invasion of lung cancer. Actually, there is no proof from a study based on a bulk tissue RNA analysis that observed increased expression of ßIGH3 derived from the cancer cells. Thus, the difference in the gene expression between T4 vs T1/T2 may be due to the difference in the stromal component, such as connective tissues, blood vessels, immune cells and necrotic cells.
TGF-ß1 overexpression was reported to be significantly correlated with a poor prognosis in lung carcinomas (22). However, our results showed that the survival rates were not significantly different between the groups of normal and elevated ßIGH3 mRNA in lung carcinoma, although the observation period was short. Using the LightCycler RT-PCR assay described here, the determination of ßIGH3 mRNA levels might provide a potential marker for advanced lung cancer. However, further studies and a longer follow-up are needed to confirm the impact of ßIGH3 on the biological behavior of the tumor.
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Acknowledgment |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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The authors thank Mrs Atsuko Miyazaki for her excellent technical assistance.
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FOOTNOTES |
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+ For reprints and all correspondence: Hidefumi Sasaki, Department of Surgery II, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. E-mail: hisasaki@med.nagoya-cu.ac.jp
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Received October 1, 2001; accepted December 25, 2001.
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