Human MOB1 Expression in Non–Small-Cell Lung Cancer

original contributio n Human MOB1 Expression in Non–Small-Cell Lung Cancer Hidefumi Sasaki, Osamu Kawano, Katsuhiko Endo, Eriko Suzuki, Haruhiro Yukiue, Yoshihiro Kobayashi, Motoki Yano, Yoshitaka Fujii Abstract PURPOSE: Human MOB1 (hMOB1) is a recently isolated gene that is a human homologue of the Schizosaccharomyces mitotic checkpoint gene MOB1. The loss of checkpoint control in mammalian cells results in genomic instability, leading to the amplification, rearrangement, or loss of chromosomes, events associated with tumor progression. We hypothesized that hMOB1 might be expressed in non–small-cell lung cancer (NSCLC). PATIENTS AND METHODS: We attempted to determine the influence of hMOB1 expression on clinicopathologic features in patients with NSCLC who had undergone surgery. Expression of hMOB1 messenger RNA (mRNA) was evaluated by reverse transcription-polymerase chain reaction in 60 NSCLCs and adjacent histologic normal lung samples using LightCycler®. RESULTS: Human MOB1/glyseraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was significantly decreased in the tumor of lung cancer tissue (3.347 ± 4.306) compared with normal lung tissue (4.833 ± 4.306; P = 0.0437), although 22 of 60 lung cancer tissue samples had > 1 tumor-normal ratio of MOB1/GAPDH mRNA expression. There was no relationship between hMOB1 gene expression and age, sex, pathologic stages, or pN status. However, decreased hMOB1/GAPDH expression was especially seen in pT1 lung cancer (tumor-normal ratio; 0.318 ± 0.328) when compared with pT4 lung cancer (1.915 ± 1.895; P = 0.0362). CONCLUSION: The decreased expression of hMOB1 mRNA might be the early phase phenomenon for tumor invasion from NSCLC. Alternatively, loss of mitotic checkpoint might play a role in oncogenesis for lung cancer. Clinical Lung Cancer, Vol. 8, No. 4, 273-276, 2007

Key words: Messenger RNA, Mitotic checkpoint, pN status, Tumor invasion

Introduction

or breast cancer.5 A variety of clinicopathologic characteristics could affect prognosis.6 Despite steadily accumulating evidence that numerous genetic markers influence the biologic behavior of NSCLC, the intrinsic nature of gene deregulation that leads small tumors to metastasize remains highly elusive.7 The abnormal expression of certain genes in cancer cells is closely related to various aspects of tumor progression, including tumor growth, invasion, and metastasis. Proper cell division requires precise coordination and execution of several events in the cell cycle, including centrosome duplication, DNA replication, mitotic spindle assembly, chromosome segregation, and cytokinesis. A failure in the execution or proper timing of any of these events could lead to chromosome segregation defects, resulting in aneuploidy or polyploidy. Such genomic instability is the hallmark of transformed cells but has also been observed in various mutant strains of yeast.8-10 MOB1 is an essential Saccharomyces cerevisiae gene required for completion of mitosis and maintenance of ploidy.11 More

Recently, lung cancer incidence has been increasing in Japan, and lung cancer has become the leading cause of cancer-related death among Japanese men1 because of its malignant behavior and lack of major advancements in treatment.2 Lung cancer was the leading indication for respiratory surgery (42.2%) in 1998 in Japan.3 More than 15,000 patients underwent surgery at Japanese institutions in 1998. The clinical behavior of non–small-cell lung cancer (NSCLC) is largely dependent on its stage.4 However, the prognosis for patients with operable NSCLC remains gloomy in comparison with that observed in gastric, colon, Department of Surgery II, Nagoya City University Medical School, Japan Submitted: April 22, 2006; Revised: June 21, 2006; Accepted: June 23, 2006 Address for correspondence: Hidefumi Sasaki, MD, PhD, Department of Surgery II, Nagoya City University Medical School, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya, 467-8601, Japan Fax: 81-52-853-6440; e-mail: [email protected]

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Clinical Lung Cancer January 2007

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Human MOB1 Expression in Lung Cancer

A

Fluorescence (F1)

Figure 1

Reverse-Transcriptase Polymerase Chain Reaction Analysis for MOB1 Using LightCycler®

15 13 11 9 7 5 3 1 –1 0 2

6

10

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26

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34

38

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46

50

B

Fluorescence -d(F1)/dT

Cycle Number 6.5 5.5 4.5 3.5 2.5 1.5 0.5 –0.5 67 68

70

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92 93

Temperature (°C) (A) MOB1 products were seen between 22 and 32 cycles. (B) Melting curve for MOB1 showed 1 peak at 79°C.

recently, mammalian homologue of MOB1 was identified and found to be a member and a putative substrate of strain family-protein phosphatase 2A complexes.12 Because human homologue BUB1, which is a yeast mitotic gene, was found to be inactivate in several human tumors, including primary colon cancer,13 we investigated human MOB1 (hMOB1) transcript in patients with NSCLC by means of a reverse-transcription polymerase chain reaction (PCR) analysis using LightCycler®. We assessed the clinocopathologic factors of hMOB1 expression in these patients.

Patients and Methods The study groups included 60 patients with NSCLC who had undergone surgery at the Department of Surgery II, Nagoya City University Medical School between January 1997 and December 1999. The lung cancers were classified according to the general rule for clinical and pathologic record of lung cancer.14 All tumor and normal lung samples were collected at resection and immediately frozen in liquid nitrogen. Reverse-Transcriptase Polymerase Chain Reaction Assays for Human MOB1 Total RNA was isolated from tumor and adjacent histologic normal lung tissue using Isogen kit according to the manufacturer’s instructions. RNA concentration was determined by spectrophotometer and adjusted to a concentration of 200 ng/mL. Human lung cancer cell line CRL185 (A549, adenocarcinoma cell line) was used. Total RNA from the cell line was also isolated. RNA (1 μg) was reverse transcribed by Superscript II enzyme with 0.5 μg oligo

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(dT).15 The reaction mixture was incubated at 42°C for 50 minutes followed by incubation at 72°C for 15 minutes. To ensure the fidelity of messenger RNA (mRNA) extraction and reverse transcription, all samples were subjected to PCR amplification with oligonucleotide primers specific for the constitutively expressed gene glyceraldehyde3-phosphate dehydrogenase and normalized. The primer sequences for the hMOB1 gene were as follows: forward primer, 5-TGGCACCTCTTCAAGAATTA-3; and reverse primer, 5-GTAGACACAGGCAATGGGTA-3 to amplify a 182 bp fragment. The cycling conditions were as follows: initial denaturation at 95°C for 10 minutes, followed by 50 cycles at 95°C for 5 seconds, 60°C for 5 seconds, 72°C for 8 seconds (Figure 1). All PCR reactions were performed using a LightCycler® FastStart DNA Master SYBR Green I kit and quantified.16 Statistical Methods Statistical analysis was done using the Stat-View software package. Differences among the means of the age and sex were examined using the Mann-Whitney U test. Differences among the means of pathologic stages, pN and pT status, and pathologic subtypes in the patients with NSCLC were examined using Fisher’s paired least significant difference test. The overall survival of patients with NSCLC was examined by the Kaplan-Meier method, and survival characteristics were compared using log rank tests. It was considered significant when the P value was < 0.05.

Results Group Characteristics The clinical and pathologic characteristics of the 60 patients with NSCLC are shown in Table 1; these included 22 cases at stage I, 13 at stage II, 23 at stage III, and 2 at stage IV. The mean age was 64.5 years (range, 42-88 years). Among the 60 patients with NSCLC, 13 (21.7%) were women and 47 (78.3%) were men. Forty had lymph node metastasis–negative disease and 20 had lymph node metastasis–positive disease. Of 60 patients, 14 had squamous cell carcinoma and 38 had adenocarcinoma (Table 1). Human MOB1 Messenger RNA Expression by Reverse Transcriptase-Polymerase Chain Reaction Assay Of the samples studied from 60 patients with NSCLC, all of the normal lung samples and 55 of 60 lung cancer samples had hMOB1 transcript. The hMOB1/glyseraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was significantly decreased in the tumor of lung cancer tissue (3.347 ± 4.306) compared with the normal lung tissue (4.833 ± 4.306; P = 0.0437; Figure 2). The tumor-normal ratio of hMOB1/GAPDH expression in men (1.226 ± 1.909) and women (1.081 ± 0.99) was not significantly different (P = 0.7947). Patient groups were further stratified according to pathologic factors.

Hidefumi Sasaki et al Table 1

Figure 2

Clinicopathologic Data of 60 Patients with Non–Small-Cell Lung Cancer

MOB1/GAPDH Messenger RNA Expression in Lung Tumor and Adjacent Normal Lung Tissues

Tumor-Normal Ratio of MOB1 Expression

Mean Age (Years)

No. of Patients (%) 64.5 ± 9.7

MOB1/GAPDH (T/N ratio)

P = 0.0437 P Value

30 25

T; 3.347 ± 2.814 N; 4.833 ± 4.306

–

Age (Years) < 60

23 (38.3)

1.76 ± 2.499

> 60

37 (61.7)

0.843 ± 0.919

Male

47 (78.3)

1.226 ± 1.909

Female

13 (21.7)

1.081 ± 0.99

I

22 (36.7)

1.168 ± 2.316

II

13 (21.7)

0.788 ± 0.809

III

23 (38.3)

1.456 ± 1.586

IV

2 (3.3)

1.129 ± 0.318

0.0959

MOB1/GAPDH mRNA Levels

Characteristic

20 15 10

3.347

4.833

5

Sex

0

Tumor

Normal

0.7947

Pathologic Stage

Figure 3 Not significant

P = 0.0362 12

T Status 9 (15)

0.318 ± 0.328

pT2

26 (43.3)

1.271 ± 2.136

pT4 vs. pT1

pT3

12 (20)

0.906 ± 0.793

0.0362

pT4

13 (21.7)

1.915 ± 1.895

Lymph Node Metastasis

MOB1/GAPDH mRNA Levels

10

pT1

pN0

MOB1/GAPDH Messenger RNA Levels in Lung Cancer According to the Pathologic T Status

8 6 4

1.915

2

40 (66.7)

0.318

1.308 ± 2.058

1.271

0

pN1

4 (6.7)

0.54 ± 0.51

pN2

16 (26.7)

1.074 ± 0.865

SCC

14 (23.3)

1.689 ± 2.805

Adenocarcinoma

38 (63.3)

1.005 ± 1.361

Others

8 (13.3)

1.184 ± 0.824

Not significant

pT1

pT2

0.906 pT3

pT4

Histology

Discussion Not significant

Abbreviation: SCC = squamous cell carcinoma

There was no difference of tumor-normal ratio of hMOB1/ GAPDH mRNA expression between groups with different pathologic stages (stage I, 1.168 ± 2.316; stage II, 0.788 ± 0.809; stage III, 1.456 ± 1.586; stage IV, 1.129 ± 0.318). The hMOB1/GAPDH overexpressed tumors appeared more frequently in tumors with pathologically negative lymph node metastases (tumor-normal ratio, 1.308 ± 2.058) than in tumors with lymph node metastases (tumor-normal ratio, 0.967 ± 0.824); however, the difference was not significant (P = 0.7419). There was no significant difference between histologic subtype and hMOB1/GAPDH gene expression. Tumor-normal ratios of hMOB1/GAPDH mRNA expression in different pathologic T status were: pT1, 0.318 ± 0.328; pT2, 1.271 ± 2.136; pT3, 0.906 ± 0.793; pT4, 1.915 ± 1.895. Thus, decreased hMOB1/GAPDH expression was especially seen at pT1 lung cancer when compared with pT4 lung cancer (P = 0.0362; Figure 3).

In the present study, the basal level of hMOB1 mRNA was analyzed for the first time in tumor samples from patients with NSCLC. hMOB1 mRNA expression was significantly decreased in NSCLC cases compared with that of paired normal lung tissue. In addition, decreased hMOB1/GAPDH expression was especially seen at pT1 lung cancer compared with pT4 lung cancer. These results suggest that decreased hMOB1 expression, alternatively the loss of checkpoint control, could play a role in oncogenesis of NSCLC. The progression of the eukaryotic cell cycle can be arrested in the presence of DNA damage or incomplete DNA replication. This mechanism, termed cell cycle checkpoint, allows a time for cells to undergo DNA repair or replication before progression into the next cell cycle stage and thus ensures high fidelity transmission of genetic information through cell generations.15 Cell cycle checkpoints have been most extensively studied in lower eukaryotes, especially in yeast systems.17 Conservation of cell cycle machinery across incredibly diverse eukaryotes is, by now, well established. Clearly, the proteins and mechanisms involved in imposing order on the cell division are of the highest importance and Clinical Lung Cancer January 2007

275

Human MOB1 Expression in Lung Cancer arose early in the course of evolution. Such similarities in proteins can be exploited to identify homologues of known proteins in different organisms. Although a functional role of hMOB1 has not yet been established, its homology to yeast checkpoint proteins suggests it might play a part in cell cycle control.11,12,18,19 It has been proposed in yeast that MOB1 play a role in the mitotic checkpoint.11,18,19 Exit from mitosis in budding yeast requires inactivation of cyclin-dependent kinases through mechanisms triggered by the protein phosphatase Cdc14. Mobilization of Cdc14 activity during anaphase depends on a group of genes (LTE1 TEM1, CDC5, CDC15, DBF1/DBF20, and MOB1) that comprise the mitotic exit network. Loss of function of the mitotic exit network causes cells to arrest caused by overexpression of non-degradable B-type cyclin.20 MOB1 physically interacts with the DBF2/Sid2 kinase in Saccharomyces cerevisiae21 and Schizosaccharomyces pombe.11,19 The hMOB1 gene is located on chromosome 2q12. Although no human disease has been associated with deletion of this specific chromosomal location, thousands of cases of human cancers are reported to have deletions spanning this region, including B-cell lymphoma,22 non-Hodgkin lymphoma,23 glioblastoma,24 and leukemia.25 The expression of hMOB1 mRNA was correlated with tumor status from NSCLC. The tumor invasiveness to mediastinal organs significantly influences the treatment and prognosis of patients with NSCLC.26 Previous reports suggested that expression of the Bub1 gene, which is another mitotic checkpoint gene, correlates with tumor proliferating activity in human gastric carcinomas.27 However, another report suggested that reduced Bub1 mRNA levels were associated with lymph node metastasis and shorter relapse-free survival after surgery.13 One explanation was that more checkpoint control might be needed for advanced stage of cancers. Thus, the clinical significance of the expression of the human mitotic checkpoint gene is controversial.

Conclusion The expression of hMOB1 mRNA was decreased in NSCLC and was correlated with tumor invasion from NSCLC. Longer follow-ups are warranted determining the role of hMOB1 in the progression of cancer.

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