Decreased Kallikrein 11 Messenger RNA Expression in Lung Cancer

original contribution Decreased Kallikrein 11 Messenger RNA Expression in Lung Cancer Hidefumi Sasaki, Osamu Kawano, Katsuhiko Endo, Eriko Suzuki, Hiroshi Haneda, Haruhiro Yukiue, Yoshihiro Kobayashi, Motoki Yano, Yoshitaka Fujii Abstract BACKGROUND: Human kallikrein 11 (hK11) is a putative serine protease of the human kallikrein gene family. A recently developed method for measuring hK11 suggested that the hK11 level was an indicator of favorable prognosis in patients with ovarian cancer. We have investigated hK11 messenger RNA (mRNA) levels in lung cancer. PATIENTS AND METHODS: This study included 64 lung cancer cases. The hK11 mRNA levels were quantified by real-time reverse-transcriptase polymerase chain reaction using LightCycler®. RESULTS: The hK11 mRNA levels were lower in tumor tissues from lung cancer (1.88 ± 6.314) compared with adjacent nonmalignant lung tissues (8.271 ± 9.002; n = 45; P = 0.0001). No significant difference was found among sex, age, clinical stages, tumor status, and lymph node metastasis. The hK11 mRNA levels were lower in moderately or poorly differentiated adenocarcinoma lung cancer (0.452 ± 1.614) compared with well-differentiated adenocarcinoma lung cancer (1.728 ± 2.829; P = 0.0281). The group with low hK11/gylceraldehyde 3-phosphate dehydrogenase levels (< 0.6) had a significantly worse prognosis compared with the group with high hK11/ gylceraldehyde 3-phosphate dehydrogenase levels (> 0.6; log-rank test, P = 0.0131; Breslow-Gehan-Wilcoxon test, P = 0.0172). CONCLUSION: Cox proportional hazard regression model (multivariate analysis) indicated that pathologic stages (P = 0.0443) and low hK11 expression levels (P = 0.0469) were the prognostic factors of lung cancers. However, additional studies and a longer follow-up are needed to confirm the impact of hK11 on the biologic behavior of the tumor. Clinical Lung Cancer, Vol. 8, No. 1, 45-48, 2006

Key words: Adenocarcinoma, Differentiation, Polymerase chain reaction, Prognosis

Introduction

at the DNA and amino acid level.4,5 All members of the hK gene family localize on chromosome 19q13.4 and encode for secreted serine proteases. Prostate-specific antigen (PSA) is a well-known member of the hK family of serine proteases and is currently used widely for diagnosis and monitoring of prostatic carcinoma.6 In addition, many other members of the same family have been overexpressed or underexpressed in other cancers.7-16 The gene encoding hK11 was first cloned by Yoshida et al.17 With the newly established kallikrein gene nomenclature, this gene is now known as hK11.18 By reverse-transcriptase polymerase chain reaction (PCR), it was demonstrated that the hK11 gene is highly expressed in many normal tissues, including stomach, uterus, salivary gland, thymus, prostate, and lung.19 The serum levels of hK11 were increased in prostate and ovarian cancer but not in lung cancers.20 Interestingly, 60% of patients with prostate and 50% of patients with ovarian cancer had high serum hK11 levels; however, no patients with lung cancer had a high serum hK11 level, and to our knowledge, there is no report on hK11 messenger RNA

Lung cancer is a leading cause of death from malignant diseases because of its high incidence, malignant behavior, and lack of major advancements in treatment strategy.1 Lung cancer was the leading indication for surgery (42.2%) in 1998 in Japan. More than 15,000 patients underwent surgical operation at Japanese institutions.2 The clinical behavior of lung cancer is largely associated with its stage. The cure of the disease by surgery is achieved only in cases representing an early stage of disease.3 Recently, the human kallikrein (hK) gene family has been expanded to include 15 members that share significant similarities Department of Surgery II, Nagoya City University Medical School, Nagoya, Japan Submitted: Feb 15, 2006; Revised: Apr 18, 2006; Accepted: Apr 19, 2006 Address for correspondence: Hidefumi Sasaki, MD, 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]

Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by CIG Media Group, LP, ISSN #1525-7304, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA 978-750-8400.

Clinical Lung Cancer July 2006

45

Kallikrein 11 Messenger RNA Levels in Lung Cancer (mRNA) expression in lung cancer. More recently, hK11 levels from ovarian carcinoma were reported to be an indicator of favorable prognostic factor.21,22 In a search of tumor markers for lung carcinoma screening and diagnostic purposes, we asked whether hK11 mRNA levels could serve as a marker to identify patients with lung cancer, especially in terms of prognostic potential. In this report, we investigated hK11 mRNA levels by real-time reverse-transcriptase PCR assay using LightCycler®. The findings were compared with the clinicopathologic features of lung cancer.

Patients and Methods The study groups included 64 patients with lung cancer who had undergone surgery at the department of surgery II, Nagoya City University Medical School between 1997 and 1999. The lung tumors were classified according to the general rule for the clinical and pathologic record of lung cancer in Japan.23 All tumor samples were immediately frozen and stored at −80°C until assayed. The clinical and pathologic characteristics of the 64 patients with lung cancer are shown in Table 1. These include 26 cases at stage I, 13 at stage II, 23 at stage III, and 2 at stage IV. The mean age was 63.6 years (range, 42-80 years). Among the patients with lung cancer, 38 (59.4%) were diagnosed with adenocarcinoma, 15 (23.4%) with squamous cell carcinoma, and 3 (4.7%) with bronchioloalveolar carcinoma.

Endogenous perioxidase was blocked with 0.3% hydrogen peroxide. For antigen retrieval, tissue sections were put in a microwave oven for 5 minutes in 10-mmol/L citrate buffer (pH 6). After blocking with Block Ace Solution, the slides were incubated with the monoclonal antibody against hK11 (1 μg/μL) at 1:50 dilutions overnight at 4°C. Envision Kit and DAB substrate were used to visualize the antibody binding, and the sections were counterstained with hematoxin. Statistical Analysis Statistical analyses were done 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 pathologic subtypes in the patients were examined using the test of Kruskal-Wallis and Fisher’s protected least significant difference test. The overall survival of patients with lung cancer was examined using the Kaplan-Meier method. Differences were examined by the log-rank test and Breslow-Gehan-Wilcoxon test for univariate analysis and Cox proportional hazard regression model for multivariate analysis. All analysis was done using the StatView® software package and was considered significant when the P value was < 0.05.

Results

46

Reverse-Transcriptase Polymerase Chain Reaction Assays for Kallikrein 11 Total RNA was extracted from lung cancer tissues and adjacent nonmalignant lung tissues using Isogen kit according to the manufacturer’s instructions. Total RNA was also extracted from the lung cancer cell line CCL185 (A549, adenocarcinoma cell line). This RNA was used as a positive control. RNA concentration was determined by spectrophotometer and adjusted to a concentration of 200 ng/mL. RNA (1 μg) was reverse transcribed by Superscript II enzyme with 0.5 μg oligo (dT).12-16 The reaction mixture was incubated at 42°C for 50 minutes and then at 72°C for 15 minutes. 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. All PCR reactions were performed using LightCycler®-FastStart DNA Master SYBR Green I kit. The primer sequences for the hK11 gene were as follows: the forward primer, 5-CATCATTGAGCACCAGAAGT-3, and the reverse primer, 5-TGCCTTGAAGAGACTGGTTA-3 (146 base pairs). The cycling conditions were as follows: initial denaturation at 95°C for 10 minutes, followed by 60 cycles at 94°C for 15 seconds, 52°C for 5 seconds, and 72°C for 6 seconds.

Human Kallikrein 11 Messenger RNA Expression In the lung cancer tissues, the mean value for hK11 mRNA level as standardized by the mRNA level of GAPDH (1.88 ± 6.314; mean ± standard deviation) was significantly lower than the samples from nonmalignant lung tissue (8.271 ± 9.002; P = 0.0001), and variations in the level were not related to age. The mRNA levels of hK11 of the same sample were as follows: stage I, 3.27 ± 9.456; stage II, 0.265 ± 0.32; stage III, 0.795 ± 1.152; stage IV, 6.774 ± 9.335 (Table 1). The hK11/GAPDH level in bronchioloalveolar carcinoma (17.233 ± 26.519) was significantly higher than the levels in other pathologic subtypes of lung cancer (adenocarcinoma, 1.064 ± 2.261; squamous cell carcinoma, 1.632 ± 3.418; other carcinoma, 0.479 ± 0.342). No significant difference between hK11/GAPDH mRNA levels was found among sex and age. Patients groups were further stratified according to clinicopathologic factors. The hK11/GAPDH mRNA levels were not significantly different among tumors with different T factors (T1, 0.45 ± 0.591; T2, 3.899 ± 9.622; T3, 0.215 ± 0.268; T4, 0.842 ± 0.977) or N factors (N0, 2.085 ± 7.281; N1, 0.667 ± 0.711; N2, 1.587 ± 3.469). In adenocarcinoma, the hK11 mRNA levels were lower in moderately or poorly differentiated adenocarcinoma lung cancer (0.452 ± 1.614) compared with well-differentiated adenocarcinoma lung cancer (1.728 ± 2.829; P = 0.0281).

Immunohistochemistry Mouse monoclonal antibody against hK11 (clone FB6MA11) was purchased from BioLinks Co. Four-micrometer sections were made from paraffin tissue blocks from lung tumors. The slides were treated with xylenes and then dehydrated in alcohol.

Relationship Between Clinical Course and Human Kallikrein 11 Levels The overall survival of the 64 patients with lung cancer was studied in reference to the hK11/GAPDH mRNA levels. The group with low hK11/GAPDH levels (< 0.6) had a signifi-

Clinical Lung Cancer July 2006

Hidefumi Sasaki et al Figure 1 hK11 mRNA Expression and Prognosis of Lung Cancers

Table 1 Clinicopathologic Data of Patients Kallikrein 11 Kallikrein 11/ GAPDH mRNA Levels

P Value

100

64

1.88 ± 6.314

0.4261 r2 = 0.058†

80

” 60

24 (37.5)

3.784 ± 10.052

> 60

40 (62.5)

0.737 ± 1.116

49 (76.6)

1. 288 ± 2.697

15 (23.4)

3.813 ± 12.217

I

26 (41.3)

3.27 ± 9.456

II

13 (20.6)

0.265 ± 0.32

III

23 (36.5)

0.795 ± 1.152

IV

2 (3.2)

6.774 ± 9.335

Total* Age (Years)

0.7392

Sex Male Female

Survival Rate (%)

Number of Patients

Factor

60 40 20

0.5208

Disease Stage

hK11 High Expression hK11 Low Expression

P = 0.0131

0

20

40

60

80

Months NS

Figure 2 hK11 Protein Expression in Lung Cancer by Immunohistochemistry

Tumor Status T1

11 (17.2)

0.45 ± 0.591

T2

26 (40.6)

3.899 ± 9.622

T3

14 (21.9)

0.215 ± 0.268

T4

13 (20.3)

0.842 ± 0.977

45 (70.3)

2.085 ± 7.281

N1

4 (6.3)

0.667 ± 0.771

N2

15 (23.4)

1.587 ± 3.469

Adenocarcinoma

38 (59.4)

1.064 ± 2.261

SCC

15 (23.4)

1.632 ± 3.418

BAC

3 (4.7)

17.233 ± 26.519

8 (12.5)

0.479 ± 0.342

Well

17 (48.6)

1. 728 ± 2.829

Moderately

10 (28.6)

0.779 ± 2.157

Poorly

8 (22.9)

0.043 ± 0.059

NS

Lymph Node Metastasis N0

NS

Pathologic Subtype

Others

NS

Differentiation Level of Adeocarcinoma

NS

Values in parentheses are percentages. *Patients’ median age was 63.6 years ± 9.6 years. †Correlation with hK11 levels for all 64 patients. Abbreviations: BAC = bronchioloalveolar carcinoma; NS = not significant; SCC = squamous cell carcinoma

cantly worse prognosis compared with the group with high hK11/GAPDH levels (> 0.6; Breslow-Gehan-Wilcoxon test, P = 0.0172; log-rank test, P = 0.0131; Figure 1). Using the Cox proportional hazard regression model for multivariate analysis, pathologic stage (P = 0.0443) and hK11 (P = 0.0469), but not age and sex, were the prognostic factors. Human Kallikrein 11 Protein Expression The immunohistochemical (IHC) approach was used to localize hK11 protein expression in 39 lung cancers samples. The

alveolar cells of the normal lung cancer samples were stained. In contrast, 11 of 39 of the lung cancer samples were stained (Figure 2). Because the tissue sections were taken from a different part for the mRNA analyses, there was no significant difference in the hK11 mRNA levels between the hK11 IHC+ (1.528 ± 3.411) and hK11 IHC– (0.893 ± 1.27) lung cancer samples (P = 0.7786). However, the IHC+ rate was higher in adenocarcinoma (10 of 26) than in the others (1 of 13).

Discussion In this report, we obtained findings that hK11 mRNA expression levels were significantly lower in lung cancer tissues than the levels in nonmalignant lung tissues. In addition, there were significantly higher hK11 expression levels in well-differentiated adenocarcinomas compared with those in moderately or poorly differentiated adenocarcinomas. Kallikreins are a subgroup of serine proteases. Kallikrein are known to play important roles in divers physiologic processes.24 The gene encoding hK11 was first cloned by Yoshida et al and was named as trypsin-like serine protease.17 With the newly established kallikrein gene nomenclature, this gene is now known as KLK11.18 The gene spans 5308 base pairs on chromosome

Clinical Lung Cancer July 2006

47

Kallikrein 11 Messenger RNA Levels in Lung Cancer 19q13.3-313.4. Furthermore, the significant similarities were found between hK11 and other serine proteases of the human kallikrein family, suggesting that this gene belongs to the same family.19 When the protein-coding sequence of hK11 was compared with that of other kallikrein-like proteins, it was found that neuropsin (hK10)13,25,26 has 51% exact matches. Moreover, KLK-L2 (hK5)27,28 has 50% exact homology and 68% matches with conservative changes. Recently, many kallikrein-like genes have been implicated in the development and/or progression of different malignancies. Human kallikrein 3 (hK3/PSA) is the best diagnostic and prognostic marker for prostate cancer to date.29 Recombinant hK2 protein has been shown to activate PSA in vitro,30 and the combination of hK2 and free PSA has recently been found to increase the discrimination between prostate cancer and benign prostatic hyperplasia in patients with moderately increased total PSA levels.31 On the other hand, normal epithelial cell−specific 1 (hK10) was found to be a tumor suppressor gene.26 For example, NES1 expression is downregulated during breast cancer progression.25 Zyme/proteaseM/neurosin (hK6) is suggested to be important in establishing breast and ovarian tumors and could function later in progression as a potential metastatic inhibitor.32 More recent data suggest that PSA might be a potent antiangiogenic molecule33 and an inducer of apoptosis and reduced cell proliferation.34,35 All these data allow us to speculate that at least some members of the hK gene family could be involved in diverse malignancies and might serve as markers of the disease. A recent report showed that serum levels of hK11 were increased in prostate (60%) and ovarian (50%) but not in lung cancers (0%).20 More recently, hK11 levels from ovarian carcinoma were reported to be an indicator of favorable prognostic factor.21,22 Bhattacharjee et al demonstrated that hK11 mRNA levels were increased in a subclass of adenocarcinoma; however, their data compared hK11 expression within adenocarcinomas.36 Although hK11 mRNA levels might be increased in the neuroendocrine subtype of lung cancer, our report did not include neuroendocrine lung carcinomas.

Conclusion Our results showed that the survival rates were significantly different between the groups of normal and decreased hK11 mRNA levels in lung carcinoma, although the results might be simply affected by the difference in the hK11 levels between the differentiation levels of cancers. However, because the observation period was short, additional studies and a longer follow-up are needed to confirm the impact of hK11 in the biologic behavior of the tumor.

References 1. Ginsberg RJ, Kris MK, Armstrong JG. Cancer of the lung. In: Vincet T. Devita, Samuel Hellman, Steven A. Roseberg, eds. Principles and Practice of Oncology. 4th ed. Lippincott Williams & Wilkins; 1993; 4:673-682. 2. Yasuda K, Ayabe H, Ide H, et al. On behalf of the Japanese Association for Thoracic Surgery. Thoracic and cardiovascular surgery in Japan during 1998. Annual report by the Japanese association for thoracic surgery. Committee of Science. Jpn J Thorac Cardiovasc Surg 2000; 48:401-415. 3. Postus PE. On behalf of the Lung Cancer Cooperative Group of the EORTC. The experience of the lung cancer cooperative group of the European Organization for

48

Clinical Lung Cancer July 2006

Research and Treatment of Cancer. Chest 1998; 113(suppl 1):28S-31S. 4. Diamandis EP, Yousef GM, Luo LY, et al. The human kallikrein gene family-implication in carcinogenesis. Trends Endocrino Metab 2001; 11:54-60. 5. Yousef GM, Diamandis EP. The new human tissue kallikrein gene family: structure, function and association to disease. Endocrin Rev 2001; 22:184-204. 6. Diamandis EP. Prostate specific antigen-its usefulness in clinical medicine. Trends Endocrinol Metab 1998; 9:310-316. 7. Obiezu CV, Scorilas A, Katsaros D, et al. Higher human kallikrein gene 4 (KLK4) expression indicates poor prognosis of ovarian cancer patients. Clin Cancer Res 2001; 7:2380-2386. 8. Kim H, Scorilas A, Katsaros D, et al. Human kallikrein gene 5 (KLK5) expression is an indicator of poor prognosis in ovarian cancer. Br J Cancer 2001; 84:643-650. 9. Hoffman BR, Katsaros D, Scorilas A, et al. Tumor specific activity of kallikrein 6 protein is a prognostic marker of ovarian carcinoma. Pro Am Assoc Cancer Res 2001; 42:46-47. 10. Magklara A, Scorilas A, Katsaros D, et al. The human KLK8 (neuropsin/ovasin) gene: identification of two novel splice variants and its prognostic value in ovarian cancer. Clin Cancer Res 2001; 7:2372-2379. 11. Luo LY, Katsaros D, Scorilas A, et al. Prognostic value of human kallikrein 10 expression in epithelial ovarian carcinoma. Clin Cancer Res 2001; 7:2372-2379. 12. Yousef GM, Scorials A, Jung K, et al. Molecular cloning of the human kallikrein 15 gene (KLK15): up-regulation in prostate cancer. J Biol Chem 2001; 276:53-61. 13. Luo LY, Rajperr-DeMeyts E, Jung K, et al. Expression of the normal epithelial cellspecific 1 (NES1; KLK10) candidate tumor suppressor gene in normal an malignant testicular tissue. Br J Cancer 2001; 85:220-224. 14. Yousef GM, Magklara A, Chang A, et al. Cloning of a new member of the human kallikrein gene family, KLK14, which is down-regulated in different malignancies. Cancer Res 2001; 61:3425-3431. 15. Tanimoto H, Underwood LJ, Shigemasa K, et al. The stratum corneum chymotryptic enzyme that mediates shedding and desquamation of skill cell is highly over-expressed in ovarian tumor cells. Cancer (Phila) 1999; 86:2074-2082. 16. Underwood LJ, Tanimoto H, Wang Y, et al. Cloning of tumor-associated differential expressed gene-14, a novel serine protease overexpressed by ovarian carcinoma. Cancer Res 1999; 59:4435-4439. 17. Yoshida S, Taniguchi M, Suemoto T, et al. cDNA cloning and expression of a novel serine protease, TLSP. Biochem Biophys Acta 1998; 1399:225-228. 18. Diamandis EP, Yousef GM, Clements J, et al. New nomenclature for the human tissue kallikrein gene family. Clin Chem 2000; 4:1855-1858. 19. Yousef GM, Scorilas A, Diamandis EP. Genomic organization, mapping tissue expression and hormonal regulation of trypsin-like serine protease (TLSP PRSS20) a new member of the human kallikrein gene family. Genomics 2000; 63:88-96. 20. Diamandis EP, Okui A, Mitsui S, et al. Human kallikrein 11: a new biomarker of prostate and ovarian carcinoma. Cancer Res 2002; 62:295-300. 21. Diamandis EP, Borgono CA, Scorilas A, et al M. Human kallikrein 11: an indicator of favorable prognosis in ovarian cancer patients. Clin Biochem 2004; 37:823-829. 22. Borgono CA, Fracchioli S, Yousef GM, et al. Favorable prognostic value of tissue human kallikrein 11 (hK11) in patients with ovarian carcinoma. Int J Cancer 2003; 106:605-610. 23. Japan Lung Cancer Society. General Rule for Clinical and Pathological Record of Lung Cancer. 5th ed. The Japan Lung Cancer Society 1999; 5:1-177. 24. Clements J. The molecular biology of the kallikreins and their role in inflammation. In: Farmer, S, ed. The Kinin System. New York, NY: Academic Press; 1997:71-97. 25. Liu XL, Wazer DE, Watanabe K, Band V. Identification of a novel serine proteaselike gene, the expression of which is down-regulated during breast cancer progression. Cancer Res 1996; 56:3371-3379. 26. Goyal J, Cowan JM, Wazer DE, et al. The role for NES1 serine protease as a novel tumor suppressor. Cancer Res 1998; 58:4782-4786. 27. Yousef GM, Diamandis EP. The new kallikrein-like gene KLK-L2: molecular characterization, mapping, tissue expression and hormonal regulation. J Biol Chem 1999; 274:37511-37516. 28. Brattsand M, Egelrud T. Purification molecular cloning, and expression of a human stratum corneum trypsin-like serine protease with possible function in desquamation. J Biol Chem 1999; 274:30033-30040. 29. Diamandis EP. Prostate specific antigen: its usefulness in clinical medicine. Trends Endocrinol Metab 1998; 9:310-316. 30. Takayama TK, Fujikawa K, Davie EW. Characterization of the precursor of prostatespecific antigen. Lung Cancer 1996; 16:47-59. 31. Stenman U. New ultrasensitive assay facilitate studies on the role of human glandular kallikrein (hK2) as a marker for prostatic disease. Clin Chem 1999; 45:753-754. 32. Anisowicz A, Sotiropoulou G, Stenman G, et al. A novel protease homolog differentially expressed in breast and ovarian cancer. Mol Med 1996; 92:624-636. 33. Fortier AH, Nelson BJ, Grella DK, et al. Antiangiogenic activity of prostate-specific antigen. J Natl Cancer Inst 1999; 91:1635-1640. 34. Balbay MD, Juang P, Llans N, et al. Stable transfection of human prostate cancer cell line PC-3 with prostate specific antigen induces apoptosis both in-vivo and in-vitro. Proc Am Assoc Cancer Res 1999; 40:225-226. 35. Lai LC, Erbas H, Lennard TWJ, et al. Prostate-specific antigen in breast cyst fluid: possible role of prostate-specific antigen in hormone-dependent breast cancer. Int J Cancer 1996; 66:743-746. 36. Bhattacharjee A, Richards WG, Staunton J, et al. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci U S A 2001; 98:13790-13795.