Expression of human telomerase subunit genes in primary lung cancer and its clinical significance

Expression of Human Telomerase Subunit Genes in Primary Lung Cancer and Its Clinical Significance Mitsuyuki Arinaga, MD, Shigeki Shimizu, MD, Kunihiko Gotoh, MD, Nobuhiro Haruki, MD, Takao Takahashi, MD, Takashi Takahashi, MD, and Tetsuya Mitsudomi, MD Department of Thoracic Surgery Aichi Cancer Center Hospital, and Department of Ultrastructure Research, Aichi Cancer Center Research Institute, Aichi Cancer Center, Nagoya, Japan

Background. Three major components of human telomerase, RNA component (hTERC), telomerase-associated protein (TEP1), and catalytic subunit (hTERT) have been cloned recently. The aim of this study was to examine the expression of these genes and to search for clinical usefulness. Methods. Expression of these genes was evaluated by reverse transcription-polymerase chain reaction in 92 human lung cancers and in 32 non-neoplastic lung tissues. In 15 patients, both telomerase activity by telomeric repeat amplification protocol assay and expression were evaluated. Results. hTERT expression was best associated with

telomerase activity with a concordance of 77%. In 92 lung cancer tissues, hTERC, TEP1, and hTERT were expressed in 100%, 93%, and 89%, respectively. Whereas most adjacent non-neoplastic lung tissues expressed hTERC and TEP1 (94% and 100%, respectively), hTERT was detected in only 1 of 32 normal lungs. However, there was no relationship between hTERT expression and clinicopathologic features. Conclusions. hTERT expression can be a surrogate for telomerase activity that may serve as a novel biomarker of lung cancer with high specificity and sensitivity. (Ann Thorac Surg 2000;70:401– 6) © 2000 by The Society of Thoracic Surgeons

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terminal fusion [4]. Human telomeres contain the repeat TTAGGG, which may be reiterated in tandem for up to 15 kb. Telomere length has been implicated in the control of cell life span and telomerase, the enzyme that elongates telomeric DNA, is believed to have an essential role in cell immortalization [4]. Cancer cells must attain the potential for immortality to allow progression to malignant states and hence require telomerase activity. In 1994, a highly sensitive polymerase chain reaction (PCR)based method for detecting telomerase activity, the telomeric repeat amplification protocol (TRAP) assay, was developed [5]. Since then, various tumor tissues have been analyzed for telomerase activity and most are positive (⬎ 80%) [6]. However, there is a minor subset of tumors that do not exhibit telomerase activity, which is also not usually detected in non-neoplastic untransformed cells, except for germ cells and those in some self-renewing tissues [4]. Recently, constituents of the human telomerase complex have been cloned. Human telomerase RNA component (hTERC), an RNA component that acts as a template to add telomeres to ends of chromosomes, was identified first [7]. Another component, telomerase-associated protein (TEP1), is a homologue of the p80 of ciliate Tetrahymena [8]. In 1997 a further element, human telomerase catalytic subunit (hTERT) was cloned [9 –11]. This protein has several sequence motifs characteristic of the catalytic region of reverse transcriptase [9 –11]. It has been shown that expression of hTERT but not of hTERC of TEP1 parallels telomerase activity as detected by the TRAP

ung cancer is the leading cause of cancer death in North America and it became the leading cause of cancer death in men in 1993 in Japan [1]. With current standard methods of diagnosis and treatment, fewer than 15% of patients with lung cancer survive their disease [2]. Recent advances in molecular biology of human cancer have revealed that lung cancer arises and develops as a consequence of accumulation of various genetic lesions occurring in such genes as ras, myc family, erbB2, p53, RB and the putative gene(s) on the short arm of chromosome 3 [3]. Genetic lesions relevant to the prognosis of patients are considered to be of great importance in making clinical decisions regarding the optimal treatment regimen, because using existing prognostic tools it is often difficult to predict which surgically managed patients are at risk for an early disease relapse or which rare advanced-stage patients may experience a favorable survival. Alternatively, studies that translate such findings into newer diagnosis of early lung cancer are also considered of importance, because these genetic changes are often detectable in an early, preinvasive stage of lung cancer [3]. The telomere is a specialized structure that caps eukaryotic chromosomes at each end, maintaining chromosome stability by protecting them from degradation and

Accepted for publication Mar 20, 2000. Address reprint requests to Dr Mitsudomi, Department of Thoracic Surgery, Aichi Cancer Center Hospital, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan; e-mail: [email protected].

© 2000 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

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assay [9, 10, 12]. Furthermore, ectopic expression of hTERT restores telomerase activity in human cells [12, 13]. In the present study, using resected lung cancer tissues, we first assessed the relationship between telomerase activity as detected by TRAP assay and expression of hTERC, TEP1 and hTERT. Specifically, we asked the question of whether hTERT expression could be a surrogate for telomerase activity. Finally we examined 92 lung cancer tissues for expression of telomerase subunit genes by reverse transcription (RT)-PCR to search for clinical implications.

Patients and Methods Patients A total of 92 tumor specimens from patients with primary lung cancer who underwent pulmonary resection at the Department of Thoracic Surgery, Aichi Cancer Center Hospital from November 1990 to September 1998 were examined. All tissue samples were frozen in liquid nitrogen as soon as possible following surgical removal and stored at ⫺70°C until use. The patients were 66 men and 26 women ranging in age from 41 to 80 years old (median 63). The lesions were 55 adenocarcinomas, 27 squamous cell carcinomas, 3 large cell carcinomas, 4 small cell carcinomas, and 3 other types. Postoperative staging was carried out in conjunction with mapping of regional lymph node metastases and determination of tumor size, and involvement of visceral pleura according to the International Union Against Cancer (UICC) TNM system published in 1986 [14]. Forty patients had stage I disease, 21 had stage II, 28 had stage IIIA, and 3 had stage IIIB. Total RNA was isolated from 92 tumors and 32 adjacent non-neoplastic lung tissues using the acid guanidium thiocyanate/cesium chloride procedure.

Reverse Transcription-Polymerase Chain Reaction First-strand complementary DNA (cDNA) was synthesized using 5 ␮g of total cellular RNA with M-MLV reverse transcriptase (GIBCO BRL, Life Technologies, Rockville, MD) and random hexanucleotide primers (Boehringer Mannheim-Yamanouchi, Tokyo, Japan). A 281-base-pair (bp) region of hTERT was amplified with the primer TERT/U1426 (5⬘-CCT CTG TGC TGG GCC TGG ACG ATA-3⬘) and TERT/L253 (5⬘-ACG GCT GGA GGT CTG TCA AGG TAG-3⬘) [12] for 32 cycles (94°C for 45 seconds, 61°C for 45 seconds, 72°C for 90 seconds). Because these primers were designed to cross intron sequences, a 5.5-kb band would be expected when the sample is contaminated with genomic DNA. Similarly, a 264-bp region of TEP1 was amplified using primers TP1.1 (5⬘-TCA AGC CAA ACC TGA ATC TGAG-3⬘) and TP1.2 (5⬘-CC CGA GTG AAT CTT TCT ACG C-3⬘) [9] for 28 cycles (94°C for 45 seconds, 55°C for 45 seconds, 72°C for 90 seconds). A 125-bp region of hTERC was amplified using primers F3b (5⬘-TCT AAC CCT AAC TGA GAA GGG CGT AG-3⬘) and R3c (5⬘-GTT TGC TGT AGA ATG AAC GGT GGA AG-3⬘) [9] for 24 cycles (same conditions

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as for hTERT). To confirm the integrity of cDNA, mRNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also amplified using the primers GAPDHs (5⬘-GTC AAC GGA TTT GGT CGT ATT-3⬘) and GAPDH as (5⬘-AGT CTT CTG GGG GCA GTG AT-3⬘) for 24 cycles (94°C for 60 seconds, 56°C for 60 seconds, 72°C for 60 seconds). PCR products were subjected to electrophoresis through 2% agarose gels and the gels were stained with ethidium bromide. Each specimen was analyzed at least twice and, in all instances, the replication confirmed the first analysis results.

Telomeric Repeat Amplification Protocol Assay For 15 patients from whom protein extracts of tumor and non-neoplastic lung were available, we also directly assessed telomerase activity by the TRAP assay originally developed by Kim and coworkers [5] using a commercial kit (TRAPeze telomerase detection kit, Oncor, Gaithersburg, MD) according to the manufacturer’s instructions. Briefly, frozen cell pellets were dissolved in 10 to 30 ␮L of 3-[(3-cholamidopropyl) dimethylammonio]-1propanesulfonate lysis buffer, incubated on ice for 30 minutes, and then centrifuged at 14,000 rpm for 20 minutes at 4°C. The supernatants were collected and the protein contents determined using a protein assay kit from BioRad Laboratories (Richmond, CA). Twomicroliter aliquots of the cell extracts (equivalent to 6 ␮g protein) were added to a 48-␮L reaction solution containing 10 pmol TS primer (5⬘-AAT CCG TCG AGC AGA GTT-3⬘), TRAP primer mix containing a 36-bp internal standard, 0.2 ␮ L of [ ␣ 32 P] dCTP (3,000 Ci/mmol, 10 mCi/mL, Amersham, Arlington, IL) and Taq polymerase (Takara Shuzo, Ohtsu, Japan). The mixture was incubated at 30°C for 10 minutes and then heated at 90°C for 90 seconds, followed by PCR amplification (30 cycles of 94°C for 30 seconds and 55°C for 30 seconds). The PCR products (25 ␮L) were subjected to electrophoresis on a 12.5% acrylamide denaturing gel. Six base-pair ladders were visualized by autoradiography. To confirm the specificity, heat inactivated reactions were run in parallel.

Statistical Analysis Comparisons of proportion were performed using the Fisher’s exact test. The Kaplan-Meier method was used to estimate the probability of survival as a function of time and survival differences were analyzed by the logrank test. All reported p values were two-sided, and those less than 0.05 were considered to be statistically significant.

Results Relationship Between Detection of Telomerase Activity and Expression of hTERT, hTEP1, or hTERC Results for telomerase activity and expression of hTERC, TEP1, and hTERT in 15 specimens from which both RNA and protein extracts from paired non-neoplastic and tumor tissues were available are summarized in Figure 1.

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Fig 1. Results of reverse transcription-polymerase chain reaction analysis of telomerase subunits and the TRAP assay for paired lung cancer and adjacent non-neoplastic lung tissues. (AD ⫽ adenocarcinoma; CS ⫽ carcinosarcoma; GAPDH ⫽ glyceraldehyde 3-phosphate dehydrogenase; hTERC ⫽ human telomerase RNA component; hTERT ⫽ human telomerase catalytic subunit; N ⫽ non-neoplastic adjacent lung tissue; SQ ⫽ squamous cell carcinoma; T ⫽ tumor tissue; TEP1 ⫽ telomerase-associated protein; TRAP ⫽ telomeric repeat amplification protocol.)

Telomerase activity was detected in 80% (12 of 15) of tumor tissues, whereas a significantly smaller number of non-neoplatstic lung tissues (27% [4 of 15]) had telomerase activity ( p ⫽ 0.0046, Fisher’s exact test). A much clearer difference was observed for hTERT expression, detected in 10 of 15 (67%) tumor tissues, but in only 1 of 15 non-neoplastic samples (0.7%) ( p ⫽ 0.0008, Fisher’s exact test). Concordance between telomerase activity and hTERT expression (ie, both positive plus both negative/ all cases) was 11 of 15 (73%) in tumor tissues and 12 of 15 (80%) in non-neoplastic tissues, or 23 of 30 (77%) when both tumor and non-neoplastic tissues were considered. Of the 7 discordant cases, 1 was hTERT positive and TRAP negative and 6 were hTERT negative and TRAP positive. In contrast, the concordance was only 16 of 30 (53%) for hTERC and TEP1 expression, because these two subunit genes were expressed in all non-neoplastic and tumor tissues examined. Thus, hTERT expression best correlated with telomerase activity as determined by TRAP. While intensities of bands tended to be stronger in tumors than in non-neoplastic tissues in the case of hTERC, they were similar for TEP1 (Fig 1).

Expression of hTERT, TEP1, or hTERC in Lung Cancer and Clinical Features In addition to the 15 patients as described above, we examined 77 further tumors (ie, 92 in total) for expression of the 3 subunit genes of telomerase to allow assessment of linkage with various clinical and pathologic features. Seventeen adjacent non-neoplastic lung tissues were also examined (ie, 32 in total). In tumor tissues, hTERT expression was detected in 82 of 92 (89%), whereas hTERC and TEP1 expression was detected in 92 of 92 (100%) and 86 of 92 (93%) tumors, respectively. In non-neoplastic

lung tissues, hTERT, hTERC, and TEP1 expression was detected in 1 of 32 (3%), 30 of 32 (94%), and 32 of 32 (100%) patients, respectively. Associations between hTERT expression and various clinical and pathologic features are summarized in Table 1. hTERT expression was not linked with such factors as age, sex, smoking status, stage, or histologic type.

Prognostic Impact of hTERT Expression We next examined the prognostic impact of hTERT expression in our cohort. Kaplan-Meier survival curves are shown in Figure 2. Five-year survival rates for hTERT positive and negative cases were 43% and 60%, respectively. However, this difference did not reach statistical significance ( p ⫽ 0.64).

Comment Previous reports suggested that hTERT is a key component of telomerase whose expression is strictly associated with telomerase activity in various systems [9, 10, 12]. Expression of hTERT has been observed at high levels in telomerase-positive cancer cell lines but not in nonneoplastic tissues, whereas neither TEP1 nor hTERC expression correlates with telomerase activity [9, 10, 12]. However, in recent studies of surgical specimens, although expression of hTERT demonstrated the closest association with telomerase activity among the three subunits, the fit was not perfect [15–18]. This finding could have been due to contamination of normal cells in tumor samples, lymphocytes in non-neoplastic samples, or alternatively RNA degradation inevitable to some extent when dealing with clinical specimens. In the present study, hTERT expression was not always associ-

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Table 1. Relationship Between Clinicopathologic Features and hTERT Expression in NSCLC Patients Characteristics Sex Male Female Age 63⬍ ⱕ63 Smoking status Never smoker Ever smoker T T1/2 T3/4 N N0 N1/2 Stage I/II III Histologic type Adenocarcinoma Squamous cell ca Large cell ca Small cell ca Other types Totals a

No. of Patients

hTERT Expression

%

pa

66 26

58 24

(88) (92)

0.7192

45 47

39 43

(87) (91)

0.5182

25 67

24 58

(96) (87)

0.2762

75 17

66 16

(88) (94)

0.6818

69 23

59 23

(88) (90)

0.7500

61 31

52 30

(85) (97)

0.1557

55 27 3 4 3 92

49 25 3 3 2 82

(89) (93) (100) (75) (67)

0.9999

p value by the Fisher’s exact test.

ca ⫽ carcinoma; hTERT ⫽ human telomerase catalytic subunit; NSCLC ⫽ non–small-cell lung cancer.

ated with telomerase activity similar to other studies mentioned above. Nonetheless, hTERT was best associated with telomerase activity, although the fact that six of Fig 2. Survival curves for patients with lung cancer stratified by expression of human telomerase catalytic subunit (hTERT) (p ⫽ 0.64). The solid line is for patients with hTERT-positive tumors (n ⫽ 82) and the hatched line is for those with hTERT-negative tumors (n ⫽ 10).

seven discordant cases had a TRAP-positive but hTERTnegative pattern suggests a lower sensitivity for the latter. However, analysis of larger number of samples revealed sensitivities for TRAP and hTERT in 12 of 15 (80%) and 82 of 92 (89%) tumors, respectively, while specificities were 11 of 15 (73%) and 31 of 32 (97%). Hiyama and colleagues [19] earlier reported detection of telomerase activity by TRAP in 109 of 136 (80%) lung cancer tissues and in 3 of 68 (4%) specimens of nonneoplastic adjacent tissues. Albanell and colleagues [20] similarly found 84 (85%) of 99 lung cancers without prior chemotherapy to be positive in contrast with 0 of 34 non-neoplastic lung samples. In our study, hTERT expression was evident in 82 (89%) of 92 lung cancers, whereas all but one of the non-neoplastic specimens were negative. This rate is comparable to those in previous studies for TRAP. Considering that RT-PCR is less laborious than performing TRAP assays, determination of hTERT expression may be better suited for clinical application. We asked whether a small subset of lung cancer that did not have a detectable hTERT expression might show unique clinical characteristics. Hiyama and colleagues [19] reported that higher levels of telomerase activity were observed in metastatic tumors and Albanell and colleagues [20] noted significant positive association with stage or proliferation potential as determined by Ki 67 immunostaining. Furthermore, Taga and coworkers [21] showed recently that telomerase activity is one of the important poor prognostic factors in non–small-cell lung cancer patients by a multivariate analysis. However, we failed to detect any significant difference in stage of patients or overall survival between patients with and without hTERT expression. Reasons for this discrepancy may include difference of TRAP and hTERT assay, lack of statistical power due to limited number of telomerasenegative patients, relatively short follow-up period, or

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presence of unidentified bias of prognostic factors. Further studies are clearly warranted. There have been efforts to apply telomerase activity as a marker for early detection of cancers. Yahata and coworkers [22] showed that telomerase activity was detected in 18 of 22 bronchial washings from patients with lung cancer, whereas cancer cells were detected by cytologic examination in only 9 of 22. Yang and coworkers [23] considered that detection of telomerase activity is useful in diagnosis of malignant pleural effusions. As mentioned earlier, hTERT expression for diagnosis of lung cancer had both high sensitivity (89%) and specificity (97%). This finding suggests that detection of hTERT in clinical samples may indeed be very useful in early detection, considering that RT-PCR is less labor intensive than TRAP assay. In conclusion, detection of hTERT expression but not that of TEP1 or hTERC by RT-PCR is a rapid and easy approach estimating telomerase activity in human lung cancers. Although we failed to detect any association with clinical features, the high sensitivity and specificity for hTERT in lung cancers suggest that it may be an ideal target of diagnosis or therapy to ultimately improve the prognosis of this deadly disease.

We thank Mitsuko Suzuki for secretarial assistance. This work was supported in part by the Aichi Cancer Research Foundation, the Charitable Trust Soyu Medical Foundation, the BristolMeyers Squibb Biomedical Research Grant Program, a Grantin-Aid (09671403) from the Ministry of Education Science and Culture of Japan, and the Mitsui Life Social Welfare Foundation.

References 1. Statistics and Information Department. Vital statistics, 1994, Japan, Vol. 3. Tokyo: Ministry of Health and Welfare, 1996: 96 –108. 2. Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics 1998. CA Cancer J Clin 1998;48:6–29. 3. Minna JD, Sekido Y, Fong KW, Gazdar AF. Molecular biology of lung cancer. In: DeVita VT, Hellman S, Rosenberg SA, eds. Cancer: principles and practice of oncology, 5th ed. Philadelphia: J B Lippincott, 1997:849–57. 4. Rhyu MS. Telomeres, telomerase, and immortality. J Natl Cancer Inst 1995;87:884–94. 5. Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011–5.

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6. Shay JW, Bacchetti S. A survey of telomerase activity in human cancer. Eur J Cancer 1997;33:787–91. 7. Feng J, Funk WD, Wang SS, et al. The RNA component of human telomerase. Science 1995;269:1236– 41. 8. Harrington L, McPhail T, Mar V, et al. A mammalian telomerase-associated protein. Science 1997;275:973–7. 9. Nakamura TM, Morin GB, Chapman KB, et al. Telomerase catalytic subunit homologs from fission yeast and human. Science 1997;277:955–9. 10. Meyerson M, Counter CM, Eaton EN, et al. hEST2, the putative human telomerase catalytic subunit gene, is upregulated in tumor cells and during immortalization. Cell 1997;90:785–95. 11. Kilian A, Bowtell DD, Abud HE, et al. Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types. Hum Mol Genet 1997;6:2011–9. 12. Nakayama J, Tahara H, Tahara E, et al. Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet 1998;18:65– 8. 13. Counter CM, Meyerson M, Eaton EN, et al. Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase. Oncogene 1998;16:1217–22. 14. Mountain C. A new international staging system for lung cancer. Chest 1986;89(Suppl):225s–32s. 15. Kanaya T, Kyo S, Takakura M, Ito H, Namiki M, Inoue M. hTERT is a critical determinant of telomerase activity in renal cell carcinoma. Int J Cancer 1998;78:539– 43. 16. Ito H, Kyo S, Kanaya T, Takakura M, Inoue M, Namiki M. Expression of human telomerase subunits and correlation with telomerase activity in urothelial cancer. Clin Cancer Res 1998;4:1603– 8. 17. Nakano K, Watney E, McDougall JK. Telomerase activity and expression of telomerase RNA component and telomerase catalytic subunit gene in cervical cancer. Am J Pathol 1998;153:857– 64. 18. Sumida T, Hamakawa H, Sogawa K, Sugita A, Tanioka H, Ueda N. Telomerase components as a diagnostic tool in human oral lesions. Int J Cancer 1999;80:1– 4. 19. Hiyama K, Hiyama E, Ishioka S, et al. Telomerase activity in small-cell and non-small cell lung cancers. J Natl Cancer Inst 1995;87:895–902. 20. Albanell J, Lonardo F, Rusch V, et al. High telomerase activity in primary lung cancers: association with increased cell proliferation rates and advanced pathologic stage. J Natl Cancer Inst 1997;89:1609–15. 21. Taga S, Osaki T, Ohgami A, Imoto H, Yasumoto K. Prognostic impact of telomerase activity in non-small cell lung cancers. Ann Surg 1999;230:715–20. 22. Yahata N, Ohyashiki K, Ohyashiki JH, et al. Telomerase activity in lung cancer cells obtained from bronchial washings. J Natl Cancer Inst 1998;90:684–90. 23. Yang CT, Lee MH, Lan RS, Chen JK. Telomerase activity in pleural effusions: diagnostic significance. J Clin Oncol 1998; 16:567–73.

INVITED COMMENTARY Doctor Arinaga and colleagues provide an elegant basic science translational research study. Much has been learned through molecular biology about carcinogenesis and pathways of resistance to chemotherapy and subsequent apoptotic cellular death by lung tumors over the last ten years. Interest has centered around methods with which the tumors preserve and elongate the telomere which cap each arm of the eukaryotic chromosomes. Dr © 2000 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

Arinaga’s team, using RT-PCR on frozen lung tumor specimens (n ⫽ 92) resected during the last few years, sought to measure the three active components of the human telomerase complex: hTERC (an RNA component which acts as a template for telomerase extension), TEP1 (telomerase associated protein), and hTERT (the human telomerase catalytic subunit) which appears to be the most important subunit and have shown in cell culture 0003-4975/00/$20.00 PII S0003-4975(00)01549-6