Reduced FEZ1/LZTS1 Expression and Outcome Prediction in Lung Cancer

Research Article

Daisuke Nonaka,1 Alessandra Fabbri,1 Luca Roz,2 Luigi Mariani,3 Andrea Vecchione,5,6 G. William Moore,7 Luca Tavecchio,4 Carlo M. Croce,5 and Gabriella Sozzi2

Departments of 1Pathology and 2Experimental Oncology, Units of 3Medical Statistics and Biometry and 4Thoracic Surgery, Istituto Nazionale Tumori, Milan, Italy; 5Kimmel Cancer Center, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania; 6Division of Surgical Pathology, II Faculty of Medicine, University La Sapienza, Hospital Sant’Andrea, Rome, Italy; and 7Baltimore Veterans’ Administration Maryland Health Care System, University of Maryland Medical System and Johns Hopkins Medical Institutions, Baltimore, Maryland

Abstract the 8p22 region has been shown to possess oncosuppressive Chromosomal deletions are often observed in lung cancers properties in chromosome transfer experiments (9–11). Genes isolated from this area include N33 (12, 13), PRLTS (14, 15), NAT1, suggesting that inactivation of tumor suppressor genes plays an important role in the development of this neoplasm. The NAT2 (16–18) etc., but there is little functional evidence for their region around chromosome 8p22 is a frequent and early involvement in human cancer. The FEZ1 gene (subsequently target of these deletions and has therefore been investigated renamed LZTS1) was isolated from 8p22 and it was shown to be for the presence of candidate genes. The FEZ1/LZTS1 gene, inactivated in a large fraction of prostate, esophageal, and breast located at 8p22, is inactivated in many cancers with 8p cancers (19). Although the mechanisms of inactivation are not deletions, including prostate, esophageal, gastric, bladder, completely elucidated and somatic mutations are uncommon, and breast cancer and the Fez1 protein has been shown to reduced expression has been reported in gastric (20), bladder (21), suppress growth of cancer cells and to regulate mitosis. To and oral cancers (22). More importantly, restoration of its elucidate the role of FEZ1 in lung cancer, we have analyzed its expression was shown to slow the growth of breast (MCF7), expression by immunohistochemistry in 103 primary lung prostate (AT6.2), and bladder (SW780) cancer cells (21, 23, 24) cancer specimens including 98 non–small cell lung cancers (57 and to reduce their tumorigenicity in vivo giving support to the adenocarcinomas, 32 squamous cell carcinomas, 7 large cell hypothesis that it may function as a tumor suppressor gene. The product of the FEZ1 gene is a 67-kDa cytoplasmic protein that carcinomas, and 2 others) and five small cell carcinomas. binds to microtubules and has been implicated in cell cycle Absence of Fez1 protein expression was observed in 27 cases cdc2 (26%) and additional 43 cases (42%) showed strong reduction control through its interaction with the p34 /cyclinB1 complex in immunoreactivity. There was a positive association between (23). Its frequent inactivation in many human cancers and its loss of FEZ1 expression and tumor grading (P = 0.0345) and a proposed mechanism of action make it therefore a good tendency toward a reduction in the mortality rate in subjects candidate as a tumor suppressor gene. with strong FEZ1 expression. Overall, these data indicate an The 8p21-23 region is also frequently deleted in lung cancers and important role for FEZ1 in lung cancer and suggest the this genetic alteration seems to be an early event in the possibility that it may serve as a novel prognostic indicator. development of all the different histologic types of this neoplasm (Cancer Res 2005; 65(4): 1207-12) (25). A previous analysis of FEZ1 expression in lung cancer done on cell lines from non–small cell lung cancers (NSCLC), small cell lung cancers (SCLC), and short-term cultures of resected NSCLC revealed loss of expression especially in NSCLC (76% of cell lines Introduction and 50% of short-term cultures), indicating that FEZ1 may be Inactivation of tumor suppressor genes is one of the most involved in lung cancer (26). In the present study, to clarify the role common genetic alteration underlying the development of many of FEZ1 in lung cancer, we carried out the first immunohisto- human carcinomas and frequently results in the insensitivity to chemical analysis of its expression in 103 primary lung cancer antigrowth signals that is considered one of the ‘‘hallmarks’’ of specimens and analyzed its possible association with the cancer (1). Loss-of-function of these genes may be the result of histopathologic features of the tumors as well as with patients’ different molecular events (either genetic or epigenetic) that often survival over an observation time of >4 years. include deletion of the chromosomal region harboring at least one of their alleles (2). Hotspots of genomic deletion may therefore indicate the areas where tumor suppressor genes are Materials and Methods more likely to reside. Chromosome 8p deletions are frequently Tissue Samples and Immunohistochemistry. This is a retrospective observed in many human cancers including prostate, colorectal, study of primary lung cancers diagnosed and treated between 1998 and bladder, head and neck, breast, and gastric carcinomas (3–8) and 2000 at the Istituto Nazionale Tumori (National Cancer Institute), Milan, Italy. Cases included in this study were those that were diagnosed as primary lung carcinomas and treated by surgical intervention for initial treatment and had adequate paraffin blocks for histologic and immuno- Request for reprints: Gabriella Sozzi, Cytogenetics and Molecular Cytogenetics histochemical analysis as well as complete clinicopathologic data. A total of Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Via Venezian 1, 103 lesions (from 100 patients) were studied. H&E-stained slides were 20133 Milan, Italy. Phone: 39-02-23902232; Fax: 39-02-23902764; E-mail: [email protected]. reviewed (available slides ranging from 2 to 36) and the histologic subtype I2005 American Association for Cancer Research. was classified according to the latest WHO classification. www.aacrjournals.org 1207 Cancer Res 2005; 65: (4). February 15, 2005

A representative paraffin block of each case was selected and all necrosis, and survival were computed using a two-tailed m2 statistic or sections that were mounted on positively charged slides were deparaffi- Fisher’s exact test where appropriate. nized according to the standard procedures followed by rehydration An exponential model was fit to individual disease-specific survival times through serial ethanol treatments. The slides were immersed in citrate to estimate and compare death rates according to FEZ1 expression, with or buffer [0.005 mol/L sodium citrate (pH 6.0)] and heated in an autoclave without adjustment for tumor grade. The mortality rates so obtained at 95jC for 30 minutes to enhance antigen retrieval. Endogenous correspond to the ratio between the number of events (cancer deaths) and peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 30 the sum of observation times (in years) over all subjects belonging to the minutes. Sections were immunostained with a 1:1,000 dilution of the same group (low or high expression). The model, however, allows direct polyclonal rabbit anti-Fez1 antibody overnight at 4jC. As previously calculation of rate ratios and corresponding confidence limits. reported (20), this antibody was raised in rabbits against glutathione S- P V 0.05 was considered statistically significant. transferase fusion Fez1 protein corresponding with nucleotides 1 to 1,128, which was expressed in Escherichia coli and purified with a glutathione column (Amersham Pharmacia, Piscataway, NJ). The Results specificity of the antibody was tested by Western Blot analysis and confirmed in immunohistochemical analyses of gastric and bladder This study included 87 males and 13 females (M/F = 6.7:1) with carcinomas (20, 21). ages ranging from 39 to 81 years (median 64 years). The primary antibody was omitted and replaced with preimmune Overall, 103 tumor samples were investigated. Out of these, there serum in the negative controls. Sections were then reacted with were 98 NSCLCs (57 adenocarcinomas, including 4 bronchioalveo- biotinylated anti-rabbit antibody and streptavidin-biotin-peroxidase. Dia- lar, 32 squamous cell carcinomas, 7 large cell carcinomas, minobenzidine was used as a chromogen substrate. Finally sections were 1 adenosquamous carcinoma, and 1 salivary gland type adenocar- washed in distilled water and weakly counterstained with Carazi cinoma) and five SCLCs (with one case of mixed SCLC and poorly hematoxylin. All sections were examined independently by two patholo- differentiated squamous cell carcinoma). Fifty-five lesions (53%) gists (D.N. and A.F.), and agreement was reached for the grade of Fez1 were stage I, 20 (19%) stage II, and 28 (27%) stage IIIA. The grading immunostaining. Fez-1 immunoreactivity was classified into four groups was grade 1 for six samples (6%), grade 2 for 33 samples (32%), and using the previously reported scoring method (20): +/+ or strong (96-100% grade 3 for 64 samples (62%). Fez1-positive cells), +/À or moderate (51-95% Fez-1 positive cells), À/+ or weak (2-50% Fez1-positive cells), and À/À or absent (<2% Fez1-positive Table 1 summarizes the data of FEZ1 immunostaining on cells). The tumors were graded as low grade (well to moderately individual lesions and the association with the clinicopathologic differentiated) and high grade (poorly differentiated). All cases of small information. Fez1 immunostaining was strong (+/+, 96-100% of cell carcinoma and large cell carcinoma were categorized as high grade. positive cells) in 15 lesions (15%), moderate (+/À, 51-95% of positive Statistical Methods. Associations of Fez1 expression with clinicopath- cells) in 18 lesions (17%), weak (À/+, 2-50% of positive cells) in 43 ologic variables, including age, sex, disease stage, histology, tumor grade, lesions (42%), and absent (À/À, <2% of positive cells) in 27 lesions

Table 1. Fez1 expression according to clinicopathologic features

Clinicopathologic features Fez1 expression

Total, n (%) À/À, n (%) À/+, n (%) +/À, n (%) +/+, n (%)

Age (y) <65 52 (50.5) 10 (37) 25 (58.1) 9 (50) 8 (53.3) z65 51 (49.5) 17 (63) 18 (41.9) 9 (50) 7 (46.7) Sex Female 14 (13.6) 3 (11.1) 6 (14) 1 (5.6) 4 (26.7) Male 89 (86.4) 24 (88.9) 37 (86) 17 (94.4) 11 (73.3) Stage I 55 (53.4) 14 (51.9) 23 (53.5) 11 (61.1) 7 (46.7) II 20 (19.4) 8 (29.6) 5 (11.6) 4 (22.2) 3 (20) IIIA 28 (27.2) 5 (18.5) 15 (34.9) 3 (16.7) 5 (33.3) Histology Adenocarcinomas 53 (51.5) 13 (48.1) 21 (48.8) 7 (38.9) 12 (80) Squamous cell carcinomas 32 (31.1) 8 (29.6) 13 (30.2) 9 (50) 2 (13.3) Bronchioalveolar 4 (3.9) 1 (3.7) 1 (2.3) 1 (5.6) 1 (6.7) Large cell 7 (6.8) 3 (11.1) 3 (7) 1 (5.6) — Small cell 5 (4.9) 2 (7.4) 3 (7) — — Other 2 (1.9) — 2 (4.7) — — Grading Grade 1 6 (5.8) 1 (3.7) 2 (4.7) 2 (11.1) 1 (6.7) Grade 2 33 (32) 5 (18.5) 13 (30.2) 7 (38.9) 8 (53.3) Grade 3 64 (62.1) 21 (77.8) 28 (65.1) 9 (50) 6 (40) Necrosis (%) Absent 42 (40.8) 7 (25.9) 19 (44.2) 5 (27.8) 11 (73.3) V20 31 (30.1) 10 (37) 10 (23.3) 8 (44.4) 3 (20) z20 30 (29.1) 10 (37) 14 (32.6) 5 (27.8) 1 (6)

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Figure 1. Immunohistochemical analysis of Fez1 expression in normal bronchial epithelium, precancerous lesions, and squamous cell carcinomas of the lung. A, strong Fez1 immunoreactivity in normal bronchial epithelium. Staining is also detected in stroma and lymphocytes. B, squamous intraepithelial neoplasia and poorly differentiated squamous cell carcinoma showing negative immunostaining (Fez1 À/À, same case as A). C, negative Fez1 staining in a poorly differentiated squamous cell carcinoma (Fez1 À/À). Fez1 staining is detected in infiltrating lymphocytes and stromal tissue. D, poorly differentiated squamous cell carcinoma with strong Fez1 cytoplasmic immunoreactivity (Fez1 +/+).

(26%). Overall, 70 samples (68%) showed a considerable reduction in Follow-up information was available for all the cases with a FEZ1 expression. Representative examples of Fez1 immunostaining period ranging from 2 to 68 months (median 52 months). Overall, in lung tumors of different histologic subtype are shown in Figs. 1 54 deaths were recorded in the study cohort and of these 48 were and 2. due to lung cancer. The median disease-specific survival was 50 Statistical analysis showed a significant association between Fez1 months. expression and tumor grade (P = 0.0345; Table 2). To avoid sparse Table 3 shows the number of cancer related deaths according to data, grade 1 and 2 lesions and Fez +/+ and +/À (high expressers) Fez1 expression. The lowest death rate of 12.6 per 100 person-years were grouped in the statistical analysis. In particular, the frequency at risk was observed in the group with strong Fez1 immunostaining of high expressers in the grade 1 to 2 lesions was 46% versus only the (+/+), versus a death rate of 17.8 per 100 person-years at risk in the 23% in the grade 3 lesions. No associations were found between Fez1 remaining patients. The corresponding death rate ratio was 0.711 (P expression and the other clinicopathologic variables tested, such as = 0.47) or 0.771 (P = 0.58) after adjusting for tumor grade. Such age, sex, tumor histology, or necrosis. findings denote a relative mortality reduction around 20% to 30% in Three cases of high-grade squamous intraepithelial neoplasia subjects with very strong Fez1 expression. The lack of statistical (SIN3) were detected either adjacent or in the same section of the significance is compatible with the low study power (around 20%) to tumor sample. All three cases showed that whereas positive Fez1 detect such a survival difference in the small series under immunostaining was present in the normal bronchial mucosa cells investigation. Fez1 expression was absent (two cases) or reduced (one case) in the high-grade precancerous lesions and perfectly matched the type of immunostaining observed in the associated tumor. Figure 1A and B Discussion shows representative Fez1 immunostaining in a sample containing Lung cancer still remains the leading cause of cancer-related normal bronchial epithelium, high-grade squamous intraepithelial deaths in industrialized countries (27). Current treatment protocols neoplasia and tumor areas. have little efficacy on advanced disease where the 5 years survival www.aacrjournals.org 1209 Cancer Res 2005; 65: (4). February 15, 2005

Figure 2. Immunohistochemical analysis of Fez1 expression in lung adenocarcinomas. A, poorly differentiated adenocarcinoma negative for Fez1 expression (Fez1 À/À) with adjacent normal bronchial epithelium with strong cytoplasmic immunoreactivity. B, reduced Fez1 expression in a poorly differentiated adenocarcinoma (Fez1 À/À) compared with the normal bronchial epithelium. C, poorly differentiated adenocarcinoma showing some residual Fez1 staining (Fez1 À/+). D, positive Fez1 immunoreactivity in a poorly differentiated adenocarcinoma with prominent bronchioloalveolar pattern (Fez1 +/+).

rate is <20% (28). A better knowledge of the molecular mechanisms The 8p21-23 region is frequently deleted in many cancers, underlying the development of this deadly neoplasm is therefore including lung cancer and the FEZ1 gene located at 8p22 has been needed to devise novel prevention and intervention strategies. In investigated as a potential tumor suppressor gene in breast (23), particular, identification of the genes that are altered during cancer prostate (24), and bladder carcinomas (21). Functional support for initiation and progression can provide valuable tools as therapeutic its oncosuppressive role has been provided by gene transfer targets or prognostic indicators. In this respect, the analysis of the experiments using plasmids or adenoviral vectors that resulted in genomic changes associated with cancer specimens has helped in inhibition of colony forming potential of transfected cancer cells the identification of the chromosomal regions where genes involved and reduction of in vivo tumorigenicity in the nude mouse model in cancer formation and progression are located (29). (21, 23, 24). Immunohistochemical analyses of primary tumors have

Table 2. Association between Fez1 expression and tumor grade

Grade Fez1 expression

Absent (À/À) Weak (À/+) Moderate/Strong (+/À and +/+) Total

Grade 1-2, n (%) 6 (15.38) 15 (38.46) 18 (46.15) 39 Grade 3, n (%) 21 (32.81) 28 (43.75) 15 (23.44) 64 Total, n (%) 27 (26.21) 43 (41.75) 33 (32.04) 103 (100)

NOTE: Statistical analysis, P = 0.0345 (Fisher’s exact test).

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8 Table 3. Cancer-related deaths according to Fez1 and a cancer-related phenotype. This observation also provides expression insights into how a molecular alteration (Fez1 inactivation) may affect cancer treatment (taxol administration) and therefore on its Fez 1 expression À/ÀÀ/+ +/À +/+ possible use as a predictor of therapeutic outcome. The possibility that Fez1-negative tumors might have a defined No. patients 27 42 17 14 biological behavior is also suggested by the analysis of cancer Died of disease 12 22 9 5 related deaths according to Fez1 expression in our cohort. Mortality rate per 17.8 12.6 Patients with tumors with high Fez1 expression had a 20% to 100 person-years at risk 30% relative mortality reduction compared with those whose Mortality rate ratio 0.711 (0.282-1.795) tumors had absent or strongly reduced expression, a trend that (95% confidence limits) remained evident even after adjusting for tumor grade. Although Mortality rate ratio adjusted 0.771 (0.302-1.965) the low study power doesn’t allow drawing firm conclusions it is for tumor grade possible therefore that lack of Fez1 expression might turn out to (95% confidence limits) be a negative prognostic factor in lung cancer. This is a promising observation because although many markers, including p53, bcl-2, Rb, K-ras, and c-erbB-2 have been investigated as predictors of outcome in lung cancer (30–32), their clinical shown absent or reduced expression in 62% (37 of 60) of transitional significance is often controversial (33), and a definitive role cell carcinoma of the urinary bladder (21) and in 44% (39 of 88) of seems firmly established only for few of them (e.g., p53 mutations gastric carcinomas (20) indicating that the gene might play an in stage I NSCLC; ref. 34). important role in different cancers. The mechanism of inactivation of the gene that can be inferred The only previous study of the expression FEZ1 in lung cancer from previous studies describing the down-regulation of Fez1 was done by reverse transcription-PCR in cell lines from 17 NSCLCs expression in cancer seems to be complex, involving DNA and 19 SCLCs with a reported loss of expression in 76% of NSCLC deletions, promoter methylation and, uncommonly, point muta- and 16% of SCLC (26). Similar frequencies were also observed by tions. The residual expression in a low percentage of cancer cells Western blot analysis of a subset of 18 cell lines representing both in some tumors raises the question of whether complete loss of histologic subtypes. The same study also showed loss of expression the protein is needed to abrogate the function of the gene or not. in three of six primary cultures of resected NSCLCs. The animal model provides some insights into this issue in that To clarify the role of FEZ1 in the development of lung carcinomas, both Fez À/À and +/À animals are similarly susceptible to the we have done the first immunohistochemical characterization of development of carcinogen-induced tumors indicating that loss of FEZ1 expression in primary lung cancers. In our study, 27 samples one Fez1 allele is sufficient to predispose to cancer.9 The (26%) were completely negative for FEZ1 expression and 43 (42%) haploinsufficient behavior of Fez1 in the mouse indicates displayed a significant reduction of the protein. Overall, 68% of the therefore that in tumors with 2% to 50% expressing cells (À/+ tested specimens showed highly reduced FEZ1 expression indicating class in our scoring system) the function of Fez1 could be totally a central role for FEZ1 inactivation in lung cancer. A significant impaired. association was found between lack of FEZ1 expression and tumor In conclusion, our analysis of 103 primary lung cancer samples grade (grade 1 to grade 2 versus grade 3; P = 0.0345) revealing that a provides evidence of an important role for Fez1 in lung cancer. The less differentiated phenotype might be related to loss of FEZ1 association of loss of expression with high-grade tumors and worst function. Interestingly, the same association was also reported for death rate ratio warrant further investigation on the possible use of bladder carcinomas (P < 0.005), and in the analysis of gastric Fez1 as a prognostic tool and as a determinant of therapeutic carcinomas a very strong correlation was found with the diffuse intervention outcome. histotype (P < 0.001) which may occur without well-defined glandular structures. It has been reported that FEZ1 interacts with microtubules, binds Acknowledgments cdc2 the mitotic kinase p34 and regulates its activity in the complex Received 9/24/2004; revised 12/2/2004; accepted 12/13/2004. Grant support: Italian Association for Cancer Research, Italian Foundation for with cyclinB1, a crucial modulator of the G2-M transition (23). The Cancer Research (G. Sozzi). function of FEZ1 as a player in G2-M checkpoint of the cell cycle has The costs of publication of this article were defrayed in part by the payment of page led to the hypothesis that loss of FEZ1 activity could lead to early charges. This article must therefore be hereby marked advertisement in accordance exit from mitosis and accumulation of chromosomal aberrations. In with 18 U.S.C. Section 1734 solely to indicate this fact. particular murine Fez1À/À cells cultured in the presence of taxol or nocodazole fail to arrest in the G2-M phase of the cell cycle and have a higher tendency to develop aneuploidy than Fez +/+ 8 A. Vecchione et al., submitted for publication. controls indicating a direct link between gene loss-of-function 9 A. Vecchione et al., submitted for publication.

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