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Carcinogenesis Advance Access originally published online on January 12, 2008
Carcinogenesis 2008 29(4):846-848; doi:10.1093/carcin/bgn006
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Fez1/Lzts1-deficient mice are more susceptible to N-butyl-N-(4-hydroxybutil) nitrosamine (BBN) carcinogenesis

Raffaele Baffa1,*, Matteo Fassan1,5, Cinzia Sevignani2, Andrea Vecchione1,6, Hideshi Ishii2,7, Enrico Giarnieri1,8, Renato V. Iozzo3, Leonard G. Gomella1 and Carlo M. Croce4

1 Department of Urology
2 Department of Microbiology and Immunology
3 Department of Pathology, Anatomy and Cell Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA
4 Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA
5 Present address: Department of Diagnostic Sciences and Special Therapies—Pathology Unit, University of Padova, 35121 Padova, Italy
6 Present address: Division of Pathology, II Faculty of Medicine, University ‘La Sapienza’, Ospedale Santo Andrea, 00185 Rome, Italy
7 Present address: Center for Molecular Medicine, Jichi Medical School, Tochigi 329-0498, Japan
8 Present address: University ‘La Sapienza’, Ospedale Santo Andrea, 00185 Rome, Italy

* To whom correspondence should be addressed. Tel: +1 215 955 9072; Fax: +1 215 503 2627; Email: r_baffa{at}mail.jci.tju.edu


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
FEZ1/LZTS1 is a tumor suppressor gene that is frequently altered in human cancers of different histotypes. We have reported previously that LZTS1 is downregulated in high-grade bladder cancer and that its restoration suppresses tumorigenicity in urothelial carcinoma cells. To further investigate the role of LZTS1 in the development of bladder cancer, we utilized heterozygous and nullizygous Lzts1 mice in a chemically induced carcinogenesis model. Fifty-eight mice consisting of 25 Lzts1+/+, 17 Lzts1+/– and 16 Lzts1–/– were treated with N-butyl-N-(4-hydroxybutil) nitrosamine (BBN). Results showed that there was a significant increase in neoplastic lesions in the Lzts1+/– (82.3%) and Lzts1–/– (93.8%) versus Lzts1+/+ (8.0%) mice after BBN treatment. No difference in cancer incidence between Lzts1+/– and Lzts1–/– was observed. Collectively, these findings indicate that loss of one or both LZTS1 alleles hampers the normal defenses of urothelial cells against carcinogens, favoring bladder cancer development. Therefore, LZTS1 may become an excellent target for gene therapy in advanced bladder carcinoma.

Abbreviations: BBN, N-butyl-N-(4-hydroxybutil) nitrosamine; PCR, polymerase chain reaction; UC, urothelial carcinoma


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Although bladder cancer is the fifth most common cancer in USA (1), the molecular mechanisms that determine neoplastic transformation in the bladder urothelium are still unknown. No specific or exclusive cytogenetic aberration has been identified for urothelial carcinoma (UC), but numerous non-random deletions, gain of chromosomes, polyploidization and formation of isochromosomes have been linked to it (2).

In 1999, we identified the FEZ1/LZTS1 (LZTS1) tumor suppressor gene on chromosome 8p22 (3). Subsequently, we demonstrated that the LZTS1 expression is often altered in human cancers with diverse histogenetic backgrounds, including those derived from prostate, breast, esophagous and stomach (3–5). Furthermore, LZTS1expression was absent or reduced in oral squamous cell (6) and in lung carcinomas (5), and, in the latter case, loss of LZTS1 expression was significantly associated with poor tumor grade and a higher mortality rate (7).

The LZTS1 gene encodes a 67 kDa leucine zipper protein, which shares homology with the cyclic adenosine 3',5'-monophosphate-responsive binding protein (3) and acts as a regulator of cell growth as well as an inhibitor of tumorigenesis (8–10). The reintroduction of LZTS1 into LZTS1 null cancer cells suppresses cell growth, through a direct interaction between LZTS1 and the main mitotic kinase, p34cdc2 (at present referred as Cdk1) (8–10). Overexpression of LZTS1 protein induced a block of the cell cycle at late S–G2/M phase (8–10). Conversely, inhibition of LZTS1 expression stimulated cell growth (8,11).

Notably, LZTS1 protein is reduced or absent in the majority of bladder cancer cell lines and primary UC samples tested (10). Restoration of protein expression in a bladder tumor cell line inhibits cell growth, alters cell cycle progression and suppresses tumorigenicity in nude mice (10). In agreement with these findings, a mutational analysis study of 54 bladder tumor samples and 34 bladder cancer cell lines discovered one new mutation (G374S) in one bladder cancer cell line and 42% of loss of heterozygosity in the 8p22 region of the tumor samples (12). In addition, it was observed a marked reduction in LZTS1 messenger RNA levels in the majority of bladder cancer cell lines (12).

Treatment of mice with N-butyl-N-(4-hydroxybutil) nitrosamine (BBN) results in the development of urinary bladder cancer that bears significant histopathological and molecular similarities to the human disease (13). For this reason, this model has been previously used to characterize the tumorigenic process of urinary bladder cancer (14). In the present study, we assessed the carcinogenic potential of BBN in Lzts1 knock-down mice to gain insight into the potential function of Lzts1 in vivo. We discovered that loss of one or both Lzts1 alleles favored development of bladder cancer induced by a classical carcinogen. Thus, LZTS1 may be an important target gene for therapeutic intervention and boosting its expression by pharmacological or gene therapy approaches might help combating bladder cancer in humans.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Animal experiments
Animal experiments were conducted under institutional guidelines established for the Animal Facility at the Kimmel Cancer Center (Thomas Jefferson University, Philadelphia, PA). For the carcinogenicity study, 58 mice consisting of 25 Lzts1+/+ (13 males and 12 females), 17 Lzts1+/– (9 males and 8 females) and 16 Lzts1–/– (8 males and 8 females) were treated with a freshly prepared solution of 0.1% BBN (TCI America, Portland, OR) in their drinking water, twice a week as described previously (14). Mice were weighed, observed daily, and after 13 weeks of BBN treatment followed by 2 weeks of tap water, the mice were killed, and complete necropsy was conducted. At necropsy, all the bladders were first opened and checked for gross lesions and then fixed in 10% buffered formalin. To genotype the mice colony, DNA was isolated from mouse tail using the DNeasy® Tissue Kit (Qiagen, Valencia, CA). We designed a set of primers for the detection of the wild-type Lzts1 DNA and for the detection of the neomycin-resistant gene cassette used to generate the knock-out mice (9) (wild-type: forward primer 5'-CTGTGCAAAGCTGCAGTGAGG-3' and reverse primer 5'-ACTTCCGCTCTCACCAGTGCTGATC-3'; mutant: forward primer 5'-CTGTGCAAAGCTGCAGTGAGG-3' and reverse primer 5'-CGCTATCAGGACATAGCGTTGGCTACC-3'). Polymerase chain reaction (PCR) was performed using the PCR Core Kit (Roche, Mannheim, Germany). Each mixtures contained 18.50 µl of Nuclease-free water (Fisher Scientific, Fair Lawn, NJ), 2.5 µl of PCR reaction buffer, 0.75 µl of deoxynucleoside triphosphate mix, 1 µl each primer and 0.25 µl of Taq DNA polymerase in a total volume of 25 µl. PCR was performed at 94°C for 5 min to activate the Taq DNA polymerase, followed by 32 cycles at 94°C for 30 s, 65°C for 30 s and 72°C for 2 min. The PCR products were electrophoresed on 1.5% agarose gel and visualized after staining the gel with ethidium bromide (Figure 1).


Figure 1
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  		Fig. 1. PCR analysis on mouse genomic DNA showing wild-type Lzts1 gene and Lzts1 gene/Neomycin cassette (mutant) products in Lzts1–/–, Lzts1+/– and Lzts1+/+ mice as indicated.

 
Histologic and statistical analysis
The whole bladder was histologically examined through the evaluation of multiple 50 µm progressive sections. Histologic urothelial lesions were classified into four categories (Table I, Figure 2) as described previously (14) and according to the tumor progression described in BBN-induced tumors as follows: (i) simple hyperplasia and mild dysplasia; (ii) moderate and severe dysplasia; (iii) in situ carcinoma and (iv) invasive carcinoma. For each case, only the most severe lesions were considered in the statisitical analysis. Severe dysplasia and carcinoma in situ were grouped together. The slides were randomized so that the three pathologists (R.B., A.V. and R.V.I.) evaluating the slides did not know the nature of the treatment received by each animal. The association between Lzts1 genotype, presence of preneoplastic lesions and bladder tumors was evaluated via the {chi}2 test. Differences were considered statistically significant at P < 0.05. All the statistical evaluations were performed with STATA software (Stata Corporation, College Station, TX).


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  		Table I. Summary of the urothelial lesions observed in the three different genotypes

 


Figure 2
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  		Fig. 2. Gross anatomy and histopathology of Lzts1 knock-out murine bladder after BBN administration. Macroscopic (A) and hematoxylin and eosion sections (B and C) of an ulcerated tumor protruding inside the peritoneum. Representative examples of a murine normal urothelium in a Lzts1+/+ mouse (D) and a moderate dysplasia and an invasive tumor with squamous features in Lzts1+/– mice (E and F). (B, E, F x100; C and D x400).

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
As we reported previously (9), our data confirmed the higher susceptibility of Lzts1-deficient mice to develop tumors under the effect of a carcinogen using heterozygous and nullizygous Lzts1-deficient mice in a chemically induced carcinogenesis model. In the Lzts1+/+ group (Table I), simple hyperplasia and mild dysplasia were detected in 20 mice (80.0%), three mice (12.0%) showed moderate to severe dysplasia and only two mice (8.0%) presented neoplastic lesions (one with in situ carcinoma and the other with invasive carcinoma). On the contrary, 14 of 17 (82.3%) Lzts1+/– and 15 of 16 (93.8%) Lzts1–/– mice harbored invasive carcinomas. Mild to moderate dysplasia was diagnosed in the remaining Lzts1+/– (3/17) and Lzts1–/– (1/16) mice. Preneoplastic lesions in the urothelium adjacent to advanced tumors were frequently observed. In most cases, as described previously (13,14), we observed squamous differentiation of neoplastic lesions. Specifically, 11 of 14 tumors in the Lzts1+/– and 12 of 15 tumors in the Lzts1–/– mice showed squamous features. Furthermore, cancer infiltrated the deeper muscle in 10 of 14 Lzts1+/– mice and 11 of 15 Lzts1–/–mice, respectively. The remaining tumors invaded the superficial muscle in both Lzts1+/– and Lzts1–/– mice. No significant histopathological differences were observed between wild-type and knock-out mice. When we used three outcome categories (Table I), the global P comparing all three groups was 1.3E-13, underlying the differences of outcomes across the genotypes. The pair-wise probabilities were as follows: wild-type versus heterozygous, P = 1.4E-9; wild-type versus homozygous–/–, P = 2.5E-14 and heterozygous versus homozygous–/–, P = NS.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The short arm of chromosome 8 is frequently deleted in human cancers and its deletion is commonly associated with a more aggressive malignant phenotype (15,16). In previous studies, we have identified the LZTS1 gene on chromosome 8p22 (3), a region which showed loss of heterozygosity in invasive UC tumors (12,17–19), and shown that the LZTS1 protein is lost or decreased in most of the UC-derived cell lines and primary human urothelial bladder cancers analyzed (10). Moreover, there was a statistical association between decrease or absence of LZTS1 and high-grade bladder tumors. By restoring LZTS1 expression in SW780 bladder cancer-derived cells, we inhibited cell growth, altered cell cycle progression and suppressed tumorigenicity in immunocompromised mice (10).

In a recent report, Knowles et al. (12) found a new mutation in A1698 bladder cancer cell line and 42% of loss of heterozygosity in the 8p22 region of a large series of bladder cancers. In the same study, a reduction in LZTS1 messenger RNA levels was also observed in the majority of bladder cancer cell lines (12). All these data strongly suggest that LZTS1 is indeed involved in the progression of UC of the urinary bladder.

The exact mechanism whereby LZTS1 contributes to cancer initiation and progression is not known. Recent data suggest that LZTS1 is an important player in the execution of normal M-phase progression (9). Moreover, the absence of Lzts1 leads to impaired chromosome segregation, cell transformation and cancer progression (9). Transgenic mice, where Lzts1 has been knocked out in the germ line, have an increased incidence of both spontaneous and chemically induced cancers (9).

Since the LZTS1 gene is often downregulated in cancer (3–7,10,11), and since BBN-induced bladder cancer is an established model to characterize the tumorigenic process for urinary bladder carcinogenesis (13,14), BBN treatment was used to determine whether Lzts1 modulates carcinogen-induced malignancy development. Treatment of mice with this carcinogen results in the development of transitional cell urinary bladder cancers with a high yield of tumor induction (13,14) and high rate of squamous differentiation (>50% of the induced tumors) (13,14,20). In this study, as in our previous experience (14), we observed an almost complete squamous commitment of the neoplastic lesions. After 13 weeks of BBN administration, the majority of Lzts1-deficient mice (82.3% Lzts1+/– and 93.8% Lzts1–/–) developed invasive bladder cancers (Figure 2). These incidences were significantly higher than those observed in the group of wild-type mice. This confirmed our previous observation that Lzts1-deficient mice develop forestomach tumors more quickly after exposure to N-nitrosomethylbenzylamine (9), further suggesting that Lzts1 absence sensitizes multiple tissues to carcinogenesis. We also confirmed that there is no difference in the incidence of neoplastic lesions between heterozygous+/– versus homozygous–/– mice (82.3 versus 93.8%). Thus, the loss of one single Lzts1 allele can predispose to bladder cancer formation. The difference in the incidence of bladder tumors between these mice is an important indication of the need for the urothelial cell to have both Lzts1 alleles intact to respond efficiently to the damaging action of a chemical carcinogen. Therefore, as observed for other tumor suppressor gene (21–23), we hypothesize that Lzts1 gene could be haploinsufficient in tumor suppression.

In conclusion, we have shown for the first time that Lzts1 null mouse is a useful and valid animal model for investigating the biology of bladder cancer. We further discovered that both Lzts1 alleles are necessary for a proper host response to chemical carcinogens such as BBN. Thus, further studies on this interesting gene are warranted to understand how Lzts1 participates in bladder carcinogenesis. Elucidation of the molecular mechanisms of LZTS1 activity might help not only the diagnosis but also the treatment of patients with this devastating disease.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Benjamin Perkins Bladder Cancer Fund and the Martin Greitzer Fund (to R.B.).


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received July 20, 2007; revised November 19, 2007; accepted December 20, 2007.


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