Deletion of leucine zipper tumor suppressor 2 (Lzts2) increases susceptibility to tumor development

Abstract

Using an Lzts2 knock-out mouse model, we characterized the biological role of Lzts2 in tumorigenesis. Both heterozygous and homozygous deletion of the Lzts2-targeted allele in mice shows an increased incidence in spontaneous tumor development, although Lzts2 homozygous knock-out mice show significantly higher incidences than heterozygous mice. Treatment of Lzts2-deficient mice with a carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine, increases the susceptibility to N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder carcinoma development. Examination of human prostate cancer tissue specimens shows a reduction of LZTS2 protein expression in prostate cancer cells. Further analyses of mouse embryonic fibroblasts isolated from Lzts2 knock-out embryos show that loss of Lzts2 enhances cell growth. These data provide the first line of evidence demonstrating that deletion of Lzts2 increases susceptibility to spontaneous and carcinogen-induced tumor development.

FIGURE 1.

Disruption of the Lzts2 gene in mouse ES cells and embryos. A, alignment of human and mouse LZTS2 protein sequences is shown. Identical amino acids are in bold type and marked with asterisks. The nuclear export signal is boxed. B, the targeting construct used to disrupt the Lzts2 gene in ES cells. A PGK-neomycin cassette flanked by loxP and FRT sites was inserted upstream of exon 2 (which contains the translation initiating codon). A third loxP site was placed downstream of exon 3 using Red/ET recombineering. The hypothetical crossovers between the endogenous Lzts2 allele and the targeting construct are indicated by the dashed lines. Correctly recombined ES clones were identified by PCR using primer sets for the Neo cassette (Neo1), flanking sequences (P2), and the downstream loxP site (P3). C, Southern blotting of E11.5 embryos using a ³²P-labeled probe from exon 4. D, Northern blotting of total RNA from E11.5 embryos using a ³²P-labeled probe covering the junction region of exons 4 and 5. E, Western blotting of total cell lysates from E11.5 mouse embryos analyzed with an LZTS2 antibody.

FIGURE 2.

Development of spontaneous tumors in Lzts2 null mice. A, a 14-month-old male Lzts2^−/− mouse was sacrificed, and necropsy showed multiple nodular masses in different organs (arrows), including the pancreas (panel A1), lung (panel A2), liver and gallbladder (panel A3), spleen (panel A4), and intestine and mesenteric lymph node (panel A5). Histological analyses of masses in the lung suggest pulmonary carcinoma (panel A6) and histiocytic sarcoma in the remaining affected organs (panel A7) are also shown. B, a 16-month-old female Lzts2^−/− mouse was grossly examined, revealing lymphadenopathy of the both mandibular and sublumbar lymph nodes and left renomegaly (panels B1 and B2). Microscopic analysis confirmed that the lymph nodes and left kidney were affected by multicentric lymphoma (panel B3), with additional microscopic involvement of the right kidney, retroperitoneum adjacent to ureters and aorta, salivary glands, liver, and lung. C, necropsy of a 17-month-old male Lzts2^−/− mouse showed a focally extensive, pale tan partially multilobulated mass in the region of the prostate gland and trigone of the urinary bladder (panels C1 and C2). Histological analysis of the mass suggests a primary histiocytic sarcoma of the prostate gland (panel C3).

FIGURE 3.

Effects of Lzts2 in cell proliferation and survival. A, Western blot analysis of FLAG-LZTS2 expression in LNCaP or LAPC4 cells after infection with pLenti-FLAGLZTS2 or control lentiviruses. The same blots were probed with an anti-tubulin antibody as a control. B, LNCaP cells were seeded into 96-well plates after 6 h of infection with LZTS2 expression lentiviruses. Cell growth was measured every other day by MTS assay. The data represent the means ± S.D. of three independent experiments. C, identical experiments performed in LAPC4 cells. D, MTS cell proliferation assay using E10.5 MEFs isolated from Lzts2. heterozygous intercrosses (+/+, n = 4; +/−, n = 4; −/−, n = 3). E, both Lzts2^+/+ and Lzts2^−/− MEFs were incubated in either Wnt3a-CM or control medium and tested by MTS assays.

FIGURE 4.

Histologic analysis of Lzts2-deficient murine urinary bladder after BBN treatment. Hematoxylin- and eosin-stained sections of representative samples of normal urothelium (A1–3), hyperplasia (B1–3), carcinoma in situ (C1–3), and noninvasive (D1–3)/invasive (E1–3) transitional cell carcinoma. The images were taken at 25×, 100×, or 200× magnification.

FIGURE 5.

Reduction of LZTS2 expression in human prostate cancer samples. Three sets of human prostate tissue samples were stained with antibodies against androgen receptor (AR) (A, D, and G), p63 (B, E, and H), or LZTS2 (C, F, and I). All sections used for immunohistochemistry were lightly counterstained with 5% (w/v) Harris hematoxylin. LZTS2 staining is much weaker in malignant glands (green arrows) versus normal glands (red arrows).

FIGURE 6.

Cellular localization and activity of β-catenin in Lzts2 null MEFs. A, MEFs were prepared from different genotype embryos at E10.5. Whole cell lysates were analyzed by Western blotting assays with either LZTS2 or β-actin antibody. B, either whole cell lysates or nuclear extracts were isolated from different genotype MEFs and analyzed by Western blotting assays for either β-catenin (β-cat), Ku86, or tubulin. C, both wild type and Lzts2 null MEFs were fixed and incubated with the anti-LZTS2 antibody followed by a second antibody conjugated with rhodamine (red). The nuclei were counterstained with DAPI (blue). D, luciferase assay of different genotype MEFs cultured in either Wnt3a-CM or L-CM. Luciferase activity is reported as relative light units (luciferase/β-galactosidase) and represented as the means ± S.D. E, quantitative RT-PCR assays were performed to detect mRNA levels of the endogenous beta-catenin downstream target genes Cyclin D1, c-Jun, and Axin2. The experiments were repeated three times using independent cDNA samples from wild type and Lzts2 null MEFs. The relative mRNA levels from each sample are presented as the mean ± S.E. of the triplicate reactions. A statistically significant difference (*, p < 0.05; **, p < 0.01) was observed between wild type and Lzts2 null MEFs for each target gene.

FIGURE 7.

Loss of Lzts2 alters β-catenin activity in mice. β-Galactosidase staining of E10.5 embryos using the Wnt activity reporter, Axin2^LacZ/+ (A–F). Increased staining was observed dorsally and in the forebrain, midbrain, hindbrain, and mandibular branches of Axin2^LacZ/+:Lzts2^−/− (A and B) and Axin2^LacZ/+:Lzts2^+/− (C and D) embryos as compared with Axin2^LacZ/+:Lzts2^+/+ (E and F) embryos. G–J, increased Wnt activity was also observed in the intestinal crypts of the colons of adult Axin2^LacZ/+:Lzts2^−/− mice (H and J) compared with Axin2^LacZ/+:Lzts2^+/+ mice (G and I).