U19/Eaf2 knockout causes lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostatic intraepithelial neoplasia

Abstract

Upregulated gene 19 (U19)/ELL-associated factor 2 (Eaf2) is a potential human tumor suppressor that exhibits frequent allelic loss and downregulation in high-grade prostate cancer. U19/Eaf2, along with its homolog Eaf1, has been reported to regulate transcriptional elongation via interaction with the eleven-nineteen lysine-rich leukemia (ELL) family of proteins. To further explore the tumor-suppressive effects of U19/Eaf2, we constructed and characterized a murine U19/Eaf2-knockout model. Homozygous or heterozygous deletion of U19/Eaf2 resulted in high rates of lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostate intraepithelial neoplasia. Within the mouse prostate, U19/Eaf2 deficiency enhanced cell proliferation and increased epithelial cell size. The knockout mice also exhibited cardiac cell hypertrophy. These data indicate a role for U19/Eaf2 in growth suppression and cell size control as well as argue for U19/Eaf2 as a novel tumor suppressor in multiple mouse tissues. The U19/Eaf2 knockout mouse also provides a unique animal model for three important cancers: lung adenocarcinoma, B-cell lymphoma and hepatocellular carcinoma.

Figure 1

U19/Eaf2 expression patterns and generation of U19/Eaf2-null mice. (a) RT–PCR detection of U19/Eaf2 mRNA and ribosomal protein L19 mRNA (control) in mouse tissues. (b) The genomic structure of mouse U19/Eaf2 and the targeting strategy. Exon 2 was replaced with a Neo gene. The positions of probes 1 and 2 for the Southern blot are indicated. (c and d) Southern blot analysis of genomic DNA, extracted from mouse tail, using the 3′-flanking probe, Probe 2 (c), and the 5′-flanking probe, Probe 1 (d). (e) PCR analysis of mouse tail genomic DNA. PCR primers amplified 508 bp from the wild-type and 231 bp from the targeted allele. M, marker size; C, control without template DNA. (f) RT–PCR analysis of U19/Eaf2 mRNA using total RNA from testes of wild-type and knockout mice. The 5′ U19/Eaf2 primer is within exon 2, and the 3′ primer is in exon 5. L19 is the control. (g) Endogenous U19/Eaf2 expression in wild-type and U19/Eaf2-null MEFs. Blots of wild-type and U19-null MEFs were probed with antibodies against U19/Eaf2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Experiments were independently done at least twice. (h) The survival curve of U19/Eaf2-null mice (n = 60) and their wild-type littermates (n = 60).

Figure 2

Cardiac enlargement in U19/Eaf2-null mice. (a) Gross morphology and hematoxylin and eosin (H&E) staining (low power and × 40) of a representative wild-type heart and enlarged U19/Eaf2-null heart from 12-month-old mice. (b) Heart/body weight ratio. The heart/body weight ratio in effected mice was at least 20% greater than that of wild-type littermates (P<0.05). (c) Quantitative analysis of cell number in wild-type and enlarged U19/Eaf2-null hearts. The relative cell number per unit area is decreased about 20% in U19/Eaf2-null heart. *P<0.05.

Figure 3

Prostatic epithelial hypertrophy, hyperplasia and dysplasia in U19/Eaf2-null mice. (a) H&E staining of 3-month-old U19^+/+ and U19^−/− ventral prostates (VP). The magnification of the objective is indicated. (b) Morphology of 6-month-old U19^+/+ and U19^−/− VPs. U19/Eaf2-null VPs displayed an increase in size and a more opaque-whitish appearance relative to wild-type. (c) Wet weight of 6-month-old U19^+/+ and U19^−/− VPs and dorsolateral prostates (DLP). Knockout VPs and DLPs had increased wet weights of approximately 30 and 20%, respectively. *P<0.05. (d) H&E staining of 6-month-old U19^+/+ and U19^−/− ventral (VP), lateral (LP), anterior (AP) and dorsal (DP) prostates. Lower power ( × 10 objective) images are shown at left and high power ( × 100 objective) images of indicated areas at right. Red arrows mark mPIN. (e) Ki67 staining (marked by red arrows) of U19^+/+ and U19^−/− VPs. The stained images ( × 100) are shown in the left panel and quantitative analysis in the right panel. *P< 0.05. (f) 5-bromo-2-deoxyuridine (BrdU) labeling (marked by red arrows) of U19^+/+ and U19^−/− VPs. The labeled images are shown in left panel ( × 100) and the quantitative analysis in the right panel. *P<0.05. (g) Epithelial cell size of U19^+/+ (wild type, WT) and U19^−/− (knockout, KO) VPs. The H&E staining images, in the left panel, were captured by Leica DFC320 3.3 megapixel color camera at the × 40 objective. The right panel shows the quantitative analysis. The length and width of individual cells were measured to calculate the cell size (length × width). *P<0.05. (h) Serum testosterone (T) in wild-type and U19/Eaf2 knockout mice.

Figure 4

Macroscopic lung adenocarcinoma, HCC and B-cell lymphoma in U19/Eaf2-null mice, 18–22 months old. (a) Morphology, hematoxylin and eosin (H&E) staining ( × 40 objective) and Ki-67 staining ( × 40 objective) of lungs from one wild-type (U19^+/+) and two U19-KO (U19^−/−) mice. One U19^−/− mouse developed lung adenocarcinoma (upper images) and the other developed poorly differentiated lung adenocarcinoma (lower images). Ki-67 staining shows more proliferation in tumor regions. (b) Morphology, H&E staining ( × 40 objectives) and Ki-67 staining ( × 40 objective) of livers from one wild type (U19^+/+) and two U19-KO (U19^−/−) mice. HCC developed in U19-null mice. Ki-67 staining shows more proliferation in tumor regions. (c) H&E staining ( × 40 objective), and Ki-67 staining ( × 40 objective) of lymphomas in the indicated organs from U19^−/− mice. Ki-67 staining shows extremely high proliferation rates in lymphoma sites. (d) CD3 and B220 staining of lymphomas in the indicated organs. (e) Macroscopic tumor incidence rate in 38 homozygous knockout (U19^−/−), 9 heterozygous (U19^+/−) and 19 wild-type (U19^+/+) mice 18–24 months old. % Mice indicates the % of mice displaying indicated phenotypes. Lung Ca, lung adenocarcinoma; HCC, hepatocellular carcinoma; multiple Ca, different types of cancers observed in the same host.

Figure 5

(a) Representative fluorescent electropherograms for microsatellite markers AV048865, RH130971 and AV130971. (b) Summary of LOH patterns of four heterozygous tumor samples, two lung adenocarcinoma, one lymphoma from spleen and one lymphoma from kidney. The markers exhibiting allelic loss are indicated in black circles.

Figure 6

(a) Effect of U19/Eaf2 overexpression on rat Dunning AT6.1 prostate cancer cell proliferation. Parental, green fluorescent protein-estrogen receptor (GFP-ER)-transfected and GFP-U19-ER-transfected AT6.1 cells were plated in six-well plates and the number of cells counted at indicated days in the absence or presence of 300 nM 4-hydroxyl-tomaxifen (4-OHT). The presence of 4-OHT inhibited the proliferation of GFP-U19-ER-transfected AT6.1 cells (P<0.01), but not the parental and GFP-ER-transfected AT6.1 cells. (b) Effect of U19/Eaf2 overexpression on AT6.1 cell proliferation in soft agar. Parental, GFP-ER-transfected and GFP-U19-ER-transfected AT6.1 cells were plated in six-well plates containing soft agar as described in ‘Materials and methods’. Top panel shows representative colonies in the presence or absence of 4-OHT. Bottom panel shows the quantitative analysis of the colony formation. The presence of 4-OHT only inhibited the colony formation of GFP-U19-ER-transfected AT6.1 cells (P<0.01). (c) Cell cycle analysis of AT6.1 cells overexpressing U19/Eaf2. No statistically significant difference in the number of G₂/M-phase cells could be detected between AT6.1 cells that overexpress U19/Eaf2 and control AT6.1 cells. Also, 4-OHT did not affect the distribution of the cells at different phases of cell cycle. Results shown as mean of three independent experiments±standard deviation.