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First published online 9 January 2008
doi: 10.1242/dev.009910

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Role of epithelial cell fibroblast growth factor receptor substrate 2 in prostate development, regeneration and tumorigenesis

Yongyou Zhang^1,*, Jue Zhang^1,*, Yongshun Lin¹, Yongsheng Lan¹, Chunhong Lin¹, Jim W. Xuan², Michael M. Shen³, Wallace L. McKeehan¹, Norman M. Greenberg⁴ and Fen Wang^1,

¹ Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M Health Science Center, 2121 W. Holcombe Blvd., Houston, TX 77030-3303, USA.
² Department of Surgery, University of Western Ontario, London, ON, N6A 4G5, Canada.
³ Departments of Medicine, and Genetics and Development, Columbia University, College of Physicians and Surgeons, Herbert Irving Comprehensive Cancer Center, 1130 St. Nicholas Avenue, Room 217B, New York, NY 10032, USA.
⁴ Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, Seattle, WA 98109-1024, USA.

View larger version (112K): [in this window] [in a new window]   Fig. 1. Disruption of Frs2 alleles in prostate epithelium. (A) Schematic of the floxed Frs2 alleles for conditional disruption. The genomic DNA containing coding exons 1-5 and adjacent introns is shown. Dashed boxes indicate non-coding exon sequence. The primers for PCR genotyping are indicated by arrows. Primers f1 and f2 amplify a 319 bp fragment from the floxed Frs2 allele. Primers f1 and f3 amplify a 261 bp fragment from the Frs2-null allele; no amplification from wild-type alleles. (B) PCR genotyping for the Frs2 conditional-null alleles. Genomic DNAs extracted from each prostatic lobe of 4-week-old mice were analyzed by PCR using the primers illustrated in A. (C) Total RNAs were extracted from prostates of different ages, and Frs2 expression was assessed by real-time RT-PCR. The data were normalized to β-actin loading controls and are expressed as mean±s.d. of at least three independent experiments. Note that Frs2 alleles were intact in the stromal compartment, which is likely to account for the basal level of Frs2 expression in Frs2cn prostates. Representative data from dorsolateral prostate are shown. (D) In situ hybridization of Frs2 expression in prostates. Enlarged image of boxed area shown on the right. Note that the expression was diminished in the epithelium of Frs2cn prostates and mature control prostates. Arrows indicate basal cells. AP, anterior prostate; DLP, dorsolateral prostate; VP, ventral prostate; M, myristylation site; PTB, phosphotyrosine-binding site; f, Frt element; p, loxP element; F/F, homozygous Frs2flox mice; CN, Frs2cn mice.

View larger version (81K): [in this window] [in a new window]   Fig. 2. Disruption of Frs2 alleles inhibits ductal branching morphogenesis in prostates. (A) The urogenital tract was dissected at the indicated times, lightly fixed and then stained with X-Gal. Stained tissues representing each prostatic lobe are shown. Note the lack of significant differences between Frs2cn (below) and control (above) prostate rudiments at E17.5 or later. (B) Prostatic lobes were dissected from mice at the indicated ages. The tissues were lightly fixed and stained with X-Gal (a,b). Note that only epithelial cells in the prostate were stained. The ductal network in each prostatic lobe from 4-week-old mice was microdissected (c-e) and the average number of tips was quantified from three prostates and is shown as mean±s.d. (f). AP, anterior prostate; DLP, dorsolateral prostate; VP, ventral prostate; B, bladder; S, seminal vesicles; U, urethra; F/F, homozygous Frs2flox mice; CN, Frs2cn mice; WT, wild-type Frs2.

View larger version (105K): [in this window] [in a new window]   Fig. 3. Depletion of FRS2 proteins in Frs2cn prostatic rudiments. (A) Prostates were collected at the indicated times, immunostained with the indicated antibodies, and visualized by confocal microscopy. The prostate epithelial cells were identified with anti-P63 antibody. Expression of FRS2 was assessed with anti-FRS2 antibody. Arrows indicate prostate epithelial cells. Note that FRS2 staining was diminished in Frs2cn prostates at postnatal day 0.5 (P0.5). (B) The ratio of p63-positive cells in the epithelial compartment at different ages was calculated from three samples. Representative mean±s.d. values of data from triplicate samples are shown. (C,D) Stability of FRS2 proteins as compared with FGFR2 in prostate epithelial cells. TRAMP-C2 cells (1x106) treated with cycloheximide were lysed at the indicated times. The abundance of FRS2 was analyzed by western blot directly from the cell lysates (containing 50 µg protein). FGFR2 in cell lysates (containing 500 µg protein) was pulled down with anti-FGFR2 antibody. The specifically bound fractions were subjected to western analyses (C). The specific bands were quantitated with a densitometer and the data presented as ratios of treated to non-treated cells (D). CHX, cycloheximide; F/F, homozygous Frs2flox mice; CN, Frs2cn mice.

View larger version (96K): [in this window] [in a new window]   Fig. 4. Compromised proliferation in Frs2cn prostates during prepubertal and pubertal growth stages. (A) Prostate tissues were collected from control and mutant mice at the indicated ages and proliferating cells assessed by immunostaining for PCNA. Note that the proliferating cells mainly reside at the distal tips of the ductal network in 1- or 2-week-old prostates, but are randomly distributed in 4-week-old prostates of both control and mutant mice. Arrows indicate proliferating tips. Representative data from the dorsolateral prostate are shown. Insets are high-magnification views from the same sections. (B) PCNA-stained cells in distal tips of 1- or 2-week-old prostates, or whole prostate (4 weeks old), were scored. The ratio of PCNA-positive to total epithelial cells was calculated from five sections per prostate. Mean±s.d. values of data from three prostates are shown. (C) Real-time RT-PCR analyses of RNAs encoding the indicated genes from 1-week-old prostates. Data were normalized to β-actin loading controls and then presented as the relative difference to the control prostates. Mean±s.d. values of data from three prostates are shown. AP, anterior prostate; DLP, dorsolateral prostate; VP, ventral prostate; F/F, homozygous Frs2flox mice; CN, Frs2cn mice.

View larger version (100K): [in this window] [in a new window]   Fig. 5. Activation of the MAP kinase pathway is compromised in Frs2cn prostates during branching morphogenesis. (A,B) Western analyses (A) of the indicated proteins from Triton-extracts of mouse prostates at the indicated ages. β-actin was used as a loading control. Note that phosphorylated FRS2 and ERK1/2 were prominent in 1- and 2-week-old prostates and were diminished in 4-week-old prostates, whereas phosphorylated AKT remained constant in the prostates. The specific bands were quantitated (B) by densitometry. (C,D) Immunostaining of phosphorylated ERK1/2 (C) and phosphorylated AKT (D) in tissue sections of 1-week-old prostates. The specific staining was visualized with confocal microscopy. Note that phosphorylated ERK1/2 was reduced in the epithelial cells, but not in the stromal cells, of Frs2cn prostates, and that both phosphorylated ERK1/2 and phosphorylated AKT were reduced in epithelial cells of Fgfr2cn prostates. Arrows indicate epithelial cells. (E) Newborn prostate rudiments cultured with 10 µM ERK1/2 (MEK1/2) or PI3 kinase inhibitor, as indicated, for 3 days. Arrows indicate lumens of the prostatic ductal structures. pERK1/2, phosphorylated ERK1/2; pAKT, phosphorylated AKT; F/F, homozygous Frs2flox mice; CN, Frs2cn mice.

View larger version (96K): [in this window] [in a new window]   Fig. 6. Compromised proliferation activities in regenerating Frs2cn prostates induced by androgens. (A,B) Expression of Frs2 in the epithelium of regenerating prostates. Expression of Frs2 was assessed by in situ hybridization (A) or real-time RT-PCR (B). Data were normalized to the abundance of 18S rRNA and are expressed as mean±s.d. of at least three prostates. The background of Frs2 expression in the conditional mutants is from the stroma. Red arrows, epithelial cells; black arrows, stromal cells. (C,D) Reduced androgen-induced proliferation in Frs2cn prostates. Androgen was restored to mice 14 days after orchiectomy to induce prostate regeneration. Tissues were harvested at the indicated days after administration of androgen. Proliferating cells were revealed by immunostaining with anti-PCNA antibody (C). Red arrows, epithelial cells; black arrows, stromal cells. (D) Representative data from the dorsolateral prostate are shown. PCNA-positive cells were scored and reported as ratios of proliferating cells to total cell numbers. Mean±s.d. values of data from at least three prostates are shown. (E) The average wet tissue weights of normal prostates before castration (Cont), 2 weeks after the operation (Cas), and 14 days after administration of androgen (Reg). Mean±s.d. values of data from three samples are shown. F/F, homozygous Frs2flox mice; CN, Frs2cn mice.

View larger version (107K): [in this window] [in a new window]   Fig. 7. Ablation of FRS2 in the prostatic epithelium inhibits tumorigenesis in the TRAMP prostatic tumor model. (A,B) Expression of Frs2 and Fgfr1 at the mRNA level was assessed in TRAMP prostates by in situ hybridization (A) and RT-PCR (B). Red arrows, epithelial cells; black arrows, stromal cells. Inset, wild-type control showing no expression of Fgfr1 in the epithelial compartment. T1 and T2, different, individual TRAMP tumors; C2, the C2 cell line derived from TRAMP tumors; PWT, prostates of 6-month-old wild-type mice; PTRAMP, prostates of 3-week-old TRAMP mice in which PIN lesions are minimal. Brain and liver were used as positive controls; β-actin as a loading control. (C) Treatment of TRAMP-C2 cells with FGF2 (10 ng/ml) for 10 minutes after serum starvation for 24 hours. Cells were lysed and the lysates analyzed by western blot with the indicated antibodies. (D) Prostate tissue sections were prepared from TRAMP mice with homozygous Frs2-null or Frs2 floxed alleles at the indicated ages and stained with Hematoxylin and Eosin (HE) or analyzed by immunohistotaining with anti-T-antigen antibody. Blue arrows, focal lesions; green arrows, normal epithelial cells. (E) PIN foci in prostates of 10-week-old TRAMP mice were identified as defined (Park et al., 2002), and the percentage area occupied by lesions quantitated. Mean±s.d. values of data from five prostates are shown. (F) Mortality of TRAMP mice with the indicated Frs2 alleles was determined from daily observation over 250 days. The percentage of mice that survived to the indicated ages is shown. pFRS2, phosphorylated FRS2; pERK1/2, phosphorylated ERK1/2; pAKT, phosphorylated AKT; F/F, homozygous TRAMP-Frs2flox mice; CN, TRAMP-Frs2cn mice.

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