Early onset of neoplasia in the prostate and skin of mice with tissue-specific deletion of Pten - PubMed (original) (raw)

Early onset of neoplasia in the prostate and skin of mice with tissue-specific deletion of Pten

Stéphanie A Backman et al. Proc Natl Acad Sci U S A. 2004.

Abstract

PTEN is a tumor suppressor gene mutated in various advanced human neoplasias, including glioblastomas and prostate, breast, endometrial, and kidney cancers. This tumor suppressor is a lipid phosphatase that negatively regulates cell survival and proliferation mediated by phosphatidylinositol 3-kinase/protein kinase B signaling. Using the Cre-loxP system, we selectively inactivated Pten in murine tissues in which the MMTV-LTR promoter is active, resulting in hyperproliferation and neoplastic changes in Pten-null skin and prostate. These phenotypes had early onset and were completely penetrant. Abnormalities in Pten mutant skin consisted of mild epidermal hyperplasia, whereas prostates from these mice exhibited high-grade prostatic intraepithelial neoplasia (HGPIN) that frequently progressed to focally invasive cancer. These data demonstrate that Pten is an important physiological regulator of growth in the skin and prostate. Further, the early onset of HGPIN in Pten mutant males is unique to this animal model and implicates PTEN mutations in the initiation of prostate cancer. Consistent with high PTEN mutation rates in human prostate tumors, these data indicate that PTEN is a critical tumor suppressor in this organ.

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Figures

Fig. 1.

Fig. 1.

Targeted conditional deletion of Pten in murine skin, thymus, and prostate. (A) Southern-blot genotypic analysis for deletion of Pten in keratinocytes and thymus. Genomic DNA extracted from cultured keratinocytes and thymus tissue was digested with _Hin_dIII and hybridized with a probe situated upstream of exon 4 to show bands for wild-type (wt, 2-kb), nonrecombined (loxP, 6-kb), and recombined (Δ4-5, 2.3-kb) Pten alleles. DNA from keratinocytes and thymi is shown in Left and Right, respectively. Genotypes for each lane are as follows: 1 and 5, PtenloxP/loxP;2, Ptenwt/loxP;3,8,and9, PtenloxP/loxP; MMTV-cre; and 4 and 7, Ptenwt/loxP; MMTV-cre mice. Lane 9 is DNA from a thymic lymphoma. (B) Western blot analysis of cultured keratinocytes. Pten expression is lost in PtenloxP/loxP; MMTV-cre keratinocytes (Mut.) compared with Ptenwt/loxP; MMTV-cre control (Con.) keratinocytes. Weak expression of Pten in mutant samples indicates incomplete Cre-mediated recombination within the keratinocyte population. Phospho-PKB levels are elevated in PtenloxP/loxP; MMTV-cre keratinocytes compared with Ptenwt/loxP; MMTV-cre control keratinocytes under basal (10% FCS), serum-starved (0.5% FCS), and serum-stimulated conditions (Stim., starved and stimulated with 10% FCS). (C) PCR analysis for Cre-mediated recombination of the Pten locus (Δ4-5, Top), genotyping for Pten-loxP (Middle), and MMTV-cre (Bottom). Genomic DNA was isolated from the prostate of mice for PCR analysis, and products were run on an agarose gel. For Δ4-5 PCR, primers situated upstream of the 5′ loxP site and downstream of the 3′ loxP site amplified an 849-bp product only upon Cre-mediated deletion of _Pten4_-5. The Pten-loxP and wt alleles yielded products of 335 and 228 bp, respectively. Genotypes for each lane are as follows: 1, Ptenwt/loxP; 2, PtenloxP/loxP; 3–4, Ptenwt/loxP; MMTV-cre; and 5–8, PtenloxP/loxP; MMTV-cre mice. Positive control in lane 9 corresponds to PCR products from genomic DNA of Ptenwt/loxP; Gfap-cre brain tissue (16). Numbers on the left indicate size of DNA marker in base pairs.

Fig. 2.

Fig. 2.

Deletion of Pten in the skin results in skin, coat abnormalities, and hamartomas. (A) Skin on the abdomen of PtenloxP/loxP; MMTV-cre mice is more wrinkled (right, white arrow) than skin on Ptenwt/loxP; MMTV-cre control littermates (left) at postnatal day 7 (P7). (B) Coat of PtenloxP/loxP; MMTV-cre mice (right) is shaggy and ruffled compared with the smooth coat of Ptenwt/loxP; MMTV-cre littermate (left) at P21. (C–L) Hematoxylin and eosin staining. Epidermis of PtenloxP/loxP; MMTV-cre mice is papillary in appearance (D). Acanthosis with hyperkeratosis and hypergranulosis are apparent in Pten mutant skin (F). Skin from control mice is shown in C and E. Black arrows denote sebaceous glands, which were 61% more abundant in the dermis of Pten mutants compared with control skin by 3 wk (n = 4, P = 0.01). (G–L) Staining of paws (G and H), hair follicles (I and J), and tongue epithelium (K and L) of Ptenwt/loxP; MMTV-cre (G, I, and K) and PtenloxP/loxP; MMTV-cre (H, J, and L) animals. Epithelium of hair follicles and tongue of PtenloxP/loxP; MMTV-cre mice is several cell layers thicker (J and L) than controls (I and K). Arrowheads denote hair follicle epithelium. Acanthosis with hypergranulosis, hyperkeratosis, and parakeratosis are present within hamartomatous growths on paws of Pten mutant mice (H). Tissues are from 3-wk-old (C–F, I, and J) and 9- to 10-wk-old (G, H, K, and L) animals. (M) Average epidermal thickness in P2, 6- and 9-wk-old PtenloxP/loxP; MMTV-cre mice (black bars) was increased compared with Ptenwt/loxP; MMTV-cre mice (white bars, n = 3 for each genotype and age, P < 0.03). (Scale bar for A = 0.5 cm; B = 1 cm; C, D, G, and H = 100 μm; E and F = 10 μm; I and J = 20 μm; and K and L = 50 μm.)

Fig. 3.

Fig. 3.

Increased proliferation of Pten-null keratinocytes and prostate. (A) Pten-null keratinocytes (solid line) outgrew Ptenwt/loxP; MMTV-cre keratinocytes (dotted line) (trypan blue exclusion). (B) Increased expansion of Pten-null keratinocytes compared with heterozygous controls over 21 days in culture (crystal violet stain). (C–F) Ki67 immunohistochemistry in epidermis (C and D) and prostate (E and F) of Ptenwt/loxP; MMTV-cre (C and E) and PtenloxP/loxP; MMTV-cre (D and F) mice. (G) Percentage of Ki67-positive basal keratinocytes (Left) is greater in Pten mutant mice (black bars, n = 3) by 3 wk compared with controls (white bars, n = 3, P = 0.01). Proliferation of basal keratinocytes from 2-day-old Pten mutant animals (black bar, n = 3) is not significantly different from controls (white bar, n = 3, P = 0.39). Pten mutant animals have a greater percentage of Ki67-positive prostate epithelial cells (Right; black bar, n = 3) compared with glands from control animals (white bar, n = 3, P = 0.05). (Scale bar for C and D = 10 μm; E and F = 20 μm.)

Fig. 4.

Fig. 4.

Prostate neoplasias in PtenloxP/loxP; MMTV-cre mice. Prostate glands from PtenloxP/loxP; MMTV-cre animals (D) are more cellular than control glands (A) by P5. (B) Glands from the prostate of a 3-wk control male are one to two cells thick with a hollow lumen. (E) PCIS from 3-wk Pten mutant animal. Neoplastic growth with cellular atypia (see text and Fig. 6_B_) fills the lumen. Smooth muscle forms a continuous layer around control glands (C). Neoplasm in Pten mutant prostate gland invades through smooth muscle (F, above asterisk). p63 is expressed in the nucleus of basal cells from control (G) and Pten mutant (J) glands (arrowheads). A minority of cells within the neoplastic growths of Pten-null prostate were p63-positive (arrows). Androgen receptor is expressed in luminal epithelial cells from Ptenwt/loxP; MMTV-cre prostate tissue (H) and in the majority of cells within prostate neoplasms of Pten mutant animals (K). (I) Up-regulation of phospho-PKB expression in Pten-null prostate epithelium compared with Ptenwt/loxP; MMTV-cre mice (I Inset). Surface area of Pten-null prostate cells (L; 62.3 ± 5.2 μm2, n = 4) was larger than control cells (L; Inset, 40.5 ± 1.5 μm2, n = 4, P = 0.007). Hematoxylin and eosin staining is shown in A, B, D, and E. Immunohistochemistry for smooth muscle actin is shown in C and F; p63 is shown in G and J. Androgen receptor is shown in H and K. Phospho-PKB is shown in I. Pan-cadherin is shown in L. (Scale bar for A and D = 20 μm, B and E = 100 μm, G–K = 50 μm, and L = 10 μm.)

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