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Conditional loss of PTEN leads to precocious development and neoplasia in the mammary gland

Gang Li^1,4, Gertraud W. Robinson², Ralf Lesche^1,*, Hilda Martinez-Diaz¹, Zhaorong Jiang³, Nora Rozengurt³, Kay-Uwe Wagner², De-Chang Wu⁴, Timothy F. Lane⁵, Xin Liu³, Lothar Hennighausen² and Hong Wu^1,

¹ Department of Molecular and Medical Pharmacology, and Howard Hughes Medical Institute,
³ Department of Pathology and Laboratory Medicine,
⁵ Jonsson Comprehensive Cancer Center, UCLA School of Medicine, 650 Circle Drive South, Los Angeles, CA 90095-1735, USA
² Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0822, USA
⁴ Institute of Radiation Medicine, Beijing, PR China
^* Present address: Epigenomics AG, Kastanienallee 24, 10435 Berlin, Germany

View larger version (73K): [in a new window]   Fig. 1. Conditional deletion of Pten in mammary gland. (A) LoxP sequences are inserted on both sides of exon 5. After crossing with MMTV-Cre+/– transgenic mice, exon 5 is deleted in the mammary gland. P1, P2 and P3 localize the PCR primers used for genotyping. The predicted PCR products are: loxp=1100 bp; wild type=1000 bp; 5=300 bp. (B) PCR screen for Cre-mediated Pten exon5 deletion in Ptenloxp/loxp/MMTV-Cre+/– mammary glands at different developmental stages. Lanes 1-4, control DNAs isolated from wild type (WT), Ptenloxp/+, Ptenloxp/loxp and Ptenloxp/5 mice; lane 5-8, DNAs prepared from 5 week virgin, 8 week virgin, 3 days after weaning, and resting multi-parous mammary glands. (C) Decreased PTEN protein expression in the Ptenloxp/loxp/MMTV-Cre+/– mammary gland. Total cell lysates from mutant (Ptenloxp/loxp/MMTV-Cre+/–, M) and control (Ptenloxp/loxp/MMTV-Cre–/–, C) mice of 6 week-old virgin (V-6wk), pregnancy day 13 (P13), lactation day 10 (L10), and post-weaning day 1 (PW1) were subjected to western blot analysis, using -PTEN antibody and -actin antibody for loading controls. (D) Deletion of PTEN in mammary epithelial cells. Immunohistochemical staining for PTEN is conducted on mutant and control mammary glands at day 13 of pregnancy. Scale bar: 20 µm.

View larger version (72K): [in a new window]   Fig. 2. Accelerated ductal extension, excessive side-branching and precocious lobulo-alveolar development in mutant mammary glands. (A) Whole-mount preparations of control and mutant mammary glands at different developmental stages. Carmine Red-stained whole mounts of inguinal mammary glands from control (a, b, e-g, k and l) and mutant (c, d, h-j, m and n) mice at 6 (a-d), 8 (e-j), 14 (k-n) weeks of age. Images in b, d, f, g, i, j, l, and n show higher magnifications of the areas bracketed in a, c, e, h, k and m. Note the lobulo-alveolar development on mutant glands (arrows in d, j, and n). Scaled arrows in a and c indicate the distances between the centers of the lymph node and the end of the TEBs. Box in a indicates the area where the number of branches was counted. (B) Quantitative representation of distances from the center of the lymph node to the far end of TEBs (top), and the number of branching points within the comparable boxed area (see Aa. 8 mm2) at different stages analyzed (n=45). (C) Northern blot analysis of ß-casein mRNA levels in control (C) and mutant (M) mammary glands. RNA was extracted from virgin mammary glands at 6 weeks (6W) and 10 weeks (10W). Northern blots were probed with cDNAs encoding – and ß-casein. One representative of two independent experiments is shown. The amount of ribosomal RNAs was used as loading controls. (D) Enlarged lobulo-alveolar in mutant mammary gland compared with control at 13 days after pregnancy.

View larger version (71K): [in a new window]   Fig. 3. Increased BrdU incorporation in the mutant mammary glands. Five-week-old (A) and 14-week-old (B) virgin control and mutant mice were pulse-labeled with BrdU. The numbers of BrdU+ cells were counted in the TEB region (for 5-week-old samples) or in the ductal areas (for 14-week-old samples). The proliferation index for 5-week-old mice was calculated as follows: [number of BrdU+-labeled cells]/[total number of cells]x100; Error bars represent standard errors of the mean, P<0.01, 2-test, n=3. For 14-week-old mice, all of the BrdU-labeled cells from the whole sections were counted; values were expressed as [number of BrdU+-labeled cells]/[100 ductal structures]. Bar: 40 µm.

View larger version (108K): [in a new window]   Fig. 4. Abnormal ductal development of transplanted mutant mammary epithelium. Whole-mount analysis of mammary epithelial transplants (A and C, control epithelium; B and D, mutant epithelium) from virgin females. At higher magnification, more and feather-like irregular side-branches are seen in mutant ductal and terminal structures (arrow in D) while control transplants display smooth ducts and normal terminal end buds (arrow in C). Scale bar: for A and B, 300 µm; for C and D, 200 µm.

View larger version (84K): [in a new window]   Fig. 5. Involution is severely affected in the mutant mammary glands. (A) HE staining of mammary glands sections. (Left panels) Mammary glands from control mice. (Right panels) Mammary glands from mutant mice. (Upper panels) Day 1 of involution; (second panels) day 3 of involution; scale bar: 40 µm; (third panels) Carmine Red-stained whole mounts of mammary glands at day 21 of involution; scale bar: 800 µm; (bottom panels) HE staining of sections from the corresponding whole-mounts samples; scale bar: 80 µm. (B) TUNEL analysis of control (upper left panels) and mutant (upper right panel) involuting mammary glands 3 days post parturition. TUNEL-positive cells are in green. Lower panels show DAPI staining of the corresponding sections. Scale bar: 40 µm. (C) Quantification of TUNEL-positive cells (left). Ten 200x magnification fields of view were randomly counted. The apoptosis index was calculated as following: ([number of TUNEL positive cells]/[total number of cells])x100. The total cell numbers per 400x magnification fields of view was also quantified and graphed (right panel). Statistical analysis was performed using Student’s t-test. Error bars represent standard errors of the mean.

View larger version (37K): [in a new window]   Fig. 6. Analysis of down-stream signaling targets of PTEN controlled pathway. (A) Northern blot analysis of ß-casein, cyclin D1, and c-myc mRNA levels in control (C) and mutant (M) mammary glands. RNA was extracted from pregnancy day 13 (P13), lactation day 10 (L10), and involution day 1, 3 and 6 glands, respectively (I-1 to I-6). One representative of two independent experiments is shown. The amount of ribosomal RNAs was used as loading control. (B-E) Western blot analysis. Total cell lysates from lactation day 10 (L10), involution day 1 (I-1), day 3 (I-3), and day 6 (I-6) glands were examined. Mutant and control are labeled as M, mutant; C, control. -actin antibody was used as loading control.

View larger version (87K): [in a new window]   Fig. 7. (A) Mutant mice develop mammary tumors early in life. (a) A whole-mount image reveals multiple nodules (arrows) in the mutant gland. (b) Virgin mutant transplants with focal ductal hyperplasia containing papillary structures (arrow). (c) Mutant glands after one pregnancy showed intra-luminal focal hyperplasia. Note the ductal epithelial cells contain multi-layers and protrude into the lumen (arrows). (d,e) Mutant gland after one pregnancy with dysplastic changes (d) and pleomorphic nuclei and mitotic figures (arrow in e). (f) Mutant gland with squamous epithelium and keratinous debris within (arrows). (g,h) Tumors from multiparous mutant mammary glands show various histological features, ranging from well differentiated fibroadenoma (g) to pleomorphic adenocarcinoma (h). Scale bar: b, 80 µm; c, 160 µm; d,f,g,h, 80 µm; e, 16 µm. (B) Up-regulated cytokeratin 5 and 6 expressions in the mutant mammary tissue transplants. Cytokeratin 5 (left panels) and cytokeratin 6 (right panels) immuno-staining (in red) of normal virgin (a,b), hyperplastic areas of mutant virgin (c,d) and term glands (e,f). All sections were counter-stained with ß-catenin antibody (in green). Cytokeratin 5 expression is low in myoepithelial cells of normal glands, and is upregulated in hyperplastic areas (arrow in c). Cytokeratin 6 is absent in normal alveoli and is expressed only in epithelial cells that protrude into the lumen of hyperplasic areas (arrows in d). Broader ranges of cytokeratin 5 and 6 expressions can be detected in the term mutant glands (e,f). Bar for a,b,e,f, 80 µm; c,d, 250 µm. (C) Tumor incidence of Ptenloxp/loxp;MMTVCre+/– (n=37) and Ptenloxp/loxp (n=21) females.