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First published online November 3, 2003
doi: 10.1242/10.1242/dev.00820

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Squamous cell carcinoma and mammary abscess formation through squamous metaplasia in Smad4/Dpc4 conditional knockout mice

Wenmei Li^1,*, Wenhui Qiao^1,*, Lin Chen¹, Xiaoling Xu¹, Xiao Yang¹, Dan Li¹, Cuiling Li¹, Steven G. Brodie¹, Michael M. Meguid², Lothar Hennighausen³ and Chu-Xia Deng^1,

¹ Genetics of Development and Disease Branch, NIDDK, NIH, 10/9N105, 10 Center Drive, Bethesda, MD 20892, USA
³ Laboratory of Genetics and Physiology, NIDDK, NIH, 10/9N105, 10 Center Drive, Bethesda, MD 20892, USA
² Department of Surgery, Neuroscience Program, Upstate Medical University, Syracuse, NY 13210, USA

View larger version (110K): [in a new window]   Fig. 7. Loss of TGFß-mediated ß-catenin degradation and TGFß responsiveness in Smad4–/– cells. (A,B) Western blot showing ß-catenin in Smad4+/+ (A) and Smad4–/– (B) cells after they were treated with 2 ng/ml of TGFß for 24 hours. Samples 1 and 2 were mammary tumor cell lines derived from Brca1 conditional knockout and MMTV-ras transgenic mice, respectively. Sample 3 was a cell line derived from a P16 mammary gland from a Smad4+/+ mouse. Sample 4 and 5 were two Smad4–/– cell lines derived independently from mammary abscesses of Smad4Co/CoWAP-Cre mice. Treatment of TGFß(+) led to a decrease in ß-catenin in all Smad4+/+ cell lines, whereas no changes were detected in both independently derived Smad4–/– cells. (C) Loss of Smad4 blocks TGFß responsiveness as Smad4–/– cells did not undergo EMT and failed to decrease ß-catenin upon TGFß treatment. (D) Timecourse response of Smad4+/+ (wild-type) cells to TGFß induced EMT and downregulation of ß-catenin. Scale bars: 100 µm for phase contract images and 39 µm for immunofluorescent images.

View larger version (65K): [in a new window]   Fig. 1. Expression of Smad4 in adult mouse tissues and conditional knockout Smad4 in mammary glands. (A,B) Northern blot analysis of Smad4 expression in adult tissues. Filters bearing poly A+ RNA (A, purchased from Clontech) and total RNA (B) were probed with a probe specific to Smad4 cDNA. Ht, heart; Br, brain; Sp, spleen; Lu, lung; Li, liver; Mu, skeletal Muscle; Ki, kidney; Te, testis. (C-G) Immunohistochemical staining using an antibody to Smad4. Primary antibody was not used in sections shown in (D,F) to serve as controls. (C,D) Sections from a wild-type virgin gland. (E,F) Sections from a P14 wild-type gland. (G) A section from an I10 mutant gland, which shows very few cells that are still positive for Smad4 (arrowheads). (H) A Smad4 conditional (Co) and deletion (De) alleles. Deletion of Smad4 exon 8 in the mammary gland is achieved by crossing the Smad4 conditional mice with WAP-Cre transgenic mice. (I) Southern blot showing Cre mediated recombination. DNAs were digested with EcoRV and probed with a 2.2 kb HpaI-EcoRV fragment (black bar). Samples are from mammary glands of Smad4Co/+ mice (the first lane) and Smad4Co/CoWAP-Cre (lane 2-6) mice at different stages, including virgin, P16.5, L2, L10 and I10. 9.5 kb, 7.2 kb and 4.3 kb fragments are wild-type (Wt), deleted (De) and conditional (Co) alleles, respectively. (J) Northern blot analysis of RNA isolated from p16 and L10 wild-type (lanes 1 and 3) and mutant (lanes 2 and 4) mice. At least three mutant and control mice at each point were examined. The intensities of bands were measured using software, IP-lab and normalized with loading controls. % expression of mutant relative to control=average intensity of mutant/average intensity of controlx100/100. Scale bar: 60 µm for C-G.

View larger version (106K): [in a new window]   Fig. 2. Mammary tumor mass formation in Smad4Co/CoWAP-Cre and Smad4Co/CoMMTV-Cre mice. (A) Morphologically denser areas detected in the fourth mammary gland of a P16.5 Smad4Co/CoWAP-Cre mouse. This female is 6 months of age and went through more than four pregnancies. (B) An enlarged view of the boxed area shown in A. Arrowhead and arrow indicate areas with different morphology, respectively. (C) Multiple tumor masses (arrows) in a third gland of a one-year-old Smad4Co/CoWAP-Cre mouse. (D,E) An 8-month-old Smad4Co/CoWAP-Cre mouse developed multiple mammary abscesses, two of which are indicated by an arrow and an arrowhead. The area indicated by the arrowhead is shown in E. (F) A curve showing percent of mammary mass Smad4Co/CoWAP-Cre (n=36), Smad4Co/CoMMTV-Cre (n=34) and control mice. The controls include Smad4Co/+WAP-Cre, WAP-Cre and Smad4Co/Co mice. About 50 controls younger than 1 year and 20 between 1 and 1.5 years of age were followed and none developed mammary tumor and/or abscess. Scale bar: 3.5 mm in A; 0.87 mm in B,C.

View larger version (133K): [in a new window]   Fig. 3. Histology of mammary tumor in Smad4Co/CoWAP-Cre mice. (A,B) Normal (A) and hyperplasia (B) area of a L10 gland from a 6-month old mouse. (C) A mammary tumor mass at early stages showing undifferentiated alveolar structures. (D) In situ lesions from a 12-month-old mouse. The arrow indicates the center of initiating abscess. (E-G) Squamous cell carcinoma (E,F) and an abscess (G), showing cancer-pearl-like keratinization and onion-skin-like structures. Notice the increasing sizes of abscesses (arrows). (The boxed areas in C and E were further analyzed in Fig. 6C-F,G-J using molecular markers.) (H,I) The mutant mice also developed other types of tumors at much lower frequencies, such as carcinosarcoma (H) and adenocarcinoma (I). Scale bars: 120 µm in A,C; 240 µm in B,F,G; 136 µm in D; 90 µm in E; 80 µm in H; 60 µm in I.

View larger version (107K): [in a new window]   Fig. 4. Conditional knockout of Smad4 results in epidermis formation in mammary glands. (A,C,E,G) Skin samples; (B,D,F,H) abscesses found in the fourth gland from a 7.5-month-old Smad4Co/CoWAP-Cre mouse. Markers used for analysis were as indicated. At least 12 abscesses were studied and similar features were observed. Scale bar: 125 µm.

View larger version (113K): [in a new window]   Fig. 5. Transdifferentiation from mammary epithelial cells to keratinocytes due to the loss of Smad4. (A,B) BrdU labeling of normal (A) and hyperplasia (B) area of a L10 gland. We have counted BrdU+ cells in 12 equivalent normal (n=6) and hyperplasia (n=6) areas. Average percentages of BrdU+ cells are 0.85±0.63 and 8.5±1 in normal and hyperplasia, with the P<0.01 by Student's t-test. (C-E) Images of early stages of transdifferentiation revealed by H&E (C), BrdU (D) and K14 (E) immunohistochemical staining. Arrows indicate the center of the lesion. (F,G) Images of an abscess at later stages detected by H&E (F) and K14 (G) immunohistochemical staining. Arrow indicates to the wall of the abscess, which is K14 positive, and arrowhead indicates an alveoli, which is K14 negative. (H-L) Micro-dissection (H-J) and PCR genotype (K,L) of dissected abscesses. (H) Prior to dissection; (I) after dissection; and (J) the dissected sample. (K) Structure of floxed allele. (L) Two independent samples (1 and 2) collected by micro-dissection, were analyzed and both showed that the abscess is caused by the Cre-mediated deletion of Smad4. Primers a/c amplify about 500 bp from the recombined allele. Primers a/c amplify about 450 bp from conditional allele, which is absent in these samples. The sequences of the primers are: a, 5'-GACCCAAACGTCACCTTCAG-3'; b, 5'-GGGCAGCGTAGCATATAAGA-3'; and c, 5'-AAGAGCCACAGGTCAAGCAG-3'. All samples were from a 7-month-old Smad4Co/CoWAP-Cre mouse. Scale bar: 180 µm in A,B; 35 µm in C-E; 60 µm in F,G.

View larger version (97K): [in a new window]   Fig. 6. ß-catenin overexpression at the earliest stage of transdifferentiation. (A) Western blot showing expression of K14, ß-catenin and E-cadherin in mammary abscesses (lanes 1-5) and adjacent normal gland (lane 6). (B) All abscesses examined, irrespective of their sizes (arrows), are positive for ß-catenin as demonstrated by this section from a 1-year-old Smad4Co/CoWAP-Cre mouse. (C-R) Analyzing squamous metaplasia at three distinct phases as described in the text: phase I (C-F, and arrowhead in G-J); phase II (arrow in G-J); and phase III (K-R). C,G are amplified from Fig. 3C,E, respectively. Antibodies used are as indicated. Arrows in C indicate undifferentiated alveolar structures. The arrow and arrowhead in G indicate the differentiated and undifferentiated alveolar structures, respectively. Notice that cells prior to differentiation (arrowhead) express only ß-catenin but not K-14, while the differentiating cells (arrow) express both. The arrow in K indicates a small, but well formed abscess that expresses both genes. The arrows in O indicate initiating metaplasia, which express K14 at high levels but E-cadherin at significantly lower levels. Scale bars: 140 µm in K-N; 70 µm in B-J,O-R.