Rb inactivation accelerates neoplastic growth and substitutes for recurrent amplification of cIAP1, cIAP2 and Yap1 in sporadic mammary carcinoma associated with p53 deficiency

Abstract

Genetically defined mouse models offer an important tool to identify critical secondary genetic alterations with relevance to human cancer pathogenesis. We used newly generated MMTV-Cre105Ayn mice to inactivate p53 and/or Rb strictly in the mammary epithelium, and to determine recurrent genomic changes associated with deficiencies of these genes. p53 inactivation led to formation of estrogen receptor-positive raloxifene-responsive mammary carcinomas with features of luminal subtype B. Rb deficiency was insufficient to initiate carcinogenesis but promoted genomic instability and growth rate of neoplasms associated with p53 inactivation. Genome-wide analysis of mammary carcinomas identified a recurrent amplification at chromosome band 9A1, a locus orthologous to human 11q22, which contains protooncogenes cIAP1 (Birc2), cIAP2 (Birc3) and Yap1. It is interesting that this amplicon was preferentially detected in carcinomas carrying wild-type Rb. However, all three genes were overexpressed in carcinomas with p53 and Rb inactivation, likely due to E2F-mediated transactivation, and cooperated in carcinogenesis according to gene knockdown experiments. These findings establish a model of luminal subtype B mammary carcinoma, identify critical role of cIAP1, cIAP2 and Yap1 co-expression in mammary carcinogenesis and provide an explanation for the lack of recurrent amplifications of cIAP1, cIAP2 and Yap1 in some tumors with frequent Rb deficiency, such as mammary carcinoma.

Figure 1. Generation and characterization of a mouse model of mammary carcinoma associated with p53 and Rb deficiency

A, Generation and characterization of MMTV-Cre transgenic mice. (Top) The MMTV-Cre transgene consists of the 1.48 kb MMTV-LTR promoter followed by the 1.1 kb Cre gene and the 1.2 kb MT-1 polyadenylation site. (Bottom) Identification of MMTV-Cre transgenic mice by PCR genotyping. 296 bp and 194 bp fragments are diagnostic for the Cre gene and mouse Rb gene, respectively. MMTV-Cre founder mice are identified in lanes 1, 4, 5, 7, and 8 (lines MMTV-Cre104Ayn, 105Ayn, 106Ayn, 107Ayn, 108Ayn, respectively). B, Survival of mice with mammary-specific inactivation of p53 (n=16, median 669 days), Rb alone (n=8, median 700 days) or p53 and Rb together (n=17, median 504 days). P for log-rank comparisons of survival curves of p53^ME−/− and p53^ME−/−Rb^ME−/− mice is 0.0058. C, Neoplasms of the mammary epithelium in p53^ME−/− and p53^ME−/−Rb^ME−/− mice. (Top) Mammary carcinomas with mainly (Left) solid pattern of growth (arrow) and dense fibrous stroma (arrowhead), (Middle) glandular pattern (arrow), (Right) spindle cell pattern with diverse cell types (arrow). H&E stain. (Middle) Lung metastasis of mammary carcinoma (arrow) (Left), H&E stain. Expression of CK8, CK5 and SMA in carcinoma cells (arrows) (Middle and Right). (Bottom) Expression of Mad2, ER and PR in carcinoma cells (arrows). ABC Elite method, hematoxylin counterstaining. Calibration bar for all images: 100 μm.

Figure 2. Mammary neoplasms respond to hormone therapy with raloxifene

A, Western blot of ERα, and PR in MCN1, MCN2, MCN3 and MCF7 cell lines. To normalize for differences in loading, the blots were stripped and reprobed with mouse anti-Gapdh monoclonal antibody. B, Effects of raloxifene on proliferation of mammary carcinoma cells as determined by BrdU incorporation and compared to control (Mean ± SD, n=3 in each group, P < 0.05, indicated as *). All MCN cell lines are p53 null and have either two (+/+) or no (−/−) functional copies of the Rb gene. C, Effects of raloxifene on tumor growth in vivo. MCN1 and MCN2 cells (10⁶) were transplanted to cleared fad pad of 4 weeks old FVB mice. According to the tumor volume measurements 12 days after transplantation, raloxifene (Raloxifene +) significantly delays the tumor growth of MCN1 (P=0.0132) and MCN2 (P=0.0088) cells as compared to control group without raloxifene treatment (Raloxifene −).

Figure 3. Genomic alterations in mammary carcinomas of p53^ME−/− and p53^ME−/− Rb^ME−/− mice

A, The log2 ratios for each chromosome in order from 1p to Xqter. (Top) Chromosomal regions with consistent gene copy number alterations (5 out of 6 samples, arrows). Carcinomas of p53^ME−/− mice: significant gain and loss are mapped to the chromosomal bands 9A1 and 12C2 - 12F1, respectively. Carcinomas of p53^ME−/− Rb^ME−/− mice: significant gain at 6A1 and 6A2. B, SKY analysis of chromosome metaphase spreads of primary tumor cells (Top, p53^ME−/−; Bottom, p53^ME−/− Rb^ME−/−). Karyotype of metaphase spread with classification pseudo-color and its corresponding inverted-DAPI. Arrow, net gain of chromosome 9A1 in tumors of p53^ME−/− mice. C, (Left) Comparison of aCGH profiles of tumors from p53^ME−/− and p53^ME−/− Rb^ME−/− mice. (Right) Chi-square test of the number of BAC in altered chromosome region of tumors from p53^ME−/− and p53^ME−/− Rb^ME−/− mice.

Figure 4. Rb inactivation promotes genomic instability

A, (Left) Western blot of E2F1, E2F3, and Mad2 in primary tumor cells from p53^ME−/− and p53^ME−/− Rb^ME−/− mice. (Right) Relative Mad2 mRNA expression in carcinomas of p53^ME−/− and p53^ME−/− Rb^ME−/− mice (Mean ± SD, 2.9 ± 2.1 versus 6.3 ± 4.0, n=10, P = 0.0411, indicated as *). B, (Top) Immunofluorescence staining (γ-tubulin) of centrosomes in primary tumor cells from p53^ME−/− and p53^ME−/− Rb^ME−/− mice. (Bottom Left) Percentage of cells with more than 2 centrosomes is higher in cells from p53^ME−/− Rb^ME−/− mice, as compared to that in cells from p53^ME−/− mice (75.8 ± 9.0 versus 49.6 ± 12.0, n=10, P = 0.0083). (Bottom Right) Tumor cells from p53^ME−/− Rb^ME−/− mice have higher average number of centrosomes per cell than that from p53^ME−/− mice (4.96 ± 0.76 versus 3.34 ± 0.90, n=10, P=0.0152,). C, (Left) Representative images of neutral comet assay with primary tumor cells from p53^ME−/− and p53^ME−/− Rb^ME−/− mice. (Right) Tumor cells from p53^ME−/− Rb^ME−/− mice have higher percentage of cell with comet tail as compared to p53^ME−/− (18.9 ± 1.3 versus 11.5 ± 3.3, n=10, P = 0.0232).

Figure 5. Copy number and expression of cIAP1, cIAP2 and Yap1 in mammary carcinomas of p53^ME−/− and p53^ME−/− Rb^ME−/− mice

A, (Top) The map of the genes in the chromosome 9A1 region under study. (Bottom) Mammary carcinomas from p53^{ME −/−} mice have higher cIAP1, cIAP2 and Yap1 gene copy number than those from p53^{ME −/−} Rb^{ME −/−} mice (Mean ± SD, n=10 in each group). cIAP1: 17.6 ± 19.2 versus 3.1 ± 3.1; cIAP2: 20.3 ± 19.7 versus 3.1 ± 2.4 and Yap1: 19.03 ± 17.96 versus 4.08 ± 2.95, respectively. * indicates P < 0.05. DNA copy number of Tmem123 and Pgr is similar between the cells from these two different types of mice (Tmem123: 1.88 ± 1.36 versus 1.78 ± 1.41 and Pgr: 1.87 ± 1.6 versus 1.72 ± 1.32). Quantitative PCR. B, (Left) Overexpression of cIAP1, cIAP2 and Yap1 in mammary carcinomas from both p53^{ME −/−} and p53^{ME −/−} Rb^{ME −/−} mice as compared to the wild-type (WT) mammary epithelium (Mean ± SD, n=10 in each group) cIAP1: 7.80 ± 8.61 (p53^{ME −/−}), 12.30 ± 15.30 (p53^{ME −/−} Rb^{ME −/−}) versus 1.05 ± 0.13 (WT); cIAP2: 6.31 ± 10.89 (p53^{ME −/−}), 5.73 ± 6.72 (p53^{ME −/−} Rb^{ME −/−}) versus 1.06 ± 0.18 (WT), and Yap1: 18.29 ± 27.12 (p53^{ME −/−}), 19.12 ± 20.15 (p53^{ME −/−} Rb^{ME −/−}) versus 1.16 ± 0.19 (WT), * indicates P < 0.05. Quantitative RT-PCR. (Right) Western blot of cIAP1 and Yap1 in MCN1, MCN2, MCN3 cells and primary culture of wild type mammary epithelium (WT). C, Expression of E2F1, cIAP1, cIAP2, and Yap1 after E2F1 knockdown by E2F1 siRNA as compared to scrambled siRNA control (Mean ± SD, n=4 in each group; P < 0.05 is indicated as*). All MCN cell lines are p53 null and have either two (+/+) or no (−/−) functional copies of the Rb gene (Top Left). Relative expression (Mean ± SD, n=4 in each group) of cIAP1 (Top Right), cIAP2 (Bottom Left) and Yap1 (Bottom Right) collected at 24 and 72 hours after treatment of mammary epithelium cells from floxed p53 (p53^L/L), Rb (Rb^L/L), or p53 and Rb (p53^L/LRb^L/L) mice with blank Adenovirus (Blank) or AdCre (P < 0.05 is indicated as *). Quantitative RT-PCR.

Figure 6. cIAP1, cIAP2, and Yap1 cooperate in mammary carcinogenesis

A, Downregulation of either cIAP1, cIAP2 or Yap1 by siRNA in primary mammary carcinoma cells leads to decreased cell proliferation (Left) and increase of apoptosis (Right) as compared to scrambled siRNA control. Both effects are more pronounced after inactivation of any two or all three genes. (Mean ± SD, n=4 in each group, P<0.05, indicated as*). B, Effect of cIAP1, cIAP2 or Yap1 knockdown on tumor growth (Mean ± SD, n=4 in each group). According to the tumor volume measurements 17 days after transplantation, downregulation of cIAP1, cIAP2 and Yap1 by siRNA in MCN1 mammary carcinoma cells decelerates tumor growth as compared to control (P = 0.014, P = 0.0056, and P = 0.0091, respectively). Combination of cIAP1 and cIAP2, cIAP1 and Yap1, or all three genes further delays the tumor growth (P = 0.0022, P = 0.0073, and P = 0.0069, n=4, respectively).