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

Cheng L, Zhou Z, Flesken-Nikitin A, Toshkov IA, Wang W, Camps J, Ried T, Nikitin AY - Oncogene (2010)

Bottom Line: Genetically defined mouse models offer an important tool to identify critical secondary genetic alterations with relevance to human cancer pathogenesis.Rb deficiency was insufficient to initiate carcinogenesis but promoted genomic instability and growth rate of neoplasms associated with p53 inactivation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853-6401, USA.

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.

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Generation and characterization of a mouse model of mammary carcinoma associated with p53 and Rb deficiencyA, 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 p53ME−/− and p53ME−/−RbME−/− mice is 0.0058. C, Neoplasms of the mammary epithelium in p53ME−/− and p53ME−/−RbME−/− 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.
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Figure 1: Generation and characterization of a mouse model of mammary carcinoma associated with p53 and Rb deficiencyA, 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 p53ME−/− and p53ME−/−RbME−/− mice is 0.0058. C, Neoplasms of the mammary epithelium in p53ME−/− and p53ME−/−RbME−/− 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.

Mentions: To avoid genetic background variations and frequent lymphomas due to Cre expression in lymphocytes and other tissues we generated mice expressing Cre under the control of MMTV-LTR (Fig. 1 A) and screened founders for exclusive expression of Cre in the mammary epithelium after their crosses with Gt(ROSA)26SorTM1sor reporter mice. One out of five tested lines, FVB/N Tg(MMTV-Cre)105Ayn expressed Cre selectively in the mammary epithelium (Suppl. Table 1 and Suppl. Fig. 1) Furthermore, no lymphomas or other non-mammary neoplasms were observed in crosses of this line with p53floxP/floxP mice. Therefore, it has been chosen for all subsequent experiments and will be described as MMTV-Cre.


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

Cheng L, Zhou Z, Flesken-Nikitin A, Toshkov IA, Wang W, Camps J, Ried T, Nikitin AY - Oncogene (2010)

Generation and characterization of a mouse model of mammary carcinoma associated with p53 and Rb deficiencyA, 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 p53ME−/− and p53ME−/−RbME−/− mice is 0.0058. C, Neoplasms of the mammary epithelium in p53ME−/− and p53ME−/−RbME−/− 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.
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Figure 1: Generation and characterization of a mouse model of mammary carcinoma associated with p53 and Rb deficiencyA, 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 p53ME−/− and p53ME−/−RbME−/− mice is 0.0058. C, Neoplasms of the mammary epithelium in p53ME−/− and p53ME−/−RbME−/− 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.
Mentions: To avoid genetic background variations and frequent lymphomas due to Cre expression in lymphocytes and other tissues we generated mice expressing Cre under the control of MMTV-LTR (Fig. 1 A) and screened founders for exclusive expression of Cre in the mammary epithelium after their crosses with Gt(ROSA)26SorTM1sor reporter mice. One out of five tested lines, FVB/N Tg(MMTV-Cre)105Ayn expressed Cre selectively in the mammary epithelium (Suppl. Table 1 and Suppl. Fig. 1) Furthermore, no lymphomas or other non-mammary neoplasms were observed in crosses of this line with p53floxP/floxP mice. Therefore, it has been chosen for all subsequent experiments and will be described as MMTV-Cre.

Bottom Line: Genetically defined mouse models offer an important tool to identify critical secondary genetic alterations with relevance to human cancer pathogenesis.Rb deficiency was insufficient to initiate carcinogenesis but promoted genomic instability and growth rate of neoplasms associated with p53 inactivation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853-6401, USA.

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.

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Related in: MedlinePlus