Human sporadic cancers have a broad repertoire of genetic changes and understanding of their contributions to major pathways defects is of critical importance. Mouse models of human cancer have been shown to serve as useful systems to facilitate identification of genetic alterations essential for carcinogenesis by comparative oncogenomic approaches (Kim et al., 2006
, Maser et al., 2007
, Zender et al., 2006
). During the past two decades it has become increasingly clear that different cancer phenotypes of mouse models may reflect distinct initiating genetic and epigenetic alterations (Cardiff et al., 2000
). Indeed, combination of p53
somatic inactivation results in formation of tumors mimicking human BRCA1-mutated ER negative basal-like cancer (Liu et al., 2007
). At the same time inactivation of p53
together with E-cadherin leads to ER negative metastatic lobular mammary carcinoma (Derksen et al., 2006
). Mammary neoplasms associated with p53 deficiency alone were reported to be either ER negative (Liu et al., 2007
) or both ER negative and positive, depending on MMTV
promoter used to express Cre in deleter mouse strains (Lin et al., 2004
Since available mammary epithelium-specific deleter strains express Cre in lymphoid and other tissues, we have established an additional MMTV-Cre strain with a highly mammary epithelium-restrictive expression pattern. Interestingly, different from other MMTV-Cre based models and likely due to transgene positional effects, the cancers forming in our model are ER positive and have no or very limited non-luminal differentiation typical for such tumor types as adenomyoepthelial, adenosquamous and basal-like carcinomas. Thus interpretation of cancer phenotypes is also likely to be affected by variations in transgene expression patterns and transformation of distinct cell lineages and their subpopulations.
Based on the expression of markers for luminal differentiation, such as ER and CK8, combined with p53 deficient status, as well as expression of Mad2 and cIAP2 (Frasor et al., 2009
), this model may represent an attractive tool for studies of human luminal subtype B breast cancers (Hu et al., 2006
, Sorlie et al., 2001
). This model is also particularly amenable to further experiments because of responsiveness of mammary carcinomas to an estrogen antagonist as well as availability of established syngeneic mammary carcinoma cell lines. Considering the 50% frequency of mammary carcinomas and long latency period of carcinogenesis in p53ME−/−
mice, they are particularly well suited for assessment of other endogenous and exogenous factors which are expected to accelerate carcinogenesis.
Our studies confirmed previous observations that sporadic inactivation of Rb
alone is insufficient for the initiation of mammary carcinogenesis (Robinson et al., 2001
). Furthermore, the use of a Cre-loxP
approach allowed direct genetic demonstration that Rb
loss-of-function, without inactivation of p107 and/or p130, leads to acceleration of mammary carcinogenesis associated with p53
inactivation. At least in part, this effect may be explained by increased proliferation rate of Rb-deficient neoplastic cells.
p53 and Rb pathways are extensively connected and their inactivation frequently cooperates during carcinogenesis presumably by abrogating E2F-induced p53-mediated apoptosis or senescence (Sherr and McCormick, 2002
, Sherr, 2004
). Our study illuminates genomic instability as another mechanism of p53 and Rb cooperation. Genomic instability is a hallmark of most human cancers. Although much attention has been focused on the role of p53 in the maintenance of genomic stability, an accumulating body of evidence indicates Rb as another important player (Knudsen et al., 2006
inactivation has been shown to promote genomic instability by uncoupling cell cycle progression from mitotic control (Hernando et al., 2004
) and by mediating DNA double strand break accumulation (Pickering and Kowalik, 2006
) in cell culture models. In vivo, Rb
loss results in ectopic cell cycle, compromises ploidy control in mouse liver (Mayhew et al., 2005
) and promotes hepatocarcinogenesis (Mayhew et al., 2007
, Reed et al., 2009
). Our study extends these observations by demonstrating higher levels of the E2F downstream target Mad2, higher rates of double strand DNA breaks and centrosome amplification and overall increase in chromosomal structural instability in mammary carcinomas deficient for both p53 and Rb. Further studies should determine if Rb
loss leads to similar consequences in the normal mammary epithelium. It also remains to be demonstrated whether observed increase in phenotypical diversity and trend towards increase of cellular polymorphism and poorer differentiations in carcinomas of p53ME−/−RbME−/−
mice are a result of elevated genomic instability associated with Rb deficiency.
Using aCGH, we identified a number of recurrent genetic alterations in our mammary carcinoma models. It is likely that each of these alterations has individual contributions to carcinogenesis. The amplification of 9A1 locus is of a particular interest because it includes protooncogenes, such as cIAP1, cIAP2
and Yap1. cIAP1
encoded proteins contain baculoviral IAP repeat (BIR) domain and are key regulators of apoptosis, cytokinesis, and signal transduction. Both genes are are commonly amplified in many human cancers (reviewed in LaCasse et al., 2008
). Yap1 contains a WW domain and binds to the SH3 domain of the tyrosine kinase Yes. It has been shown to be expressed in common solid tumors (Steinhardt et al., 2008
) and to have tumorigenic properties in both liver (Zender et al., 2006
) and breast cancer (Overholtzer et al., 2006
). Considering the high frequency of 9A1 locus amplification in neoplasms of p53ME−/−
mice, this model may prove to be very valuable for further studies of mechanisms involved in amplification of human orthologous genes on chromosome 11q22.
Amplification of both cIAP1
was observed in 4 out of 7 tumors in a transplantable mouse model of liver cancer based on transduction of p53 deficient fetal hepatoblasts with Myc retrovirus (Zender et al., 2006
). At the same time, amplification of cIAP1
, together with matrix metalloproteinase MMP13, was observed in 5 out of 41 osteosarcomas (Ma et al., 2009
amplification was detected in one out of 15 mammary tumors of MMTV-Cre Brca1floxP/−
mice. However, amplification of Yap1
was detected in none of over 100 sporadic human breast cancers (Overholtzer et al., 2006
). Our observations of E2F-mediated control of cIAP1, cIAP2 and Yap1 may explain how dysfunctional Rb/E2F pathway may substitute for recurrent amplification of these genes. Since alterations in the Rb pathway are quite common in mouse and human tumors, including mammary carcinomas, this mechanism may also explain differences in frequencies of cIAP1, cIAP2
amplification among various tumor types.
Our results demonstrate that cIAP1, cIAP2 and Yap1 overexpression is critical for mammary carcinogenesis associated with p53
mutations. It is of interest that overexpression of cIAP2 has been recently reported to be associated with luminal subtype B of breast cancer (Frasor et al., 2009
). Further studies will examine whether tumors of this subtype also overexpress cIAP1 and Yap1. Cooperation among cIAP1, cIAP2 and Yap1 in promoting tumorigenicity observed in our work is consistent with previously reported cooperation between cIAP1 and Yap1 in hepatocarinogenesis (Zender et al., 2006
). Recently a broad variety of IAP molecule antagonists has been developed (LaCasse et al., 2008
). Our results indicate that their application may be particularly effective in combination with downregulation of Yap1.
In summary, we established a new mouse model of sporadic ER positive luminal mammary carcinoma associated with p53 inactivation. We demonstrated that Rb deficiency accelerates mammary carcinogenesis and leads to increased genomic instability and to a different spectrum of recurrent genomic alterations. Of particular interest, genes of chromosome band 9A1, namely cIAP1, cIAP2 and Yap1, were shown to be important for mammary carcinogenesis. We proposed the E2F-mediated mechanism of their regulation and established cooperation among all three genes in promoting neoplastic growth. These observations provide the basis for further elucidation of cooperation of these genes in human cancers and lay the ground for rational development of therapeutic approaches in preclinical settings.