The C3(1) 5′ region of the rat prostatic steroid binding protein has been previously used in our laboratory to direct the expression of SV40-T/t-antigens to the prostate and mammary glands of mice (Maroulakou et al. 1994
). SV40-T/t-antigens induce cellular transformation in large part through its ability to bind to and inactivate p53 and Rb, two tumor suppressor genes critical for cell cycle regulation and genome stability. SV40-T/t-antigen binds p53, functionally inactivating it, as well as sequestering the tumor suppressor gene from degradation, thus increasing its expression in the nucleus Likewise, it also binds to unphosphorylated Rb, sequestering the active form from binding to E2F, thus abrogating the cell cycle checkpoint (Ali and DeCaprio 2001
). A significant portion of human breast and many other human cancers have been shown to harbor mutations that effectively inactivate or mutate these tumor suppressor genes leading to abnormalities in cell cycle control, proliferation, and apoptosis (Ludlow 1993
; Nielsen et al. 1997
; Troester et al. 2006
). In the present study, transgenic rats were produced that developed mammary carcinomas recapitulating important features of triple-negative or Her2-positive tumors.
Although the pathogenesis of breast cancer remains poorly understood, gene expression profiling has identified at least five basic molecular subtypes of human breast cancer: basal, Her2 positive, luminal A, luminal B, and normal breast subtypes (Dabbs et al. 2006
; Perou et al. 1999
; Sorlie et al. 2001
). The basal subtype in particular has been shown to be associated with hormone (ERα
-, PR-) independence, negative Her2/neu (c-ErbB2) status, and a poor prognosis (Dabbs et al. 2006
; Doane et al. 2006
; Skoog et al. 1987
). These tumors express markers present on normal basal cells (or myoepithelial cells) of the breast, including CK5/6, CK14, CK17, vimentin, and Her-1 (Dabbs et al. 2006
; Gusterson et al. 2005
; Livasy et al. 2006
; Nielsen et al. 2004
; Ribeiro-Silva et al. 2005
). Although molecular categorization of the heterogeneous collection of breast cancers has been integrated into many articles, this categorization remains controversial. For example, some tumors that cluster within the Her2 type of breast cancers may lack Her2 protein expression, and ER and Her2 type breast cancers often fall into the luminal B subgroup (Weigelt and Reis-Filho 2009
). Furthermore, the use of cytokeratins to characterize the origins of human breast cancer has suggested that basal breast cancers are positive for CK14, CK17, and CK5, and negative for the luminal markers CK8, CK18, and CK19. However, both luminal and basal markers may be expressed in some human breast cancers (Gusterson 2009
; Gusterson et al. 2005
). Normal rat mammary epithelium in this model consistently expressed CK18, and was negative for CK14, consistent with a previous report (Dundas et al. 1991
). However, given that it is found in the human TDLU (Gusterson 2009
; Gusterson et al. 2005
), this could be reexamined in the rat.
The rat model recapitulated several important aspects of human breast cancer not generally observed in the C3(1)/Tag mouse model. In human breast cancer, as part of the multifactorial Nottingham prognostic index, histologic phenotype often correlates with malignant behavior and prognosis (Ellis et al. 1992
; Elston and Ellis 2002
). In the mouse model, tumors are often tubular adenocarcinomas, the most common human histologic subtype (Shibata et al. 1998
). However, in the rat model, several histologic subtypes of mammary tumors were more readily observed ranging from tubular or tubuloacinar adenocarcinoma to scirrhous carcinoma, a tumor type in human breast cancer that has a poor prognosis (Kitagawa et al. 2004
; Nozoe et al. 2007
) with a prominent stromal component. These data indicate that the rat model may recapitulate more phenotypic heterogeneity as seen in human breast cancer.
Secondly and most importantly, tumors from the rat model recapitulate the immunophenotypes of triple negative (ER–, PR–, Her2–) or Her2-positive cancers observed in sub-sets of human breast cancers. Although several GEM models including the Brca+/−
, and some chemically induced tumor models display basal-type characteristics and certain molecular features similar to the human basal-type, triple-negative tumors (Bennett and Green 2008
; Deeb et al. 2007
; Herschkowitz et al. 2007
; Xu et al. 1999
), the C3(1)/Tag mouse mammary cancer model was identified as having the strongest molecular relationships to human basal-type, triple-negative breast cancer (Deeb et al. 2007
; Herschkowitz et al. 2007
). Although expression profiling could not be performed on the rat tumors, it seems likely that they share similar molecular signatures of the triple-negative tumors as documented for the mouse model.
The rat mammary tumors were also consistently negative for α
SMA and multifocally positive for CK18, a luminal marker. Although these tumors as a group expressed markers of both luminal and basal phenotypes, it must be remembered that the basal immunophenotype and triple negative status are not synonymous. Approximately 20% of human triple-negative breast cancers express luminal markers. Triple negative status is based on clinical assays for ER, PR, and Her2 in human breast cancer, whereas the basal phenotype has been primarily based upon gene expression profiling (Anders and Carey 2008
). While most triple negative tumors have a basal phenotype, and most basal breast cancers are hormone receptor and HER2 negative, a proportion of triple negative do not express basal markers, and alternatively, some basal breast cancers are hormone receptor positive (Bertucci et al. 2008
; Rakha et al. 2008
). Therefore, in human triple negative breast cancer, there is significant overlap between biological and clinical characteristics of sporadic triple-negative and basal-like cancers (Reis-Filho and Tutt 2008
). Furthermore, basal-type carcinomas may express α
SMA by immunohistochemistry, and this is more frequently associated with development of in situ carcinoma (Lerma et al. 2007
). Additionally, Brcaco/co
mouse model tumors are generally considered basal tumors, although some luminal marker expression can be observed (Herschkowitz et al. 2007
), as we have observed in the rat tumors. Alternatively, the tumors may represent triple negative tumors that possess neither a distinct luminal nor basal immuno-phenotype. Importantly, early precancerous lesions of ADH and DCIS were negative for ERα
and PR, in contrast to the mouse model, which often has hormone receptor positive precancerous lesions that gradually lose ERα
immunoreactivity as they progress to carcinoma (Yoshidome et al. 1998
). Histologic types of triple negative breast cancers are similar to those seen in basal breast cancer, most commonly infiltrating ductal carcinoma (Sasaki and Tsuda 2009
; Thike et al. 2010
). Most basal-type tumors are characterized by solid architecture, pushing margins, central to comedo-type necrosis, cellular pleomorphism with poorly differentiated nuclear features, high nuclear-cytoplasmic ratio and mitotic index, and scant stromal content, and central geographic or comedo-type necrosis (Banerjee et al. 2006
; Dabbs et al. 2006
; Rakha et al. 2008
) The tumors in the C3(1)/Tag transgenic rat were characterized by a variable histologic phenotype, but contained large solid areas consistent with features of the basal subtype noted above.
Although expression of the SV40 T/t-antigen transgene using the C3(1) promoter consistently produced mammary precancerous lesions and carcinomas in rats, several other lesions related to transgene expression were also noted. These included atypical hyperplasias (parotid salivary gland, nasal submucosal glands, and Harderian gland) and tumors (pituitary adenomas, pancreatic islet cell carcinomas, thyroid follicular carcinoma and parotid salivary carcinoma) in very young animals. Lesions such as pituitary adenomas and islet cell tumors, otherwise considered age-related background pathology in the rat, occurred in animals at 3–4 months of age. This rapid onset of mammary and non-mammary neoplasia led to the early death and poor reproductive performance of the founder lines and their offspring.
The presence of these non-mammary lesions indicates that the 5′ flanking region of the C3(1) promoter is able to direct significant levels of expression of the SV40 early region to multiple epithelial tissues in the rat in a manner more promiscuous than when introduced into the mouse germline. It is important to note that prostatic precancerous lesions and neoplasia, common in the mouse model, were not present in the rat model at the time of their early death. It is possible that prostatic lesions may have developed had the rats survived longer. Alternatively, although the C3(1) gene is an endogenous rat gene expressed at extremely high levels in the rat prostate, a critical androgen response element contained within the first intron of the promoter was excised during the construction of the transgene, likely resulting in the loss of expression in the rat prostate (Maroulakou et al. 1994
). It remains possible that additional hormone and/or prostate-specific regulatory elements not contained within the transgenic construct resulted in the lack of prostate expression. Therefore, the 5′ flanking region of the C3(1) promoter appears to contain regulatory elements that specify more general epithelial expression.
In summary, by utilizing the rat C3(1) promoter to drive the expression of SV40 T/t-antigens, we developed transgenic rats that recapitulated the multi-step progression of mammary carcinoma from pre-invasive through invasive carcinoma with features of triple negative breast and Her-positive basal breast cancer. Triple-negative breast cancer in particular remains a form of the disease for which few extremely effective therapies exist and for which improved pre-clinical models are very much needed. As the characterization of triple negative breast cancer is based on the immunophenotype of ER, PR, Her2 negative, and the basal phenotype has been based upon gene expression profiling, these tumor types may overlap but are not synonymous. And while it is controversial in terms of how these classifications may change clinical management of these types of breast cancer, it is hoped that through the use of models such as the C3(1)/Tag transgenic rat, further insights on potential therapeutic molecular targets may lead to more personalized medicine to combat this complex disease.
Unfortunately, the transgene used in this study resulted in very high levels of expression in the mammary glands leading to aggressive tumors developing in young animals that were subsequently unable to generate offspring to allow for the propagation of this model. However, it may be possible to alter the promoter region in future iterations to potentially reduce promiscuous transgene expression and increase mammary specificity. An additional possibility would be to directly target the mammary ductular epithelium with an SV40 Tag-expressing construct through the utilization of mammary duct infusion techniques developed by Gould et al. (1991) using retroviral vectors (Wang et al. 1991
). This technique would circumvent many of the problems associated with pronuclear injection (Smits et al. 2007
). Given the potential advantages of a rat model of triple-negative mammary cancer compared to existing mouse models, this would be an important step forward in studying the biology of triple-negative tumors.