Mouse Model Of Mammary Tumorigenesis Research Articles

Transcriptome analyses of mouse and human mammary cell subpopulations reveal multiple conserved genes and pathways

IntroductionMolecular characterization of the normal epithelial cell types that reside in the mammary gland is an important step toward understanding pathways that regulate self-renewal, lineage commitment, and differentiation along the hierarchy. Here we determined the gene expression signatures of four distinct subpopulations isolated from the mouse mammary gland. The epithelial cell signatures were used to interrogate mouse models of mammary tumorigenesis and to compare with their normal human counterpart subsets to identify conserved genes and networks.MethodsRNA was prepared from freshly sorted mouse mammary cell subpopulations (mammary stem cell (MaSC)-enriched, committed luminal progenitor, mature luminal and stromal cell) and used for gene expression profiling analysis on the Illumina platform. Gene signatures were derived and compared with those previously reported for the analogous normal human mammary cell subpopulations. The mouse and human epithelial subset signatures were then subjected to Ingenuity Pathway Analysis (IPA) to identify conserved pathways.ResultsThe four mouse mammary cell subpopulations exhibited distinct gene signatures. Comparison of these signatures with the molecular profiles of different mouse models of mammary tumorigenesis revealed that tumors arising in MMTV-Wnt-1 and p53-/- mice were enriched for MaSC-subset genes, whereas the gene profiles of MMTV-Neu and MMTV-PyMT tumors were most concordant with the luminal progenitor cell signature. Comparison of the mouse mammary epithelial cell signatures with their human counterparts revealed substantial conservation of genes, whereas IPA highlighted a number of conserved pathways in the three epithelial subsets.ConclusionsThe conservation of genes and pathways across species further validates the use of the mouse as a model to study mammary gland development and highlights pathways that are likely to govern cell-fate decisions and differentiation. It is noteworthy that many of the conserved genes in the MaSC population have been considered as epithelial-mesenchymal transition (EMT) signature genes. Therefore, the expression of these genes in tumor cells may reflect basal epithelial cell characteristics and not necessarily cells that have undergone an EMT. Comparative analyses of normal mouse epithelial subsets with murine tumor models have implicated distinct cell types in contributing to tumorigenesis in the different models.

Igf1r as a therapeutic target in a mouse model of basal-like breast cancer

Considering the strong association between dysregulated insulin-like growth factor (IGF) signaling and various human cancers, we have used an expedient combination of genetic analysis and pharmacological treatment to evaluate the potential of the type 1 IGF receptor (Igf1r) for targeted anticancer therapy in a mouse model of mammary tumorigenesis. In this particular strain of genetically modified animals, histopathologically heterogeneous invasive carcinomas exhibiting up-regulation of the Igf1r gene developed extremely rapidly by mammary gland-specific overexpression of constitutively active oncogenic Kras* (mutant Kras(G12D)). Immunophenotyping data and expression profiling analyses showed that, except for a minor luminal component, these mouse tumors resembled basal-like human breast cancers. This is a group of aggressive tumors of poor prognosis for which there is no targeted therapy currently available, and it includes a subtype correlating with KRAS locus amplification. Conditional ablation of Igf1r in the mouse mammary epithelium increased the latency of Kras*-induced tumors very significantly (approximately 11-fold in comparison with the intact model), whereas treatment of tumor-bearing animals by administration of picropodophyllin (PPP), a specific Igf1r inhibitor, resulted in a dramatic decrease in tumor mass of the main forms of basal-like carcinomas. PPP also was effective against xenografts of the human basal-like cancer cell line MDA-MB-231, which carries a KRAS(G13D) mutation.

The Mammary Progenitor Marker CD61/β3 Integrin Identifies Cancer Stem Cells in Mouse Models of Mammary Tumorigenesis

The cells of origin and mechanisms that underpin tumor heterogeneity in breast cancer are poorly understood. Here, we have examined three mouse models of mammary tumorigenesis (MMTV-wnt-1, MMTV-neu, and p53(+/-)) for changes in their epithelial cell hierarchy during the preneoplastic and neoplastic stages of tumor progression. In preneoplastic tissue, only MMTV-wnt-1 mice showed a perturbation in their epithelial subpopulations. In addition to an expanded mammary stem cell pool, repopulating cells capable of yielding extensive mammary outgrowths in vivo were revealed in the committed luminal progenitor population. These findings indicate that wnt-1 activation induces the appearance of aberrant progenitor cells, and suggest that both mammary stem and progenitor cells can serve as the cellular targets of wnt-1-induced tumorigenesis. In tumors arising in MMTV-wnt-1 tumors, the luminal epithelial progenitor marker CD61/beta3 integrin identified a cancer stem cell (CSC) population that was highly enriched for tumorigenic capability relative to the CD61(-) subset. CD61 expression also defined a CSC subset in 50% of p53(+/-)-derived tumors. No CSCs, however, could be identified in the more homogeneous MMTV-neu/erbB2 model, suggesting an alternative model of tumorigenesis. Overall, our findings show the utility of the progenitor marker CD61 in the identification of CSCs that sustain specific mammary tumors.

Early neoplastic and metastatic mammary tumours of transgenic mice detected by 5-aminolevulinic acid-stimulated protoporphyrin IX accumulation

A photodynamic technique for human breast cancer detection founded upon the ability of tumour cells to rapidly accumulate the fluorescent product protoporphyrin IX (PpIX) has been applied to transgenic mouse models of mammary tumorigenesis. A major goal of this investigation was to determine whether mouse mammary tumours are reliable models of human disease in terms of PpIX accumulation, for future mechanistic and therapeutic studies. The haeme substrate 5-aminolevulinic acid (5-ALA) (200 mg kg−1) was administered to mouse strains that develop mammary tumours of various histological subtypes upon expression of the transgenic oncogenes HRAS, Polyoma Virus middle T antigen, or Simian Virus 40 large T antigen in the mammary gland. Early neoplastic lesions, primary tumours and metastases showed consistent and rapid PpIX accumulation compared to the normal surrounding tissues, as evidenced by red fluorescence (635 nm) when the tumours were directly illuminated with blue light (380–440 nm). Detection of mouse mammary tumours at the stage of ductal carcinoma in situ by red fluorescence emissions suggests that enhanced PpIX synthesis is a good marker for early tumorigenic processes in the mammary gland. We propose the mouse models provide an ideal experimental system for further investigation of the early diagnostic and therapeutic potential of 5-ALA-stimulated PpIX accumulation in human breast cancer patients.

Dietary Depletion of Vitamin E and Vitamin A Inhibits Mammary Tumor Growth and Metastasis in Transgenic Mice

We showed previously that dietary antioxidant depletion enhances tumor reactive oxygen species (ROS) and apoptosis, resulting in a reduction in brain tumor size in the TgT(121) transgenic mouse model, a nonmetastatic tumor model. Here, in a transgenic mouse model of mammary tumorigenesis with defined rates of tumor growth and lung-targeted metastasis, we determined the ability of dietary antioxidant depletion to inhibit tumor growth and metastasis. Compared with control mice fed a standard diet, antioxidant-depleted mice exhibited tumor-targeted generation of ROS manifested by increased levels of oxidatively modified DNA/RNA (8- hydroxy-2'-deoxyguanine, 8-hydroxyguanine) and lipid peroxidation (4-hydroxy-2-nonenal) in primary and metastatic tumor foci. In addition to increased tumor-targeted ROS, the number of apoptotic cells was increased approximately 500% (P < 0.01) and terminal dUTP nucleotide DNA end-labeling-positive cells 200% (P < 0.01) in mice fed the antioxidant-depleted diet, whereas the percentage of tumor cells undergoing mitosis was >50% lower than in controls (P < 0.01). The proportional distribution of small (<1.5 cm) and large (> or = 1.5 cm) primary mammary tumors differed. The mice fed the antioxidant-depleted diet had more small primary tumors (P <0.05) and fewer large primary tumors (P < 0.05). Importantly, they also had fewer lung metastatic tumor foci compared with mice fed the control diet (4.5 +/- 1.3 vs. 15.8 +/- 8.5 foci/lung, P < 0.01). These findings may be important in understanding the role of dietary antioxidant vitamins in tumor growth and metastasis.

The Splicing Factor U1C Represses EWS/FLI-mediated Transactivation

EWS is an RNA-binding protein involved in human tumor-specific chromosomal translocations. In approximately 85% of Ewing's sarcomas, such translocations give rise to the chimeric gene EWS/FLI. In the resulting fusion protein, the RNA binding domains from the C terminus of EWS are replaced by the DNA-binding domain of the ETS protein FLI-1. EWS/FLI can function as a transcription factor with the same DNA binding specificity as FLI-1. EWS and EWS/FLI can associate with the RNA polymerase II holoenzyme as well as with SF1, an essential splicing factor. Here we report that U1C, one of three human U1 small nuclear ribonucleoprotein-specific proteins, interacts in vitro and in vivo with both EWS and EWS/FLI. U1C interacts with other splicing factors and is important in the early stages of spliceosome formation. Importantly, co-expression of U1C represses EWS/FLI-mediated transactivation, demonstrating that this interaction can have functional ramifications. Our findings demonstrate that U1C, a well characterized splicing protein, can also function in transcriptional regulation. Furthermore, they suggest that EWS and EWS/FLI may function both in transcriptional and post-transcriptional processes.

Amplification of the neu/erbB-2 oncogene in a mouse model of mammary tumorigenesis

The neu (c-erbB-2, Her-2) protooncogene is amplified and overexpressed in 20-30% of human breast cancers. Although transgenic mouse models have illustrated the role of Neu in the induction of mammary tumors, Neu expression in these models is driven by a strong viral promoter of questionable relevance to the human disease. To ascertain whether expression of activated Neu under the control of the endogenous promoter in the mammary gland could induce mammary tumors we have generated mice that conditionally express activated Neu under the transcriptional control of the intact endogenous Neu promoter. Expression of oncogenic neu in the mammary gland resulted in accelerated lobulo-alveolar development and formation of focal mammary tumors after a long latency period. However, expression of activated Neu under the normal transcriptional control of the endogenous promoter was not sufficient for the initiation of mammary carcinogenesis. Strikingly, all mammary tumors bear amplified copies (2-22 copies) of the activated neu allele relative to the wild-type allele and express highly elevated levels of neu transcript and protein. Thus, like human erbB-2-positive breast tumors, mammary tumorigenesis in this mouse model requires the amplification and commensurate elevated expression of the neu gene.

Chemical carcinogen-induced tumorigenesis in parous, involuted mouse mammary glands.

Chemical carcinogen-induced tumorigenesis in parous, involuted mouse mammary glands.

Expression of v-src in mammary epithelial cells induces transcription via STAT3

Transgenic mouse models of mammary tumorigenesis and analyses of human breast tumour samples have indicated a role for Src proteins in the tumorigenic process. The downstream effectors of Src function in mammary epithelial cells are less well understood. STAT proteins constitute a family of transcription factors whose activation by cytokine and non-cytokine receptors leads to tyrosine phosphorylation, dimerization and translocation from the cytoplasm to the nucleus. In the nucleus they activate the transcription of specific genes by binding to consensus DNA elements. STATs 1 and 3 can be activated by both cytokine and non-cytokine receptors, and bind as homodimers or heterodimers to viral simian sarcoma virus (sis)-inducible elements such as that found in the c-fos promoter. Here we report that one of the downstream effectors of Src function in mammary epithelial cells is STAT3. We demonstrate that v-src expression in mammary epithelial cells induces Tyr-705 phosphorylation, nuclear translocation and DNA binding of STAT3. Furthermore, we demonstrate that v-src can induce STAT3-dependent transcription. These observations are the first direct evidence that v-src can regulate transcription through the activation of STAT proteins, and add a further level of complexity to the understanding of the mode of action of v-src.