Transgenic overexpression of IGF-IR disrupts mammary ductal morphogenesis and induces tumor formation

Overexpression and hyperactivation of the type I insulin-like growth factor receptor (IGF-IR) has been observed in human breast tumor biopsies. In addition, in vitro studies indicate that overexpression of IGF-IR is sufficient to transform cells such as mouse embryo fibroblasts and this receptor promotes proliferation and survival in breast cancer cell lines. To fully understand the function of the IGF-IR in tumor initiation and progression, transgenic mice containing human IGF-IR under a doxycycline-inducible MMTV promoter system were generated. Administration of 2 mg/ml doxycycline in the animals' water supply beginning at 21 days of age resulted in elevated levels of IGF-IR in mammary epithelial cells as detected by Western blotting and immunohistochemistry. Whole mount analysis of 55-day-old mouse mammary glands revealed that IGF-IR overexpression significantly impaired ductal elongation. Moreover, histological analyses revealed multiple hyperplasic lesions in the mammary glands of these 55-day-old mice. The formation of palpable mammary tumors was evident at approximately 2 months of age and was associated with increased levels of IGF-IR signaling molecules including phosphorylated Akt, Erk1/Erk2 and STAT3. Therefore, these transgenic mice provide evidence that IGF-IR overexpression is sufficient to induce mammary epithelial hyperplasia and tumor formation in vivo and provide a model to further understand the function of IGF-IR in mammary epithelial transformation.     

Geminin overexpression induces mammary tumors via suppressing cytokinesis

Aneuploidy plays an important role in the development of cancer. Here, we uncovered an oncogenic role for geminin in mitotic cells. In addition to chromatin, tyrosine phosphorylated geminin also localizes to centrosome, spindle, cleavage furrow and midbody during mitosis. Geminin binding to Aurora B prevents its binding to INCENP, and thus activation leading to lack of histone H3-(serine 10) phosphorylation, chromosome condensation failure, aborted cytokinesis and the formation of aneuploid, drug resistance cells. Geminin overexpressing human mammary epithelial cells form aneuploid, aggressive tumors in SCID mice. Geminin is overexpressed in more than half of all breast cancers analyzed. The current study reveals that geminin is a genuine oncogene that promotes cytokinesis failure and production of aneuploid, aggressive breast tumors when overexpressed and thus a worthy therapeutic target (oncotarget) for aggressive breast cancer.     

Mammary-specific Ron receptor overexpression induces highly metastatic mammary tumors associated with beta-catenin activation

Activated growth factor receptor tyrosine kinases (RTK) play pivotal roles in a variety of human cancers, including breast cancer. Ron, a member of the Met RTK proto-oncogene family, is overexpressed or constitutively active in 50% of human breast cancers. To define the significance of Ron overexpression and activation in vivo, we generated transgenic mice that overexpress a wild-type or constitutively active Ron receptor in the mammary epithelium. In these animals, Ron expression is significantly elevated in mammary glands and leads to a hyperplastic phenotype by 12 weeks of age. Ron overexpression is sufficient to induce mammary transformation in all transgenic animals and is associated with a high degree of metastasis, with metastatic foci detected in liver and lungs of >86% of all transgenic animals. Furthermore, we show that Ron overexpression leads to receptor phosphorylation and is associated with elevated levels of tyrosine phosphorylated beta-catenin and the up-regulation of genes, including cyclin D1 and c-myc, which are associated with poor prognosis in patients with human breast cancers. These studies suggest that Ron overexpression may be a causative factor in breast tumorigenesis and provides a model to dissect the mechanism by which the Ron induces transformation and metastasis.     

Identification and testing of a gene expression signature of invasive carcinoma cells within primary mammary tumors

We subjected cells collected using an in vivo invasion assay to cDNA microarray analysis to identify the gene expression profile of invasive carcinoma cells in primary mammary tumors. Expression of genes involved in cell division, survival, and cell motility were most dramatically changed in invasive cells indicating a population that is neither dividing nor apoptotic but intensely motile. In particular, the genes coding for the minimum motility machine that regulates beta-actin polymerization at the leading edge and, therefore, the motility and chemotaxis of carcinoma cells, were dramatically up-regulated. However, ZBP1, which restricts the localization of beta-actin, the substrate for the minimum motility machine, was down-regulated. This pattern of expression implicated ZBP1 as a suppressor of invasion. Reexpression of ZBP1 in metastatic cells with otherwise low levels of ZBP1 reestablished normal patterns of beta-actin mRNA targeting and suppressed chemotaxis and invasion in primary tumors. ZBP1 reexpression also inhibited metastasis from tumors. These experiments support the involvement in metastasis of the pathways identified in invasive cells, which are regulated by ZBP1.     

Aurora A overexpression induces cellular senescence in mammary gland hyperplastic tumors developed in p53-deficient mice

Aurora A mitotic kinase is frequently overexpressed in various human cancers and is widely considered to be an oncoprotein. However, the cellular contexts in which Aurora A induces malignancy in vivo are still unclear. We previously reported a mouse model in which overexpression of human Aurora A in the mammary gland leads to small hyperplastic changes but not malignancy because of the induction of p53-dependent apoptosis. To study the additional factors required for Aurora A-associated tumorigenesis, we generated a new Aurora A overexpression mouse model that lacks p53. We present evidence here that Aurora A overexpression in primary mouse embryonic fibroblasts (MEFs) that lack p53 overrides postmitotic checkpoint and leads to the formation of multinucleated polyploid cells. Induction of Aurora A overexpression in the mammary glands of p53-deficient mice resulted in development of precancerous lesions that were histologically similar to atypical ductal hyperplasia in human mammary tissue and showed increased cellular senescence and p16 expression. We further observed DNA damage in p53-deficient primary MEFs after Aurora A overexpression. Our results suggest that Aurora A overexpression in mammary glands is insufficient for the development of malignant tumors in p53-deficient mice because of the induction of cellular senescence. Both p53 and p16 are critical in preventing mammary gland tumorigenesis in the Aurora A overexpression mouse model.     

Biochemical characteristics of mouse mammary tissues, preneoplastic lesions and tumors

A number of metabolizing systems are measured in normal, preneoplasticand neoplastic mouse mammary tissues derived under three differentconditions. These biochemical functions are considered to beimportant in the activation and detoxi fication of carcinogensand other xenobiotics and have been linked to the process ofrat liver hepatocarcinogenesis. The cytochrome P450-dependentenzyme aminopyrine N-de methylase, consistently depressed inhepatocarcinogenesis models in mouse and rat, does not showa significant change among normal, preneoplastic and neoplasticmammary tissues. Glutathione and the enzymes of glutathionemetab olism and utilization (e.g. glutathione-S-transferasesand -glutamyl transferase), active in the detoxification ofxenobiotics, show no significant differences in carcinogen-inducedtumors or in their homologous preneoplasias compared to controltissue. There is no increase in the anionic glutathione-S-transferase,a principal marker in rat hepato carcinogenesis. The only observedbiochemical change was a significant decrease in -glutamyl cysteinesynthetase the glutathione synthetic enzyme, in the carcinogen-inducedpre neoplastic and neoplastic line compared to control. Alsoinorganic glutathione peroxidase was lower in the preneo plastic,but not in the neoplastic tissues.     

The chemopreventive effect of mifepristone on mammary tumorigenesis is associated with an anti-invasive and anti-inflammatory gene signature

Progesterone receptor (PR) antagonists are potent antitumor agents in carcinogen and progestin-dependent mammary tumorigenesis models through both PR- and non-PR-mediated mechanisms. The PR antagonist mifepristone/RU486 has been used primarily as an abortifacient possessing high affinity for both the PR and glucocorticoid receptors (GR). To determine whether mifepristone would be effective as a chemopreventive agent, we assessed its effect on progestin/7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in wild-type (WT) and estrogen receptor-α-positive (ER(+)) transgenic mice expressing the dominant-negative Pax8PPARγ (Pax8) fusion protein. Mifepristone administered at a dose of 2.5 mg significantly delayed mammary tumorigenesis in WT, but not in Pax8 mice, whereas, a three-fold higher dose almost completely blocked tumorigenesis in both WT and Pax8 mice. The sensitivity of WT mice to 2.5 mg mifepristone correlated with an expression profile of 79 genes in tumors, 52 of which exhibited the opposite response in Pax8 mice, and corresponded primarily to the downregulation of genes associated with metabolism, inflammation, and invasion. These results suggest that the chemopreventive activity of mifepristone in WT mice correlates with a specific gene expression signature that is associated with multiple nuclear receptor signaling pathways.     

Morphology of MXT mouse mammary tumors. Correlation with growth characteristics and hormone sensitivity

The transplantable MXT mouse mammary tumor has been a useful tool for studying endocrine mechanisms underlying mammary tumor growth. It is our experience, however, that this model is unstable during serial transplantation. This paper analyses this variability from the viewpoints of histology and estrogen receptor content and indicates that these parameters should always be checked before planning experimental work. It is advised that a more homogeneous material is needed and that this goal should be achieved by clonal selection before transplantation.     

C-kit overexpression correlates with KIT gene copy numbers increases in phyllodes tumors of the breast

Breast Cancer Research and Treatment - We determined c-kit expression in the stroma and epithelia of benign, borderline, and malignant phyllodes tumors (PTs), respectively, as well as the...     

Intraductally administered pegylated liposomal doxorubicin reduces mammary stem cell function in the mammary gland but in the long term, induces malignant tumors

Previously, we have shown that the intraductal (i.duc) administration of pegylated liposomal doxorubicin (PLD) to Her2/neu transgenic mice is associated with mammary tumor regression and prevention. Exploring the mechanism underlying the protection afforded by PLD, we studied: the effects of i.duc PLD-treatment with a subsequent pregnancy on outgrowth of tumors in Her2/neu mice; whether the i.duc PLD antitumor effect was mediated partially through changes in normal mammary stem cells (MaSCs); and the long-term safety of i.duc PLD into the normal mouse mammary gland. Her2/neu mice were treated with two i.duc injections of PLD given four weeks apart; pregnancy was induced and mice were followed up for changes in physiology, and tumor formation. We found that all pups born to i.duc PLD-treated Her2/neu mice died without weight gain within 7 days after birth. Despite an additional pregnancy, compared to vehicle control PLD-treated Her2/neu mice had a significantly longer latency and lower frequency of tumor development. Mammary epithelial cells isolated from untreated and i.duc PLD-treated 6–8 months-old multiparous FVB/N mice were analyzed for their repopulating ability in mammary fat pads of naïve recipients. Mice were also monitored for abnormalities in mammary gland morphology and function, including tumor formation. PLD-treated FVB/N mice displayed histomorphologic changes and a significant reduction in the outgrowth potential of cells from the mammary glands. Thus, i.duc PLD administration altered the mammary gland structurally and functionally by reducing the MaSC population, which could compromise milk production. Followed long term, i.duc PLD-treated FVB/N mice developed malignant mammary tumors, confirming similar published findings on doxorubicin injected into the mammary gland of rats. Unless there are fundamental species differences in PLD metabolism in rodents and humans, this finding seriously limits the consideration of i.duc PLD use in the clinic for treatment or prevention of breast cancer.     

Cutaneous human papillomaviruses down-regulate AKT1, whereas AKT2 up-regulation and activation associates with tumors

Epithelial tumorigenesis has been linked to AKT up-regulation. Human papillomaviruses (HPV) cause anogenital cancers and anogenital HPV infection up-regulates AKT activity. Mounting evidence points to a role for cutaneous HPVs as etiologic factors in skin tumorigenesis. High-risk cutaneous beta HPVs have been linked to carcinogenesis in immunosuppressed patients, and high-risk cutaneous HPV8 genes enhance tumorigenesis in transgenic mice. We find that, in contrast to anogenital HPVs, cutaneous HPV8 early genes down-regulate epidermal AKT activity by down-regulating AKT1 isoform levels. This down-regulation occurs before papilloma formation or tumorigenesis and leads to cutaneous differentiation changes that may weaken the epidermal squame for viral release. We find that, in viral warts (papillomas) and HPV gene-induced epidermal tumors, AKT activity can be activated focally by up-regulation and phosphorylation of the AKT2 isoform. In squamous cell carcinomas (SCC), AKT1 down-regulation is also common, consistent with a viral influence, whereas AKT2 up-regulation is widespread. Activation of up-regulated AKT2 by serine phosphorylation associates with high-grade tumors. Our data suggest that AKT2 up-regulation is characteristic of SCC and that coincident AKT2 activation through serine phosphorylation correlates with malignancy. These findings highlight differences between the effects of anogenital and cutaneous HPV on epithelial AKT activity and furthermore show that AKT isoforms can behave differently during epidermal tumorigenesis. These findings also suggest AKT2 as a possible therapeutic tumor target in SCC.     

p16(Ink4a) overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors

p16(Ink4a) is a protein involved in regulation of the cell cycle. Currently, p16(Ink4a) is considered a tumor suppressor protein because of its physiological role and downregulated expression in a large number of tumors. Intriguingly, overexpression of p16(Ink4a) has also been described in several tumors. This review attempts to elucidate when and why p16(Ink4a) overexpression occurs, and to suggest possible implications of p16(Ink4a) in the diagnosis, prognosis and treatment of cancer.     

Bifidobacterium longum as a delivery system for gene therapy of chemically induced rat mammary tumors

A fundamental obstacle in cancer gene therapy is the specific targeting of therapy directly to a solid tumor, and no systemic delivery system yet exists. A strain of domestic bacteria, Bifidobacteriumlongum, which is nonpathogenic and anaerobic, selectively localized to and proliferated in 7,12dimethylbenz[a]anthraceneinduced rat mammary tumors after systemic application. We further ascertained the tumor specificity of genetically engineered, as well as wildtype, Bifidobacterium longum. This is the first demonstration that Bifidobacterium longum can be utilized as a specific gene delivery vector for gene therapy on solid breast tumors.     

High incidence of mammary tumors in mice with inherited asplenia carriers for the nude gene.

A colony of mice suffering from dominant hemimelia associated with agenesis of the spleen has been developed and characterized during the past 7 years. The hereditarily asplenic (Dh/+) mice have a very low incidence (9%) of spontaneous mammary tumors (SMT). Asplenic (Dh/+) females were mated with mice homozygous (nu/nu) for hereditary athymia (nude) having a BALB/c background. BALB/c females heterozygous for the nu gene and with spleen (nu/+,+/+) have a moderate incidence (12%) of SMT, whereas nu/+,Dh/+ breeders have a drastic increase in the incidence of SMT to 46% when bred under identical conditions. Since all parent strains have a very low incidence of SMT, it appears that the spleen agenesis is a major factor accounting for an earlier and higher incidence of SMT in hereditarily asplenic (nu/+,Dh/+) mice than in normal (nu/+,+/+) siblings. The SMT express mammary tumor virus antigen(s) and possess estrogen, progesterone, and glucocorticoid receptors. The SMT rapidly metastasize and kill the host within 30 to 45 days. The BALB/c asplenic mice with SMT represent a unique model relevant to human breast cancer and for study of the function of the spleen in the development of solid tumors in general and of SMT in particular.     

Hormone-induced protection against mammary tumorigenesis is conserved in multiple rat strains and identifies a core gene expression signature induced by pregnancy

Women who have their first child early in life have a substantially lower lifetime risk of breast cancer. The mechanism for this is unknown. Similar to humans, rats exhibit parity-induced protection against mammary tumorigenesis. To explore the basis for this phenomenon, we identified persistent pregnancy-induced changes in mammary gene expression that are tightly associated with protection against tumorigenesis in multiple inbred rat strains. Four inbred rat strains that exhibit marked differences in their intrinsic susceptibilities to carcinogen-induced mammary tumorigenesis were each shown to display significant protection against methylnitrosourea-induced mammary tumorigenesis following treatment with pregnancy levels of estradiol and progesterone. Microarray expression profiling of parous and nulliparous mammary tissue from these four strains yielded a common 70-gene signature. Examination of the genes constituting this signature implicated alterations in transforming growth factor-beta signaling, the extracellular matrix, amphiregulin expression, and the growth hormone/insulin-like growth factor I axis in pregnancy-induced alterations in breast cancer risk. Notably, related molecular changes have been associated with decreased mammographic density, which itself is strongly associated with decreased breast cancer risk. Our findings show that hormone-induced protection against mammary tumorigenesis is widely conserved among divergent rat strains and define a gene expression signature that is tightly correlated with reduced mammary tumor susceptibility as a consequence of a normal developmental event. Given the conservation of this signature, these pathways may contribute to pregnancy-induced protection against breast cancer.     

Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling

We have developed animal models of breast cancer that allow the direct examination of the behavior of individual green fluorescent protein-expressing carcinoma cells in live nonmetastatic and metastatic primary tumors in situ. We have combined this model with multiphoton microscopy to image differences in cell behavior within the primary tumor. Differences in cell behavior between nonmetastatic and metastatic cells in culture and within live primary tumors were correlated with results from cDNA microarray analyses to identify potentially important genetic determinants for breast cancer invasion and metastasis. Using multiphoton microscopy, we found five major differences in carcinoma cell behavior between the nonmetastatic and metastatic primary breast tumors involving extracellular matrix, cell motility, and chemotaxis. Behavioral differences were correlated with seven categories of molecules that were differentially expressed and related to these behaviors. We have found that extracellular matrix composition, actin nucleation factors, molecules involved in mechanical stability and survival, and cell polarity and chemotaxis showed large and consistent differences in gene expression. We conclude that aligning cell behavior in vivo with patterns of gene expression can lead to new insights into the microenvironment of carcinoma cells in the primary tumor and the molecular mechanisms behind cell behavior.