William J. Muller

Transforming growth factor beta signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis.

The influence of transforming growth factor β (TGF-β) signaling on Neu-induced mammary tumorigenesis and metastasis was examined with transgenic mouse models. We generated mice expressing an activated TGF-β type I receptor or dominant negative TGF-β type II receptor under control of the mouse mammary tumor virus promoter. When crossed with mice expressing activated forms of the Neu receptor tyrosine kinase that selectively couple to the Grb2 or Shc signaling pathways the activated type I receptor increased the latency of mammary tumor formation but also enhanced the frequency of extravascular lung metastasis. Conversely, expression of the dominant negative type II receptor decreased the latency of Neu-induced mammary tumor formation while significantly reducing the incidence of extravascular lung metastases. These observations argue that TGF-β can promote the formation of lung metastases while impairing Neu-induced tumor growth and suggest that extravasation of breast cancer cells from pulmonary vessels is a point of action of TGF-β in the metastatic process.

The phosphatidyl inositol 3-kinase signaling network: implications for human breast cancer

The phosphatidyl inositol 3-kinase (PI3K)/Akt pathway is activated downstream of a variety of extracellular signals and activation of this signaling pathway impacts a number of cellular processes including cell growth, proliferation and survival. The alteration of components of this pathway, through either activation of oncogenes or inactivation of tumor suppressors, disrupts a signaling equilibrium and can thus lead to cellular transformation. The frequent dysregulation of the PI3K/Akt pathway in human cancer has made components of this pathway attractive for therapeutic targeting; however, a more comprehensive understanding of the signaling intricacies is necessary to develop pharmacological agents to target not only specific molecules, but also specific functions. Here, we review a series of experiments examining the contribution of molecules of this signaling network including PI3K, phosphatase and tensin homolog deleted on chromosome 10, integrin-linked kinase and Akt and address the significance to human breast cancer.

Effect of conditional knockout of the type II TGF-beta receptor gene in mammary epithelia on mammary gland development and polyomavirus middle T antigen induced tumor formation and metastasis.

Transforming growth factor-beta (TGF-beta) isoforms are growth factors that function physiologically to regulate development, cellular proliferation, and immune responses. The role of TGF-beta signaling in mammary tumorigenesis is complex, as TGF-beta has been reported to function as both a tumor suppressor and tumor promoter. To elucidate the role of TGF-beta signaling in mammary gland development, tumorigenesis, and metastasis, the gene encoding type II TGF-beta receptor, Tgfbr2, was conditionally deleted in the mammary epithelium (Tgfbr2MGKO). Loss of Tgfbr2 in the mammary epithelium results in lobular-alveolar hyperplasia in the developing mammary gland and increased apoptosis. Tgfbr2MGKO mice were mated to the mouse mammary tumor virus-polyomavirus middle T antigen (PyVmT) transgenic mouse model of metastatic breast cancer. Loss of Tgfbr2 in the context of PyVmT expression results in a shortened median tumor latency and an increased formation of pulmonary metastases. Thus, our studies support a tumor-suppressive role for epithelial TGF-beta signaling in mammary gland tumorigenesis and show that pulmonary metastases can occur and are even enhanced in the absence of TGF-beta signaling in the carcinoma cells.

Comparison of the effectiveness of adenovirus vectors expressing cyclin kinase inhibitors p16INK4A, p18INK4C, p19INK4D, p21WAF1/CIP1 and p27KIP1 in inducing cell cycle arrest, apoptosis and inhibition of tumorigenicity

Cell cycle regulatory proteins are important candidates for therapeutic tumour suppressors. Adenovirus vectors were constructed to overexpress cyclin kinase inhibitors p16INK4A, p18INK4C, p19INK4D, p21WAF1/CIP1 and p27KIP1 under the control of the murine cytomegalovirus immediate early gene promoter. These vectors directed the efficient expression of each of the cyclin kinase inhibitors and induced growth arrest, inhibited DNA synthesis, and prevented phosphorylation of the retinoblastoma protein (pRb) in cell lines expressing functional pRb. In pRb-deficient cells, expression of the cyclin kinase inhibitors was not effective in inhibiting DNA replication or growth arrest. Interestingly, three of the cyclin kinase inhibitors, p16, p18 and p27 were found to induce apoptotic death in transduced HeLa and A549 cells. When the vectors were tested for their ability to inhibit tumorigenicity in a polyomavirus middle T antigen model of murine breast carcinoma, expression of the cyclin kinase inhibitors resulted in a delay in tumour formation that varied from several weeks for the p19 expressing vector to greater than 25 weeks for the p27 expressing vector. When tumours were injected directly with the adenovirus vectors expressing the cyclin kinase inhibitors, only treatment with the vector expressing p16 resulted in a delay in tumour growth.

Transgenic mouse models of human breast cancer

The pathogenesis of human breast cancer is thought to involve multiple genetic events, the majority of which fall into two categories, gain of function mutations in proto-oncogenes such as c-myc, cyclin D1, ErbB-2 and various growth factors which are involved in supporting cell growth, division and survival, and loss of function mutations in so called ‘tumor suppressor’ genes, such as p53, which are involved in preventing unrestrained cellular growth. A number of mouse systems exist to address the significance of these mutations in the pathogenesis of breast cancer including transgenic mice expressing high levels of a specific gene in target tissues and knockout mice in which specific genes have been ablated via homologous recombination. More recently, the combination of these techniques to create bigenics as well as the use of ‘knockin’ and conditional tissue specific gene targeting strategies have allowed the models more reflective of the human disease to be devised. Studies with these models have not only implicated particular genetic events in the progression of the disease but have emphasized the complex, multi-step nature of breast cancer progression. These models also provide the opportunity to study various aspects of the pathogenesis of this disease, from hormonal effects to responses to chemotherapeutic drugs. It is hoped that through the combined use of these models, and the further development of more relevant models, that a deeper understanding of this disease and the generation of new therapeutic agents will result.

Mammary epithelial-specific expression of the integrin linked kinase (ILK) results in the induction of mammary gland hyperplasias and tumors in transgenic mice

The integrin linked kinase (ILK) is a cytoplasmic effector of integrin receptors, involved in the regulation of integrin binding properties as well as the activation of cell survival and proliferative pathways, including those involving MAP kinase, PKB/Akt and GSK-3β. Overexpression of ILK in cultured intestinal and mammary epithelial cells has been previously shown to induce changes characteristic of oncogenic transformation, including anchorage-independent growth, invasiveness, suppression of anoikis and tumorigenicity in nude mice. In order to determine if ILK overexpression can result in the formation of mammary tumors in vivo, we generated transgenic mice expressing ILK in the mammary epithelium, under the transcriptional control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). By the age of 6 months, female MMTV/ILK mice developed a hyperplastic mammary phenotype, which was accompanied by the constitutive phosphorylation of PKB/Akt, GSK-3β and MAP kinase. Focal mammary tumors subsequently appeared in 34% of the animals at an average age of 18 months. Given the focal nature and long latency of the tumors, however, additional genetic events are likely required for tumor induction in the MMTV/ILK mice. These results provide the first direct demonstration of a potential oncogenic role for ILK, which is upregulated in human tumors and tumor cell lines.

Signal transduction in mammary tumorigenesis: a transgenic perspective.

A number of genes have been implicated in breast cancer development, yet few have been demonstrated to play causative roles in mammary tumor formation. The advent of transgenic mouse and embryonic stem cell technologies now permits manipulation of the mouse genome in such a way as to temporally and spatially control a gene products expression. Thus, the basic researcher now can directly assess the involvement of particular genes in tumorigenesis and disease progression and, in the process, to develop mouse models of human genetic disease. The utility of such technologies is emphasized in transgenic mice expressing genes thought to play important roles in the initiation and progression of mammary carcinomas. As these transgenic strains have been the subject of several reviews, here we focus on two mouse mammary tumor models, Polyomavirus middle T antigen and the Neu/ErbB-2 receptor tyrosine kinase, which are most amenable to study specific signaling pathways in process of mammary tumorigenesis.

Angiogenesis impairment in Id-deficient mice cooperates with an Hsp90 inhibitor to completely suppress HER2/neu-dependent breast tumors.

Id proteins bind basic helix–loop–helix transcription factors and function as dominant negative inhibitors of gene expression. Id1 and Id3 are required for the recruitment of bone marrow-derived endothelial cell precursors and tumors transplanted into Id-deficient mice demonstrate impaired angiogenesis. Mouse mammary tumor virus–neu mice were bred with Id1–/–Id3+/– mice to ascertain the role of Id1 and Id3 in mammary tumorigenesis in a more physiologically relevant model. In mammary tumors from these mice, Id1 and Id3 expression was restricted to the vascular endothelium. Id1 and Id3 deficiency did not prevent or delay tumor formation but did alter tumor phenotype. The tumors that developed in the Id-deficient mice were larger and cystic with a viable rim of tumor cells surrounding a nonviable core of cellular debris. The Hsp90 chaperone protein is required for cellular survival under condition of environmental stress and for the stability of the neu oncogene. 17-Allylamino-17-demethoxygeldanamycin, an Hsp90 inhibitor, was used to treat these mice. Whereas 17-allylamino-17-demethoxygeldanamycin only modestly delayed the growth of established mammary tumors in WT mice for Id, tumor suppression was dramatically more effective in an Id1- or Id3-deficient background. These data suggest that tumorigenesis can occur in a background of defective angiogenesis but that tumors developing in such an environment may be especially sensitive to inhibitors of neu and stress-activated survival pathways. Thus angiogenesis inhibitors in combination with inhibitors of Hsp90 function should be evaluated for the treatment of advanced breast cancer.

Clinical and In Vitro Evaluation of Cidofovir for Treatment of Adenovirus Infection in Pediatric Hematopoietic Stem Cell Transplant Recipients

Post-hematopoietic stem cell transplantation (HSCT) adenovirus infections were identified in 31 of 204 consecutive pediatric HSCT patients, 18 of whom had severe manifestations of infection. Cidofovir treatment led to clinical improvement in 8 of 10 patients with severe infection and to virologic clearance in 9 patients. In vitro susceptibility to cidofovir was demonstrated in 12 clinical adenovirus isolates. Cidofovir is a promising treatment option for this population.

Identification of a Stat3-Dependent Transcription Regulatory Network Involved in Metastatic Progression

High levels of activated Stat3 are often found in human breast cancers and can correlate with poor patient outcome. We employed an activated ErbB2 mouse model of breast cancer to investigate the in vivo role of Stat3 in mammary tumor progression and found that Stat3 does not alter mammary tumor initiation but dramatically affects metastatic progression. Four-fold fewer animals exhibited lung metastases in the absence of Stat3 and a 12-fold reduction in the number of lung lesions was observed in animals bearing Stat3-null tumors when compared with the wild-type cohort. The decreased malignancy in Stat3-deficient tumors is attributed to a reduction in both angiogenic and inflammatory responses associated with a Stat3-dependent transcriptional cascade involving CCAAT/enhancer binding protein delta.