Eran R. Andrechek

Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type–specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.

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.

Anchorage-independent cell growth signature identifies tumors with metastatic potential

The oncogenic phenotype is complex, resulting from the accumulation of multiple somatic mutations that lead to the deregulation of growth regulatory and cell fate controlling activities and pathways. The ability to dissect this complexity, so as to reveal discrete aspects of the biology underlying the oncogenic phenotype, is critical to understanding the various mechanisms of disease as well as to reveal opportunities for novel therapeutic strategies. Previous work has characterized the process of anchorage-independent growth of cancer cells in vitro as a key aspect of the tumor phenotype, particularly with respect to metastatic potential. Nevertheless, it remains a major challenge to translate these cell biology findings into the context of human tumors. We previously used DNA microarray assays to develop expression signatures, which have the capacity to identify subtle distinctions in biological states and can be used to connect in vitro and in vivo states. Here we describe the development of a signature of anchorage-independent growth, show that the signature exhibits characteristics of deregulated mitochondrial function and then demonstrate that the signature identifies human tumors with the potential for metastasis.

ErbB2 Is Required for Muscle Spindle and Myoblast Cell Survival

ABSTRACT Signaling mediated by ErbB2 is thought to play a critical role in numerous developmental processes. However, due to the embryonic lethality associated with the germ line inactivation of erbB2, its role in adult tissues remains largely obscure. Given the expression of ErbB2 at the neuromuscular junction, we have created a muscle-specific knockout to assess its role there. This resulted in viable mice with a progressive defect in proprioception due to loss of muscle spindles. Interestingly, a partial reduction of ErbB2 levels also reduced the number of muscle spindles. Although histological analysis of the muscle revealed an otherwise normal architecture, induction of muscle injury revealed a defect in muscle regeneration. Consistent with these observations, primary myoblasts lacking ErbB2 exhibit extensive apoptosis upon differentiation into myofibers. Taken together, these results illustrate a dual role for ErbB2 in both muscle spindle maintenance and survival of myoblasts.

Centrosome abnormalities, recurring deletions of chromosome 4, and genomic amplification of HER2/neu define mouse mammary gland adenocarcinomas induced by mutant HER2/neu

The conditional expression of activated HER2/neu gene under its endogenous promoter in the mammary epithelium of the mouse results in accelerated lobular development and focal mammary tumors. Carcinogenesis, however, requires amplification and considerably increased expression levels of oncogenic neu. Deducing from the multiple genetic aberrations required for human breast cancer to develop, we hypothesized that in addition to the over-expression of an activated HER2/neu, secondary aberrations would occur. We have therefore conducted a genomic screen for chromosomal imbalances and translocations using comparative genomic hybridization and spectral karyotyping. The results reveal a moderate degree of chromosomal instability and micronuclei formation in short-term cultures established from primary tumors. Genomic instability appears to be linked to the amplification of functional centrosomes, a phenomenon that we frequently observed in other tumor types. Seventy per cent of the tumors revealed genomic amplification of HER2/neu, often in the form of double minute chromosomes, which correlated with recurring loss of mouse chromosome 4D-E, a region that is orthologous to distal human chromosome 1p. It is likely that this region contains putative tumor suppressor genes whose inactivation is required for tumor formation in this model of human breast cancer.

Tyrosine kinase signalling in breast cancer: Tyrosine kinase-mediated signal transduction in transgenic mouse models of human breast cancer

The ability of growth factors and their cognate receptors to induce mammary epithelial proliferation and differentiation is dependent on their ability to activate a number of specific signal transduction pathways. Aberrant expression of specific receptor tyrosine kinases (RTKs) has been implicated in the genesis of a significant proportion of sporadic human breast cancers. Indeed, mammary epithelial expression of activated RTKs such as ErbB2/neu in transgenic mice has resulted in the efficient induction of metastatic mammary tumours. Although it is clear from these studies that activation these growth factor receptor signalling cascades are directly involved in mammary tumour progression, the precise interaction of each of these signalling pathways in mammary tumourigenesis and metastasis remains to be elucidated. The present review focuses on the role of several specific signalling pathways that have been implicated as important components in RTK-mediated signal transduction. In particular, it focuses on two well characterized transgenic breast cancer models that carry the polyomavirus middle T(PyV mT) and neu oncogenes.

Genetic heterogeneity of Myc-induced mammary tumors reflecting diverse phenotypes including metastatic potential

Human cancers result from a complex series of genetic alterations, resulting in heterogeneous disease states. Dissecting this heterogeneity is critical for understanding underlying mechanisms and providing opportunities for therapeutics matching the complexity. Mouse models of cancer have generally been used to reduce this complexity and focus on the role of single genes. Nevertheless, our analysis of tumors arising in the MMTV-Myc model of mammary carcinogenesis reveals substantial heterogeneity, seen in both histological and expression phenotypes. One contribution to this heterogeneity is the substantial frequency of activating Ras mutations. Additionally, we show that these Myc-induced mammary tumors exhibit even greater heterogeneity, revealed by distinct histological subtypes as well as distinct patterns of gene expression, than many other mouse models of tumorigenesis. Two of the major histological subtypes are characterized by differential patterns of cellular signaling pathways, including β-catenin and Stat3 activities. We also demonstrate that one of the MMTV-Myc mammary tumor subgroups exhibits metastatic capacity and that the signature derived from the subgroup can predict metastatic potential of human breast cancer. Together, these data reveal that a combination of histological and genomic analyses can uncover substantial heterogeneity in mammary tumor formation and therefore highlight aspects of tumor phenotype not evident in the population as a whole.

Targeted disruption of ErbB2/Neu in the mammary epithelium results in impaired ductal outgrowth

The ErbB2 receptor tyrosine kinase has been implicated as a critical growth factor receptor in both normal development and cancer. Amplification and overexpression of this receptor is observed in 20–30% of all human breast cancers and is inversely correlated with patient survival. Studies with transgenic mice have established that elevated expression of erbB2 in mammary epithelium can directly induce mammary carcinomas. Although these studies confirmed a role for ErbB2 in breast cancer induction, the precise role of ErbB2 in normal mammary gland development remained to be elucidated due to the embryonic lethality associated with the null mutation. Here, we demonstrate that the mammary-specific ablation of erbB2 through Cre-mediated recombination leads to a striking ductal elongation defect. In addition to the observed elongation defect, we noted that branching in the adult mammary gland was also reduced. Despite these perturbations in virgin mammary gland morphogenesis, targeted disruption of erbB2 had little impact on the ability of these animals to lactate. Taken together, these observations indicate that erbB2 plays a critical role in the initial stages of mammary gland morphogenesis.

Accelerated mammary tumor development in mutant polyomavirus middle T transgenic mice expressing elevated levels of either the Shc or Grb2 adapter protein.

ABSTRACT The Grb2 and Shc adapter proteins play critical roles in coupling activated growth factor receptors to several cellular signaling pathways. To assess the role of these molecules in mammary epithelial development and tumorigenesis, we have generated transgenic mice which individually express the Grb2 and Shc proteins in the mammary epithelium. Although mammary epithelial cell-specific expression of Grb2 or Shc accelerated ductal morphogenesis, mammary tumors were rarely observed in these strains. To explore the potential role of these adapter proteins in mammary tumorigenesis, mice coexpressing either Shc or Grb2 and a mutant form of polyomavirus middle T (PyV mT) antigen in the mammary epithelium were generated. Coexpression of either Shc or Grb2 with the mutant PyV mT antigen resulted in a dramatic acceleration of mammary tumorigenesis compared to parental mutant PyV mT strain. The increased rate of tumor formation observed in these mice was correlated with activation of the epidermal growth factor receptor family and mitogen-activated protein kinase pathway. These observations suggest that elevated levels of the Grb2 or Shc adapter protein can accelerate mammary tumor progression by sensitizing the mammary epithelial cell to growth factor receptor signaling.

Patterns of cell signaling pathway activation that characterize mammary development

Previous work has detailed the histological and biochemical changes associated with mammary development and remodeling. We have now made use of gene expression profiling, and in particular of the previously described signatures of cell signaling pathway activation, to explore the events associated with mammary gland development. We find that there is elevated E2F-specific pathway activity prior to lactation and relatively low levels of other important signaling pathways, such as RAS, MYC and SRC. Upon lactation and continuing into the involution phase, these patterns reverse with a dramatic increase in RAS, SRC and MYC pathway activity and a decline in E2F activity. At the end of involution, these patterns return to that of the adult non-lactating mammary gland. The importance of the changes in E2F pathway activity, particularly during the proliferative phase of mammary development, was confirmed through the analysis of mice deficient for various E2F proteins. Taken together, these results reveal a complex pattern of pathway activity in relation to the various phases of mammary gland development.