Steven Frisch - US grants

Analysis of the mechanisms and components underlying the coupled cellular processes of signal transduction, transcriptional response and cell growth is the major career interest of the applicant for this research grant. Of particular interest is the mechanism by which oncogene proteins regulate these processes. The activated oncogene is essentially a mutant gene, that provides a phenotype revealing continually new aspects of the behavior, design and interactions of regulatory molecules. In this framework, the proposed research promises to yield new insights concerning the interaction of the E1A oncogene with two dissimilarly regulated genes, interstitial collagenase (CL) and type IV collagenase (T4). The proteolytic enzymes encoded by these genes play an important role in tumor metastasis. E1A protein will be used as a critical probe to analyze their regulation. Specifically, an enhancer element of the CL gene promoter, the TPA- Regulatory Element, was found by this investigator to be an E1A-regulated enhancer: E1A represses its activity in one cell line, but activates it in another. The identification of factors that respond to E1A positively or negatively will be achieved by functionally assaying proteins from one cell system in the background of the other. Assays for this purpose will be of two general types, in vitro (e.g., in vitro transcription) and in vivo (transfection or microinjection.) The T4 gene regulatory region will be analyzed further, as both enhancer and silencer elements have been found. One E1A-repressible enhancer element that binds an AP2-like transcription factor will be characterized in more detail, and the mechanism of E1A repression will be analyzed.

DESCRIPTION (provided by applicant): Cell migration and invasion are critical aspects of tumor metastasis that are incompletely understood. Recently, we have found that the apoptosis regulator, caspase-8, enhances cell migration and invasion of tumor cells, as well as modulating other aspects of cytoskeletal regulation, including calpain activation, Rac activation, generation of lamellipodia, and cell adhesion. We propose to elucidate the mechanisms whereby caspase-8 controls these processes. In the first specific aim, we will determine how caspase-8 controls calpain activation, particularly the effects of caspase-8 on the phosphorylation and activation of calpains by adhesion complexes. In specific aim 2, we will examine the hypotheses that caspase-8 enhances Rac activation by stimulating the calpain-mediated cleavage of guanine nucleotide exchange factors (GEFs) for Rac and/or by regulating the p85 subunit of PI3- Kinase. Specific aim 3 utilizes a genetically defined and well-characterized mouse model for human breast cancer to test the effects of caspase-8 on tumor metastasis. Caspase-8 expression is retained in most human tumor types. It may coordinate the opposing processes of apoptosis vs. cell motility signaling, which are carried out by the mature or unprocessed forms of the enzyme, respectively. It is important to understand the ramifications of this novel function of caspase-8 for oncogenesis and the treatment of human cancer.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Anoikis is defined as apoptosis that is inhibited by extracellular matrix; it was discovered by our laboratory and reported in a 1994 Journal of Cell Biology paper. We are focusing on the mechanisms by which the oncogenic EMT (epithelial-to-mesenchymal transition) confers resistance to anoikis upon carcinoma cells. In particular, we are investigating the role of ankyrins, cytoskeletal proteins that link the actin cytoskeleton to various transmembrane adhesion receptors including E-cadherin. We hypothesize that ankyrin expression in normal epithelial cells sets up a transcriptional program that permits cells to die in response to cell-matrix detachment. Conversely, we posit that the loss of ankyrin that occurs frequently in carcinoma cells establishes an anoikis-resistance gene expression program. This project addresses these hypotheses