David G. Menter - US grants

THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. The malignant progression of melanoma is often accompanied by overexpression of the low-affinity p75 neurotrophin receptors. The most aggressive melanomas also frequently exhibit brain colonization properties. I am using brain-colonizing melanoma variants that express p75 in association with metastatic potential as a model system for analyzing the effects of neurotrophins on metastatic behavior. My working hypothesis is that p75-mediated trophism may promote the invasion and survival of melanoma cells in the neurotrophin rich microenvironment of the brain. I have most recently shown that neurotrophins affect the metastatic properties of human melanoma cells by (1) enhancing malignant melanoma chemoinvasion and (2) promoting malignant melanoma cell survival. Importantly, the low-affinity p75 receptor expression by certain brain- colonizing human melanomas directly correlates with their metastatic potentials. These observations prompted me to develop the following specific aims: (1) I will determine the exact roles of p75 in invasion and survival by modifying the expression of p75 in melanoma cells using: a) fluorescence activated cell sorting; b) p75 antisense oligo-nucleotides; c) p75 expression constructs; or d) genetic ablation of p75 in mouse melanoma. (2)1 will determine the relative roles of the various neurotrophins in promoting invasion and survival of melanoma cells in vitro. These studies will involve Transwell(TM) invasion assays and survival assays developed in my laboratory to examine the effects of neurotrophin-3, brain-derived neurotrophic factor and neurotrophin-4/5 on melanoma invasion and survival. (3) Depending on the success of the in vitro studies, I will examine the role of p75 during invasion and survival of selected human melanoma variants following injection into nu/nu mice. Animals will be sacrificed and tumors reestablished in culture as well as paraffin embedded for immunohistochemical characterization of p75. My results are the first to suggest the importance of trophic mechanisms in the progression of metastatic melanoma. Similar examples of trophic survival mechanisms may occur during tumor dormancy, cell survival in the necrotic regions of tumors, and cryptic survival of micrometastases. Further understanding of trophic mechanisms in melanoma progression may provide an important basis for the future research, discoveries and therapeutic approaches for not only malignant melanoma but also other malignant diseases.

DESCRIPTION (provided by applicant): Selenium (Se) is a highly promising compound for the chemoprevention of prostate cancer. Although intensively studied, the molecular mechanisms responsible for the chemopreventive effectiveness of Se remain unclear. We hypothesize that a loss of redox-protective activity in the prostate results from the functional suppression of key redoxselenoproteins (RoxSePs; e.g. glutathione peroxidase, GPx and thioredoxin reductase, TxR). This process occurs during aging or low dietary Se intake leaving prostate tissues defenseless against the oxidative damage responsible for carcinogenesis. To test this hypothesis, we will produce recombinant mouse models that lack factors involved in co-translationally generating selenoproteins. Selenoproteins are co-translationally formed when other proteins that recognize selenocysteine insertion sequences (SECIS) insert selenocysteine at opal stop codon (UGA) positions instead of terminating the protein chain. One such protein, SECIS binding protein 2 (SBP2), is our primary target. The SBP2 gene will be targeted to simultaneously eliminate all selenoproteins, this will avoid compensation by multiple RoxSeP isoforms and functional overlap by various RoxSePs. Standard targeting methods are unlikely to work since selenocysteyl-tRNA gene knockout (KO) mice (-/-) exhibit embryonic lethality. Lethality, however, can be overcome by controlling tissue-specific targeting and KO timing. To achieve these goals we will use a specialized Cre/Iox recombination system involving Cre-ERT. In the Cre-ERT fusion protein, Cre is fused with a mutated estrogen receptor ERT that is active only when 4-hydroxytamoxifen (4-OH-T) is bound and then excises DNA between adjacent Iox sites. The prostate-tissue-specific probasin (PB) promoter will be inserted in front of the Cre-ERT molecule to target SBP2 only in the mouse prostate. We will use the resulting 4-OH-T inducible PB-Cre-ERT/floxed SBP2 (Iox-SBP2-lox) mice to study selenoprotein function during early and late-stage prostate carcinogenesis. Aim 1: Will isolate and characterize the SBP2 gene. An SBP2 cDNA clone will be used to screen a mouse 129S genomic library. Sequencing will be performed to determine the intron-exon boundaries and the most useful exon for targeting. Aim 2: Will generate recombinant mice to study the role of SBP2 in the prostate. The PB-Cre-ERT/floxed SBP2 mice generated will be given 4-OH-T at the appropriate stage to induce recombinant excision of SBP2. Aim 3: Will examine the SBP2 (-/-) mouse phenotype and the role of SBP2 gene loss during carcinogenesis. Prostate cells containing PB-Cre-ERT/floxed SBP2 will be placed into tissue-culture and examined for the lack of 75Se isotope incorporation into GPx, and TxR using antibodies generated in our lab. These animals are needed to perform pre-clinical evaluation of promising chemopreventive selenocompounds in the presence or absence of RoxSePs during prostate carcinogenesis.