The objective of this application is to identify the molecular target(s) by which specific amino acid dependency modulates the viability and invasiveness of human androgen-independent prostate cancer calls. We hypothesize that specific amino acid-regulated invasion is dependent on the inhibition of FAK and its binding partners. We further hypothesize that specific amino acid-regulated induction of apoptosis is due to the modulation and/or interference in cross talk between the MEKJERK survival pathway and the Akt pathway leading to loss of mitochondrial integrity with consequent activation of effector caspases.
The specific aims are: 1) Identify if FAK/Cas/Crk or Rho/Ras pathway is the molecular target(s) of specific amino acid restriction in integrin-mediated attachment/invasion. 2) Determine how specific amino acid restriction modulates and/or interferes with the cross talk between the MEK/ERK and Akt survival pathways. 3) Determine the role(s) of BH123 and/or BH3 proteins of the Bcl-2 protein family on mitochondrial outer membrane permeabilization (MOMP), mitochondrial release of cytochrome c, and apoptosis-inducing factor (AIF), and subsequent activation of caspases, and 4) Determine if dietary tyrosine and phenylalanine restriction and methionine restriction will inhibit growth and metastasis of prostate cancer xenografts. Specialized techniques utilized in the application involve cell attachment, migration/invasion, and wounding assays and immunoprecipitation and Western blot analysis. The cellular location of various signaling molecules will be examined with confocal immunofluorescence microscopy. Gene transfection experiments will be used to determine the role of certain cell signaling molecules. Intracellular amino acids will be determined by high pressure liquid chromatography and apoptosis will be measured by flow cytometry. A major benefit from the proposed research proposed is that it will expand knowledge into newer pathways of apoptosis research specific for prostate cancer cells as well as enhance understanding of the mechanisms underlying the anticancer activity of tyrosine/phenylalanine and methionine restriction. This is especially important research since there still is no satisfactory drug for treatment of androgen-independent, metastatic human prostate cancer. This research could serve as the basis for future development of more specific antimetastatic, anti-invasive, apoptosis-based therapies for human prostate cancer.
