The overall objective of our research is to capitalize on our novel finding that the calpain (Cp)/calpastatin (Cs) network associates with the endoplasmic reticulum (ER) and Golgi apparatus (GA) in normal, transformed and tumor cells. It is well-established that the Cp/Cs network plays a vital role in cell adherence, shape change and movement, although the precise mechanism remains to be clarified. The fact that a substantial amount of activatable Cp and its regulatory molecules firmly associate hydrophobically with subcellular membranes suggests this association plays a critical role Cp activation or/and function. Thus, it is reasonable to assume that cell adhesion, shape change and migration/invasion are dependent on the ability of Cp to associate with organelles, where it would be in position to cleave substrates as well be shuttled to the plasma membrane (PM). Once associated with the PM it can bind with membrane microdomains (rafts) and subsequently focal adhesions. With this as background we hypothesize that inhibition of tumor cell migration and invasion can be achieved by treating these cells with targeted cell penetrating peptides (CPPs) linked to Cp peptides that disrupt the association of Cp with subcellular membranes and subsequent cell function. To achieve this, the following experiments will be done.
We will identify the amino acid sequence in the CpII catalytic subunit that allow engagement to the ER and GA. To achieve this, recombinant DNA technologies will be used to investigate the three regions of the CpII catalytic subunit that could serve as regulators of its association with membranes. Using a variety of molecular and cellular assays, mutated CpII proteins will be analyzed for their ability to associate with the ER and GA.. CpII membrane associations and the direct link of this activity to cell migration and cell survival would suggest this membrane association parameter might serve as an ideal target for therapeutic intervention into tumor cell migration. We hypothesize that inhibition of CpII membrane association in tumor cells will inhibit their migratory potential, limit the proliferative potential and promote programmed cell death (apoptosis). To investigate this, blocking peptides will be synthesized to the domain(s) identified in the above as requisite for CpII membrane interaction. These peptides will be introduced into tumor cells using cell penetrating peptide (CPP) technology, and the impact of these peptides on cell migration and cell survival will be assayed utilizing a variety of cellular and enzymatic assays (adhesion, spreading, migration/invasion, and apoptosis assays).
Inhibition of Lung cancer Cell Migration/Invasion by Cell Penetrating Peptides.
11/30/09-12/1/12. KLCR Grant.
Roszman T.L., Brooks W.H. and Noonan D. Targeting tumor dispersal through the use of novel calpain cell penetrating peptides. In preparation.
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Goebel J., Forrest K., Wills-Krap M., Roszman T.L. Tublin polymerization modulates interleukin-2 receptor signal transduction in human T cells. J Receptors and Signal Transduction 26:87-106, 2006.
Hood J.L., Brooks W.H., Roszman T.L., Differential compartmentalization of the calpain/calpastatin network with the endoplasmic reticulum and golgi apparatus. J Biol Chem. 279:43126-43135, 2004.
Hood J.L., Logan B.B., Sinai A.P., Brooks W.H., Roszman T.L. Association of the calpain/calpastatin network with subcellular organelles. Biochem Biophy Res Commun. 310:1200-12, 2003.
Goebel J., Forrest K., Flynn D., Rao R., Roszman T.L., Lipid rafts major histocompatibility complex molecules and immune regulation. Hum Immunol. 63:831-20, 2002.
Morford L.A., Forrest, K., Logan B., Overstreet L.K., Goebel J., Brooks W.H., Roszman T.L., Calpain II colocalizes with detergent-isoluble rafts on human and Jurkat T-cells. Biochem Biophys Res Commun. 295:540-6, 2002.
Goebel J., Forrest K., Morford L., Roszman T.L. Differential localization of IL-2 and -15 receptor chains in membrane rafts of human T cells. J Leukoc Biol. 72:199-206, 2002.
Goebel J., Stevens E., Forrest K., Roszman T.L. Daclizumab (Zenapax) inhibits early interleukin-2 receptor signal transduction events. Transpl Immunol. 8:1153-159, 2000.
Rock M.T., Dix A.R., Brooks W.H., and Roszman T.L. β1 integrin-mediated T-cell adhesion and cell spreading are regulated by calpain. Exp.Cell Res. 261:260-270, 2000.