The liver is a frequent metastatic site of primary malignant tumors, suggesting that the liver itself provides a prometastatic microenvironment for growth of cancer cells. Hepatic stellate cells (HSC) are liver specific pericytes. Stimulated by tumor derived factors such as transforming growth factor beta (TGF- ), they transdifferentiate into myofibroblasts (MFB) that are thought to promote metastatic growth through multiple mechanisms, including growth factor and cytokine production, extracellular matrix deposition and stromal cell recruitment. Thus, a better understanding of the mechanisms governing HSC activation is of therapeutic significance for liver metastases. Our long-term objectives are to understand mechanisms that regulate HSC activation and metastatic growth in the liver. IQ motif containing GTPase activating protein 1 (IQGAP1) and vasodilator stimulated phosphoprotein (VASP) are two cytoskeleton regulatory proteins, not previously recognized as participants in growth factor dependent HSC activation. We have implanted tumor cells into murine livers and have found that IQGAP-/- mice developed more liver metastases and have higher densities of MFB within their metastases as compared to IQGAP+/+ mice. In vitro, knockdown of IQGAP1 by siRNA potentiates MFB transdifferentiation of HSC. Additionally, we have identified that both TGF- receptor II (T2R- II) and VASP bind to the carboxyl terminus of IQGAP1 (a.a. 1503-1657), and that knockdown of VASP inhibits MFB transdifferentiation in vitro. These novel data support our central hypothesis: IQGAP1 suppresses TGF- 1 dependent activation of quiescent HSC into MFB, and this process is counterbalanced by VASP. Alterations in this counterbalance influence HSC activation within the hepatic tumor microenvironment thereby influencing metastatic growth.
The Specific Aims are: (1) to test that IQGAP1 inhibits TGF- 1 mediated HSC activation by binding to T2R-II and inhibiting T2R-II function. IQGAP1 and T2R-II mutants, kinase activity assays, and in vitro protein binding assays will be utilized to test this hypothesis. (2) to test that VASP promotes TGF- 1 mediated HSC activation by sequestering IQGAP1 from T2R-II in a phosphorylation dependent manner thereby antagonizing IQGAP1 inhibition of T2R-II function. Immunoprecipitation, immunofluorescence, kinase activity assays, VASP phosphomutants, and isothermal titration calorimetry will be used to test this hypothesis. (3) to determine that alterations in the counterbalance of IQGAP1 and VASP in HSC influence HSC activation in vivo and metastatic growth in the liver. This will be tested by two complementary animal liver metastasis models together with a state of the art in vivo tumor imaging technique. Thus, this proposal will delineate novel mechanisms whereby IQGAP1 and VASP act in concert to regulate TGF- 1 mediated HSC activation in vitro and in vivo and metastatic tumor growth, thereby, significantly advancing our understanding of TGF- 1 signaling, HSC biology and the prometastatic microenvironment of the liver.
Hepatic stellate cells are liver specific pericytes and their activation within the hepatic tumor microenvironment is a prominent modifier of metastatic tumor growth in the liver. This proposal tests novel mechanisms whereby the cytoskeleton regulatory proteins IQGAP1 and VASP act in concert to regulate the TGF- 1 mediated HSC activation process and metastatic tumor growth in the liver. Mechanisms proposed may present important targets for reducing metastasis progression in the liver.
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