Functional and molecular analysis of HGF/Met signaling in germinal center B-cells and B-cell neoplasia
01 / 2001 - 12 / 2004
The c-met proto-oncogene encodes the receptor tyrosine kinase for Hepatocyte Growth Factor/Scatter Factor (HGF). Genetic studies in vivo have implicated HGF and Met in embryogenesis and organogenesis. Furthermore, Met plays a key role in growth, invasion, and metastasis of several human tumors. In vitro studies have established a regulatory role for HGF/Met in a wide variety of cellular responses, including mitogenesis, motogenesis, and morphogenesis of epithelial cells. Interestingly, we have recently found that Met is specifically expressed on B cells localized in the germinal centers (GC) of secondary lymphoid tissue, where the B cells differentiate into memory B cells or plasma cells. HGF is also produced at these sites of antigen-specific B cell differentiation, thus establishing a paracrine signaling mechanism. Using mice repopulated with Met knock-out B cells, we were able to establish a role for Met in antigen-specific B cell differentiation. The rapid clonal expansion (proliferation) and affinity selection (apoptosis) that GC B cells undergo make these cells a prime target for malignant transformation. Indeed, about half of the non-Hodgkin's lymphomas (NHLs) are related to GC B cells. We and others have observed overexpression of Met in a subset of these NHLs. In addition, in most cases of multiple myeloma (MM), the malignant counterpart of plasma cells, both Met and HGF are expressed, thus constituting an autocrine loop. - Purpose: The above findings indicate that HGF/Met signaling plays an important role in antigen-specific B cell differentiation and may be involved in lymphomagenesis; however, the mechanism by which Met regulates these processes remains to be established. In both processes, the regulation of adhesion and migration (invasion and metastasis), as well as proliferation and apoptosis (autonomous tumor growth), plays a critical role. Therefore, we propose to investigate which of these fundamental cellular responses are controlled by HGF/Met in B cells, which signal transduction mechanisms underlie these responses, and which of these signaling pathways and responses are involved in HGF/Met-controlled antigen-specific B cell differentiation and lymphomagenesis. - Plan of investigation: 1. Study the effect of HGF stimulation on adhesion, migration, proliferation, and apoptosis of fresh tonsillar Met+ (GC) B cells, NHL-, and MM-cell lines, and investigate the functional consequence of creation or disruption of the autocrine HGF/Met-loop in MM by withdrawal of HGF or stable expression of HGF, wild type, oncogenic, constitutively active, or dominant negative Met. 2. Analyze the effect of HGF/Met on the activation of different signaling molecules, including several kinases and GTPases, and establish the function of the responsive signaling molecules in the distinct cellular responses by partially complementary pharmacological and genetic approaches, including the use of transiently or stably transfected as well as specific gene-deficient knock-out B cells. 3. Investigate the role of HGF/Met and the distinct cellular responses in B cell lymphomagenesis in vivo, by: a) investigating Met+ NHL and MM patient material (and cell lines) for amplification or point mutation of the Met gene; b) analyzing these primary tumors by in situ visualization of the activity of signaling pathways by means of immunohistochemistry, using (phospho-)specific antibodies against signaling molecules of which the phosphorylation, translocation, or expression serves as a critical read-out for the distinct HGF/Met responses; c) studying the dissemination, metastasis, and tumorigenicity in SCID mice of NHL or MM cells in which, by stable transfection of HGF and/or Met, or signaling molecule mutants, an autocrine loop is created or interrupted, or distinct cellular responses are specifically abrogated. - Results and relevance: We anticipate that this project will provide important information, both on a cellular and molecular level, as to how HGF/ Met signaling regulates antigen-specific B cell differentiation and contributes to B cell neoplasia. These results may provide novel means of diagnosis and treatment of cancer.