HGAL, a Lymphoma Prognostic Biomarker, Regulates Lymphocyte and Lymphoma Cell Motility by Modulation of RhoA Signaling Pathway.

Abstract 316

Identification of genes whose expression correlates with clinical outcome is of paramount importance for designing upfront patient-tailored treatments that will potentially improve the outcome of lymphoma patients. Furthermore, new molecular prognostic biomarkers may lead to the discovery of novel pathophysiological mechanisms underlying lymphoma pathogenesis, potentially highlighting unique biological processes regulated by these proteins in lymphoma cells and in their normal cellular counterparts. HGAL is a novel germinal center (GC)-specific gene whose expression predicts survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). Recently, we have demonstrated that HGAL is involved in negative regulation of lymphocyte migration, thus potentially constraining lymphocytes to the GC and preventing lymphoma dissemination; however, the molecular mechanism of HGAL effects on cell motility is unknown. Herein, we demonstrated that HGAL serves as a physiological regulator of the RhoA signaling pathway. HGAL over-expression or siRNA-mediated knock-down leads to increased or decreased levels of GTP-bound RhoA, respectively, but does not affect levels of GTP-bound CDC42 and RAC1. HGAL-induced activation of RhoA results in inhibition of lymphocyte and lymphoma cell motility and chemotaxis by activation of its downstream effector ROCK leading to: a) phosphorylation of myosin regulatory light chain (MRLC) that regulates actin-activated Mg-ATPase activity of myosin II; b) phosphorylation of myosin phosphatase (myosin PPTase) subunit MYPT1 inhibiting myosin light chain dephosphorylation; and c) phosphorylation of cofilin leading to cytoskeletal reorganization, as reflected by increased formation of focal adhesions and stress fibers. Opposite effects on RhoA downstream effectors are observed upon knock-down of HGAL expression. Furthermore, HGAL over-expression or knock-down modulate additional functions of RhoA, including transcriptional activation by serum response factor and RhoA transforming potential, as assessed by foci formation in NIH 3T3 cells. Co-immunoprecipitation experiments demonstrated that the effect of HGAL on RhoA is indirect and is mediated by RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate. Moreover, we delineate the structural domains of HGAL and PDZ-RhoGEF that mediate the interaction between these proteins. Over-expression of HGAL mutants lacking the PDZ binding domain, mediating the interaction between HGAL and these RhoGEFs, fails to induce activation of RhoA and does not inhibit lymphoma cell motility and chemotaxis. Similarly, knock-downs of PDZ-RhoGEF and/or LARG reverse the inhibitory effects of HGAL on lymphoma cell motility and chemotaxis. These observations reveal a novel molecular mechanism underlying the inhibitory effects of HGAL on the motility of normal GC lymphocyte and GC-derived lymphoma cells. Since HGAL is specifically expressed only during the GC stage of B lymphocyte differentiation, these findings highlight the uniqueness and complexity of cell processes occurring during the GC reaction. They further elucidate the pathophysiologic mechanism underlying the favorable outcome of DLBCL and cHL patients whose tumors express high levels of HGAL protein.