טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSoueid-Baumgarten Sharon
SubjectCharacterizing the Role of Lysyl Oxidase Like protein-2
(LOXl2) in Tumor Progression
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Gera Neufeld


Abstract

 The lysyl oxidase gene family members function as extracellular matrix modulating enzymes. It was found that a member of this family, Lysyl Oxidase like protein-2 (Loxl2), is highly expressed in metastatic breast cancer derived cell lines but not in the non-metastatic MCF7 cell line. Loxl2 expressing MCF7 cells induced deposition of collagen fibers and production of fibrotic foci in tumors that developed following the implantation of these cells in mice. These changes were accompanied by an increase in invasiveness. In order to determine if the enzymatic activity of Loxl2 is required for the induction of these effects, a point mutation was made in order to create the Loxl2-Y689F mutant. Loxl2-Y698F was completely inactive in comparison to the Loxl2 wild type (WT). MCF7 cells were infected with a lentivirus vector containing Loxl2-WT or Loxl2-Y689F cDNA. Neither Loxl2-WT nor Loxl2-Y689F affected the proliferation of MCF7 cells in-vitro. MCF7 cells expressing Loxl2-WT or Loxl2-Y689F were implanted in nude mice. Tumors derived from MCF7 cells expressing Loxl2-WT developed more rapidly than tumors derived from cells expressing the inactive Loxl2 mutant. In our experiment, we did not detect invasiveness. However, we found that the enzymatic activity of Loxl2 is important for the formation of necrotic and fibrotic foci in the resulting tumors, since tumors derived from MCF7 cells expressing Loxl2-WT contained more fibrotic and necrotic foci than tumors derived from MCF7 cells expressing Loxl2-Y689F.

A binding assay, using 125I-Loxl2, revealed that Loxl2 binds specifically to cell surface receptors on HUVEC cells. The inactive Loxl2-Y689F inhibited the binding of Loxl2 to HUVEC, implying that the enzymatic activity is not required for receptor recognition. Neither heparin nor heparinase treatment inhibited the binding of Loxl2 to HUVEC. The binding of Loxl2 to the HUVEC was sensitive to changes in pH. A wash with 1.5M NaCl, intended to remove membrane associated proteins prior to 125I-Loxl2 binding, did not affect 125I-Loxl2 binding to HUVEC, suggesting that the receptor is an integral cell membrane protein. Heat denaturized Loxl2 still retained about 80% of its binding ability, suggesting that Loxl2 is a heat resistant enzyme. Scatchard analysis of Loxl2 binding experiments revealed that the Loxl2 receptors display a relatively high affinity and are quite abundant on cell surface.

When performing a cross-linking experiment, we detected a cross-linking product, which is yet to be determined whether this band contains the Loxl2 receptor.