טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBrekhman Vera
SubjectMolecular Mechanisms Mediating Lysyl Oxidase Like Protein
2 (LOXL2) Activity in Tumor Progression
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Gera Neufeld
Full Thesis textFull thesis text - English Version


Abstract

Previous studies have shown that expression of LOXL2 in MCF7 cells enhances invasiveness of these cells in an in vivo tumor model. To further characterize the influence of LOXL2 on the invasion process we developed a three-dimensional system that allows quantitative monitoring of the invasion of cancer cells. In this assay cancer cells are seeded as a monolayer between two connective tissue layers and allowed to migrate out of the monolayer. At different time points, the composite gels are sectioned and the number of invaded cells and the distance they passed through from the original monolayer is measured.

In order to explore the role of LOXL2 in cancer cells invasiveness we inhibited LOXL2 expression in MDA-MB-231, LM2-4 and HT1080 cells. Using our invasion assay, we demonstrated that inhibition of LOXL2 expression in these cells strongly diminished their invasiveness in vitro.

To analyze the tumorigenic potential of MDA-MB-231 breast cancer cells in which the expression of LOXL2 was inhibited, these cells were allowed to form tumors in the mammary fat pads of female nude mice. Tumors induced by LOXL2 silenced cells grew at a slower rate then tumors developing from control cells and displayed a significant reduction of 50% in tumor weight.

To better understand the molecular mechanisms of LOXL2 activity in tumor progression, we performed a comparative gene expression analysis of control and LOXL2 overexpressing MCF7 cells in order to identify genes that are involved in the aggressive phenotype of MCF7-LOXL2 cells. We identified a gene called Receptor Activity-Modifying Protein 3 (RAMP3) that was strongly up-regulated following LOXL2 overexpression.  LOXL2 silencing in tumor cells has diminished the RAMP3 transcript level. We found that reexpression of RAMP3 could partially restore the invasive capacity and mesenchymal features of the LOXL2-silenced cells.

Our present study suggests that RAMP3 can affect the behavior of tumorigenic cells independently of LOXL2. MDA-MB-231 cells in which the expression of RAMP3 was inhibited assumed an endothelial cells-like shape, had diminished concentration and cellular distribution of the mesenchymal marker vimentin. Silencing of RAMP3 in MDA-MB-231 and LM2-4 cells resulted in a statistically significant inhibition of these cells' invasiveness in an in-vitro invasion assay, suggesting that RAMP3 contributes to the invasive potential of metastatic cells.