Abstract A112: Cell differentiation and downregulation of MET receptor as novel approaches for rhabdomyosarcoma treatment

Abstract

Introduction: The molecular basis for loss of cell differentiation in the process of carcinogenesis is not well understood but differentiation status of the given tumor strongly influences its progression and metastatic ability and therefore the overall patient survival. Rhabdomyosarcoma (RMS) is one of most frequent soft tissue tumor occurring in children and adolescent. The precise molecular mechanism responsible for the disruption of myogenesis, characteristic for RMS tumors, is not fully understood. Activation of MET receptor has been shown to influence proliferation, survival and migration of RMS cells and MET is rapidly downregulated at the onset of myogenic differentiation. However, the influence of MET receptor on differentiation status of RMS has not been studied at all. Objectives: We have focused on the induction of differentiation as a novel approach for RMS treatment and looked at the role of MET receptor in this process. Materials and Methods: The expression of muscles specific genes (MyoD, Myogenin) was evaluated using RT-PCR and western blot in differentiated RMS cells and RMS cells with downregulated MET receptor. Number of apoptotic cells was studied by Annexin V binding assay. In order to study the differentiation potential of MET negative cells, RMS animal model in NOD-SCID mice was developed. We studied tumor morphology (H&E staining), maturity (desmin staining) and proliferation rate (Ki67 staining). Results: We found that in control cells (poorly maturated RH30 cell line), the expression of MyoD (present in undifferentiated muscle cells) was high whereas during differentiation it decreased. On the other hand, Myogenin expression (present in more matured muscle cells) markedly increased at the same time. We noticed increased number of apoptotic cells in differentiation medium with 2 % of horse serum. We also obtained RMS cell lines with stable downregulation of MET receptor. The expression of differentiation related genes in MET negative cells was similar to serum differentiated RMS cells. MyoD expression was almost completely reduced in these cells. Analysis of tumor lesions formed in NOD-SCID mice showed that tumors with downregulation of MET were much more differentiated and their proliferation rate was much lower. MET negative tumors were also less metastatic in comparison to wild type controls. These data are in concordance with decreased expression of MyoD in vitro and suggest higher differentiation level of MET negative tumors in vivo. Summary: In this study, for the first time, we have shown that differentiation of RMS cells is connected to the decrease of expression and signaling of MET receptor. These findings might have the significant clinical implication for the treatment of RMS cells because they suggest that induction of differentiation of RMS cells by e.g. blocking MET receptor might have influence on the aggressiveness/metastatic potential of these tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A112.