Participation of the mitofusin 2 protein (mfn2) in cell immortalization and transformation

Identifier:  TFG:2941

Handle: https://hdl.handle.net/20.500.11797/TFG2941

Authors:  Páramo Castillejo, Idoia

Abstract:
Mitofusin 2 (Mfn2) is a mitochondrial outer membrane GTPase, key in the mitochondrial fusion process; whose deficiency has been associated with some types of cancer. During the process of cancer biogenesis, a set of gene expression, epigenetic and metabolic changes originate in the cell that lead to cell immortalization and transformation. The metabolic changes originated in the process of cell immortalization and transformation increase the use of amino acids, lipids and glucose by different metabolic routes, through mechanisms of cellular metabolic reprogramming. Mfn2 seems to play a relevant role in metabolic reprogramming. Our laboratory has shown that the loss of Mfn2 function leads to an increase in the utilization of amino acids, such as glutamine and cystine, by the cell. In this project we are interested in investigating in which step of the cell immortalization and/or transformation process, the mitochondrial protein Mfn2 would play a relevant role in leading to a metabolic change required to sustain cell proliferation. To this end, we generated a series of immortal cell lines from adult human and mouse skin primary cultures. The results of this work showed that the immortalization of human and mouse parental fibroblasts produces a phenotypic change, characterized by; decreased cell size, the formation of cell clusters, and increased proliferation. Interestingly, the immortalization of parental human fibroblasts through the expression of SV40L or Ras G12V, produces an increase in the expression of the Mfn2 protein. Similar results were observed in mouse fibroblasts immortalized with SV40L. In order to investigate the participation of Mfn2 in the immortalization process, we generated knockdown human cells in the expression of the Mfn2 protein, using a lentiviral vector expressing a shRNA directed to the Mfn2 mRNA. Our studies with crystal violet showed that the silencing of Mfn2 in immortal cells promotes cell proliferation, indicating that this protein could be involved in enhancing the cellular transformation process. On the other hand, during immortalization with SV40L, the expression of proteins involved in the Krebs cycle, such as the C subunit of the enzyme succinate dehydrogenase (SDHC), and in the metabolism of amino acids, such as asparagine synthetase (ASNS), whose genes are activated during the metabolic reprogramming of amino acids.