Caracterización de EMX1 y EMX2 como nuevos genes supresores de tumores en sarcoma

Resumen

In this thesis the biology of the EMX1 and EMX2 genes is studied and they are proposed as the transcription factors responsible for the reduction of cancer stem cell populations (CSCs) that are involved in the sarcomagenesis process, regardless of the neural or mesodermal embryological sarcoma origin. Our work shows that EMX1/EMX2 act as tumor suppressors in sarcomas by repressing the activity of the stem cells properties regulatory genes (OCT4, SOX2, KLF4, MYC, NANOG, NES and PROM1) and the effectors of the canonical Wnt pathway (CTNNB1, TCF4, MYC, WNT1 and CCDN1). It has been proven that the EMX1 or EMX2 overexpression reduces the tumorigenic properties while the reduction of their levels increases these properties. Specifically, in the context of EMX genes overexpression there is a reduction in the proliferative capacity, the clonability, the number of holoclones (CSCs) and the tumorsphere formation capability. Indicating that the self-renewal and maintenance capacities of CSCs are reduced in primary derived sarcoma cell lines and their xenografts in vivo. Furthermore, it has been observed that there are null or very reduced expression levels of EMX1/EMX2 in the cancer stem cells embedded in the sarcoma origin tissue, allowing the self-renewal of the tumor. These results have been validated in murine knock out (KO) models of Emx1 and Emx2, in which an in vivo sarcomagenesis model has been performed. It has been observed that the Emx1 and Emx2 KO induced sarcoma is more aggressive, more infiltrative and with greater capacity for tumor self-renewal, since there is an stem cell genes expression increase. Therefore, it has been shown that EMX genes decrease the expression of the stem cell regulatory properties genes and the stem cell phenotype. Additionally, it has been shown that the canonical Wnt pathway is one of the mechanisms that explains the relationship of EMX1/EMX2 and the stem cells genes in sarcoma. This relationship was validated by the negative correlation between EMX1/EMX2 and the positive effectors of the Wnt pathway confirmation in silico in sarcoma patient databases and in vitro, in primary derived sarcoma cell lines. In addition, the reversion to a more aggressive phenotype was verified in the EMX1 and/or EMX2 overexpression and the constitutive activation of the Wnt pathway. This correlated with the reversion to a phenotype with stem cell properties increased, establishing the relationship between the Wnt pathway, the stem cell genes and the EMX transcription factors. Finally, in general terms, EMX genes are not expressed in tumors and in sarcoma, possibly due to a methylation increase of the EMX1 and EMX2 promoters. This, together with other references in lung and gastric cancer, allows the EMX1 and EMX2 genes to be suggested as possible predictive biomarkers of response in sarcoma. In sarcoma databases it has been shown how EMX high levels patients groups are those with a better overall survival, so the EMX overexpression is related to a better prognosis. These results allow us to establish that sarcoma self-renewing populations originate from neural or mesodermal precursors where the EMX1 and/or EMX2 expression were silenced. The processes of cellular differentiation during the ontogenetic development produce the silencing of EMX genes by hypermethylation of their promoters. This would allow the constitution of sarcoma from NESTIN (NES) positive cell populations or other stem cell genes and activation of the Wnt pathway, in a tumorigenic context where the necessary genetic and/or epigenetic mechanisms are given. All this together indicates that the EMX1 and EMX2 genes negatively regulate these tumor renovating populations or cancer stem cells, acting as tumor suppressors in sarcoma.