The inhibitor of differentiation genes expression and association with epithelial-to-mesenchymal markers in phenotypes of breast canceran in vitro and clinicopathological study

Resumen

Inhibitor of Differentiation (ID) proteins are a family of four (ID1-4) bHLH transcription factors that lack the DNA binding domain. They act by forming dimers with other transcriptional regulators and inhibiting their interaction with DNA. They play a crucial role during embryonic development and later in the adulthood, their expression is mostly restricted to a few populations of stem cells. In the last decades, many authors have described their re-activation and participation in tumor development, angiogenesis and EMT although the results are still controversial. In the first chapter of this research work, the role of ID genes as prognostic markers in breast cancer was evaluated. We studied the mRNA expression of the four ID genes and four markers of EMT by qRT-PCR in a clinical series of 307 primary breast carcinomas previously stratified in immunophenotypes. In addition, the expression of all these genes was measured in breast cancer cell lines and mammospheres. Overexpression of at least one ID gene was found in 48.9% of the studied samples. ID1 and ID4 were overexpressed mostly in TNBL and HER2-enriched subtypes, whereas ID2 and ID3 were overexpressed more frequently in luminal tumors. High ID1 and ID4 was associated with larger tumor size, histological grade 3, presence of necrosis and vascular invasion, and poorer outcome. Multivariate analysis revealed that ID4 and vascular invasion were independent factors for DFS. Regarding EMT markers, high levels of SNAI1 were associated with the overexpression of the four ID genes. Additionally, ID1 overexpression was positively related to TWIST1, and the overexpression of ID2 and ID3 was more frequently paired with tumors that conserve CHD1 expression. In vitro studies showed high expression of the four ID genes in all cell lines. However, when mammospheres were formed, mRNA levels of ID genes decreased, in contrast to SNAI1 and TWIST1, which mostly increased. In the second chapter of this thesis, we aimed to (a) describe the mechanisms of action of a small molecule pan-ID antagonist, (b) define its main targets and (c) investigate potential pathways of acquire resistance. Treatment with AGX51 led to Id protein loss, increase in ROS accumulation, cell cycle arrest, and cell death in all tumor cell lines tested. Here, we used an antioxidant compound in different cell lines to demonstrate that ROS are the main responsible of cell death following treatment with AGX51. A model of cultured quiescent cells not expressing ID proteins served to show that the main target of AGX51 are these proteins. Experiments with AGX-derivatives also supported these results. Finally, three mutagenizing agents were used in order to generate mutations that confer resistance to treatment with AGX51. Treatment with ENU gave rise to two clones apparently resistant to AGX51 effects. Based on our in vitro and clinicopathological studies, we conclude that ID1 and ID4 may act as biomarkers of worse prognosis in patients with breast cancer, and seem to be involved in the initiation of EMT mechanism. Therefore, they are potential targets for the development of novel drugs. In line with this, AGX51 arises as a potent anti-ID compound that has anticancer effects.