Relación entre las proteínas parp-1 y hif-1α durante la respuesta a hipoxia tumoral

Abstract

Cancer is one of the most relevant diseases due to its social and economic impact. In 2018, 18.1 million new cases were diagnosed, and 9.6 million people died because of the disease. Considering that the number of patients is expected to increase up to a 60% on 2040, and the number of deaths up to a 70% compared to 2018, the interest in treating this disease is currently at its highest. Cancer involves the disruption of multicellular coexisting routes. The cancer cell accumulates mutations that embarks it on a dedifferentiation process, in which single-cell survival pathways are overexpressed and become independent of multicellular regulation. Two of the main known survival routes are object of study on this Doctoral Thesis, those are the hypoxia response pathway and the DNA damage response pathway. During tumor progression, it is common to observe an induction of oxidative stress, as well as the appearance of areas with low oxygen concentration. In this aggressive microenvironment, selective forces will be generated selecting clones that overexpress both pathways, facilitating cell survival and disease progression. The response to the hypoxic situation is one of the main events observed during the malignancy progression. When a tumor grows beyond a few cubic millimetres, oxygen limitation occurs in the central areas of the tumor mass. In this context, the stabilization and activation of the family of transcription factors known as hypoxia-inducible factors (HIFs) occurs, being HIF-1α one of the best-known members of this family. These transcription factors induce genes related to activities related with the adaptation to the hypoxic context, such as the activation of angiogenesis, enhancement of glycolysis, inhibition of oxidative respiration and global repression of transcription, among others. On the other hand, one of the main components of the response to DNA damage (DDR) is the PARP-1 protein. In response to DNA damage and by consuming ATP and NAD+, PARP-1 generates a polymer of poly(ADPribose). Then PARP-1 binds this polymer to different proteins (including itself) modifying its activity and stability. PARP-1 participates in other activities independent of the DDR, such as the regulation of transcription, the conformation of chromatin and the response to inflammation, among others. As indicated in the title of this Doctoral Thesis, in this work we explore the “Relationship between PARP-1 and HIF-1α proteins during tumor hypoxia”. In our work we observe the overexpression of PARP-1 and HIF- 1α in melanoma, both at the protein level (in melanoma in situ), and at the mRNA level (in cutaneous, uveal and acral melanomas). On the other hand, we analyse in different tumor cell lines how treating with PARP inhibitors or with silenced PARP-1 causes a loss of stability on HIF-1α. The opposite happens when silencing the PARG protein (which degrades the polymer), this leads to an increase on the stability of HIF-1α. Using different fluorescent probes for the determination of reactive oxygen species (ROS) we verify in tumor and non-tumor cell lines how ROS induction occurs during early hypoxia. When treating with antioxidants, we observe the same effect described when inhibiting PARP, the accumulation of poly (ADP-ribose) and the stability and activity of HIF-1α decrease in both cases. This demonstrates how ROS production connects with the activation of PARP-1, necessary for the stabilization and activation of HIF-1α during early hypoxia. Studies using different domains of HIF-1α have allowed us to observe how it is the C-terminus domain of HIF-1α the one that is susceptible of regulation via PARP-1. Using pull-down and immunoprecipitation assays we demonstrate how the C-ter domain of HIF-1α binds to the selfmodifying region of PARP-1. Using an in vitro PARylation assay we verified how, consequence of this physical interaction, the C-ter of HIF-1α is modified by polymer in at least two amino acid sequences. To conclude, we have performed a ChIP-Seq analysis in cells with PARP-1 WT or PARP-1 knockout obtained via CRISPR / Cas9. This assay allows us to observe how in the absence of PARP-1, HIF-1α reduces its binding to the DNA and how this loss of recruitment occurs in a way that is dependent on the gene function. In the same way, we observe how the reduction of HIF-1α occurs mainly in the promoter areas, especially one kilobase around the origin of transcription of the gene. We also verify how, even when HIF-1α binds to its promoters, the absence of PARP-1 interferes with the correct expression of the genes. Considering all the results described, we demonstrate the existence of a relationship between the DDR pathway and the response to hypoxia that could have a translational potential, since the HIF proteins lack specific inhibitors, while PARP inhibitors are already approved in the clinic.