Relación entre las proteínas parp-1 y hif-1α durante la respuesta a hipoxia tumoral
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Show full item recordEditorial
Universidad de Granada
Director
Oliver Pozo, Francisco JavierDepartamento
Universidad de Granada. Programa de Doctorado en BiomedicinaMateria
Proteínas Hipoxia tumoral
Date
2021Fecha lectura
2021-04-26Referencia bibliográfica
Martí Martín-Consuegra, Juan Manuel. Relación entre las proteínas parp-1 y hif-1α durante la respuesta a hipoxia tumoral. Granada: Universidad de Granada, 2021. [http://hdl.handle.net/10481/70440]
Sponsorship
Tesis Univ. Granada.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.