Untitled - Red Temática de investigación cooperativa en cáncer

P-39
NOVEL IDENTIFICATION OF EXOSOMES IN UTERINE ASPIRATES AND ITS
UTILITY AS A SOURCE FOR NEW BIOMARKERS
CAMPOY I 1 , MARTÍNEZ GARCÍA E 1 , DEVIS JAUREGUI L 1 , GARCÍA A 1 , CASTELLVÍ J 1 , CABRERA S 1 ,
GIL-MORENO A 1 , ABAL M 2 , REVENTÓS J 1 , COLÁS E 1
1
HOSPITAL VALL D´HEBRÓN, BARCELONA (RD06/0020/0058)
2
CHUS, SANTIAGO DE COMPOSTELA
Background and aim: Exosomes are 40-100 nm extracellular vesicles released from a
variety of cell types. Our aim is to use exosomes as a source to identify new sensitive and
specific biomarkers for the detection of endometrial cancer through a non-invasive method,
isolating them from the uterine aspirate, which is the nearest and the easiest-to-access
body fluid in contact with the endometrium. To date, reports on exosomes coming from
uterine aspirates remain undescribed.
Methods: We standardized the protocol for the isolation of exosomes (both from uterine
aspirates and from media of cancer cell lines in culture) by ultracentrifugation-based techniques;
we characterized them by electron microscopy and immunoblot. We performed a
pilot study by LC-MS/MS isolating, quantifying, digesting and analyzing its protein fraction.
Computational analysis was performed by using Ingenuity Pathways Analysis.
Results: Membrane-vesicles were observed in uterine aspirates by electronic microscopy
by negative staining. Also, western blot analysis of the isolated exosomes revealed the presence
of known exosome markers, such as CD81, TSG-101 and other membrane proteins,
such as Annexin 2, both in uterine aspirates and cultured cell lines. LC-MS/MS analysis
enabled the identification of 153 proteins in the uterine aspirate exosomes. From the list of
proteins identified, we recognized several proteins that belong to the exosome biogenesis
and also endometrial cancer related proteins. IPA analysis identified Cellular Movement,
Immune Cell Trafficking, Cell-To-Cell Signaling and Interaction as the top networks, Cancer
as the top disease by Bio Function classification and Cell-To-Cell Signaling and Interaction
as the top Molecular and Cellular Function.
Conclusion: Our data demonstrates that the identification of the protein content of exosomes
isolated from uterine fluids is feasible and promising for the identification of critical
molecules involved in endometrial cancer, which can be used as diagnostic markers.
P-40
INHIBITORY MECHANISMS OF THE RESPONSE TO TGF-B BY THE THYROID
HORMONE RECEPTORS
ALONSO MERINO E 1 , MARTIN OROZCO R 1 , RUIZ LLORENTE L 1 , FANJUL RODRIGUEZ LF 1 , MARTI-
NEZ IGLESIAS O 1 , ARANDA IRIARTE A 1
1
INSTITUTO DE INVESTIGACIONES BIOMÉDICAS “ALBERTO SOLS”, MADRID (RD06/0020/0036)
The thyroid hormone receptors, encoded by the TRα and TRβ genes, are ligand-dependent
transcription factors that belong to the nuclear receptor superfamily, being the 3,5,3’-triiodothyronine
(T3) their main ligand. In addition to the role of these receptors in growth,
development and metabolism, there is increasing evidence that they can also inhibit transformation
and act as tumor suppressors.
We now have analysed the possibility that TRs could block responses to the transforming
growth factor beta (TGFβ). This regulatory cytokine exerts tumor-suppressive effects in
normal cells, but on the other hand, cancer cells evade this repression and TGFβ can promote
some tumorogenic processes such as cell proliferation, cell invasion, immune regulation
and microenvironmet modification.
In this work we demonstrate that TR, activated by T3 binding, can block transactivation
by TGFβ of reporter plasmids containing a Smad Binding Element (SBE) and repress transcription
of endogenous TGFβ target genes. We have also demonstrated that transfection
of Smad transcription factors stimulate the activity of the reporter plasmid in the absence
of TGFβ, and that T3 inhibits this activation. In immunofluorescence assays we have seen
that reversion of the TGFβ response is not mainly due to inhibition of Smads translocation
to the nucleus. However, in chromatin immunoprecripitation assays (ChIP), we have observed
that T3 inhibits TGFβ–dependent recruitment of Smads to target genes promoters
containing SBEs.
In addition, T3 reduces acetylated histones and increases histone decetylase 3 (HDAC3)
recruitment to TGFβ target genes. The hormone is also able to block TGFβ-dependent
proliferation and migration. These results demonstrate that T3 blocks transcriptional responses
to TGFβ, and suggest that some of the anti-tumorigenic and anti-invasive actions of
the thyroid hormone receptors can involve repression of the activity of the TGFβ signalling
pathway.
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