Untitled - Red Temática de investigación cooperativa en cáncer

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METHODS FOR RNA AND miRNA PURIFICATION IN EXOSOMES FROM
URINE OF PROSTATE CANCER PATIENTS
OLIVAN M 1 *, RIGAU M 1 *, MONTES M 1 , SEQUEIROS T 1 , ALTADILL T 2 , GARCIA M 1 VIEGAS
M 2 , REVENTÓS J 1 , DOLL A 1
1
BIOMEDICAL RESEARCH UNIT, RESEARCH INSTITUTE, VALL D’HEBRON UNIVERSITY HOSPITAL,
BARCELONA (RD06/0020/0058)
2
TRANSBIOMED S.L. PARC CIENTÍFIC DE BARCELONA
* EQUALLY CONTRIBUTING
Urine has become one of the most attractive biofluids in biomedical research. Urinary exosomes
have been recently described as carriers of genetic information and a potential
source of new cancer biomarkers, specifically for prostate cancer (PCa). Analysing the
transcriptome in secreted PCa exosomes in urine has the advantages of being both noninvasive
and informative as to the overall tumor malignancy status, including tumor mRNA
and miRNA levels to be used for PCa diagnosis.
Biomarker discovery approaches in urine have been hindered by concerns for reproducibility
and inadequate standardization of protocols. Reproducible procedures for the preparation
of RNA and miRNA samples isolated from urinary exosomes are essential for meaningful
transcriptomic analyses.
Here we tried to optimize RNA and miRNA extraction from urinary exosomes. In order to
evaluate the efficacy of purification we performed RTqPCR for different housekeeping genes
and miRNAs.
Our results show that urinary exosomes serve as excellent reservoir for nucleic acids, as
they maintained stable along the time, and therefore can be suitable for PCa biomarker
discovery.
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P95HER2/90-100kDa INDUCED SIGNALING LEADS TO APOPTOSIS ELUCI-
DATING A POSSIBLE MOLECULAR SWITCH BETWEEN OIS AND APOPTOSIS
BERNADÓ MORALES C 1 , ARRIBAS J 1
1
VALL D’HEBRÓN INSTITUT D’ONCOLOGIA (VHIO), BARCELONA (RD06/0020/0022)
HER2, an ErbB family tyrosine kinase receptor, is overexpressed in 15%-25% of breast
cancer tumors and its overexpression correlates with the severity of the malignancy. A
subtype of HER2-positive tumors of particularly poor clinical outcome expresses, in addition
to full-length HER2, a heterogeneous series of HER2 c-terminal fragments (CTFs), collectively
known as p95HER2. So far, only membrane bound fragments have been shown to be
active: the p95HER2/90-100kDa, generated by proteolytic cleavage, and the p95HER2/100-
115kDa, generated by alternative initiation of translation. Expression of p95HER2/100-
115kDa leads cells to senescence, an essentially irreversible cell cycle arrest, contributing to
tumor growth and metastasis formation by secreting many pro-tumoral and pro-metastatic
factors.
In this work we address the functional characterization and the mechanism of activation of
p95HER2/90-100kDa concluding thatp95HER2/90-100kDa, unlike HER2 and the previously
characterized p95HER2/100-115kDa, is not able to signal in an autonomous manner in cells
expressing low levels of other HER receptors. In fact, we show that p95HER2/90-100kDa is
activated upon heterodimerization with HER3.
Functionally, expression of p95HER2/90-100kDa, although being very similar to
p95HER2/100-115kDa, leads to cell death. Analysis of different apoptosis markers has
shown that the expression of the p95HER2/90-100kDa induces apoptosis, and only a few
fraction of the cells enter in senescence. Although both senescence and apoptosis restrain
tumor growth, senescent but not apoptotic cells may contribute to tumor invasion. Therefore,
the unexpected different outcome between the two p95HER2 active forms provides us a
useful tool to define a possible switch between apoptosis and senescence.
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