Untitled - Red Temática de investigación cooperativa en cáncer

P-24
NOX4 PLAYS A TUMOR SUPRESSOR ROLE IN HEPATOCELLULAR
CARCINOMA CELLS
Crosas-Molist E 1 , Bertran E 1 , Fernando J 1 , Fabregat I 1, 2
1
IDIBELL, BARCELONA (RD06/0020/0097)
2
UNIVERSIDAD DE BARCELONA, BARCELONA
Introduction and AIMS: NOX4 has been implicated in a variety of physiological processes,
including cellular senescence, apoptosis, survival, migration, endoplasmic reticulum
stress, and differentiation (Brown and Griendling, Free Radic Biol Med. 2009). In liver cells,
NOX4 upregulation is necessary for TGF-beta-induced suppressor effects, in particular, for
cell death (Carmona-Cuenca et al., J Hepatol. 2008; Caja et al., Cancer Res. 2009). In order
to analyze whether NOX4 may play a general tumor suppressor role in liver tumorigenesis,
we have analyzed the effect of NOX4 knock-down in human HCC (hepatocellular carcinoma)
cell lines.
Results: Stable silencing of NOX4 conferred an increased proliferative capacity to HCC
cell lines, as proved through a DNA synthesis assay and by impedance measurement of
attached cells through time (Roche xCELLigence System). Caspase-3 activity assay was performed
in order to analyze basal cell death, showing decreased apoptosis in silenced cells
when compared to control cells. Interestingly,NOX4 silencing increased migration capacity.
This finding was in accordance to a diminished cell-ECM (Extracellular Matrix) adhesion
capacity, important for individual migration. Cell-to-matrix adhesion was analyzed by impedance
measurement in different substrates such as an inert matrix, collagen I, collagen
IV and fibronectin. Results showed a decreased adhesion capacity of the silenced cells in
all the substrates studied. Moreover, Vinculin and p-FAK immunofluorescence showed a
different pattern in NOX4 knock-down cells in comparison to control cells. Finally, stable
silencing of NOX4 increased the tumorigenic capacity of HCC cells after subcutaneous injection
of them into Balb c/Nude mice.
Conclusion: Stable silencing of NOX4 in HCC cells confer them a more malignant phenotype,
since they show increased cell proliferation and decreased basal apoptosis. Simultaneously,
a more invasive phenotype is observed, with decreased cell-to-cell contact and
cell-to-matrix attachment, and increased migration. In summary, NOX4 may play a relevant
tumor suppressor role in liver tumorigenesis.
P-25
AhR BINDING TO Alu ELEMENTS X14S, X36S AND X45S MODULATES THE
EXPRESSION OF STEMNESS-RELEVANT GENES Oct4, Nanog, Shh AND Sox2
GONZÁLEZ RICO FJ 1 , ÁNGEL CARLOS ROMÁN 2 , PEDRO M. FERNÁNDEZ SALGUERO 1
1
UNIVERSIDAD DE EXTREMADURA, BADAJOZ (RD06/0020/1016)
2
INSTITUTO CAJAL, CSIC
Eukaryotic genomes are organized into expression domains whose activity depends on
local chromatin structure. To accomplish that, functional gene clusters are defined by the
binding of transcription factors to conserved regulatory sequences. Transposable elements
are no longer considered parasite mobile sequences; instead they seem to regulate gene
expression by modulating genome organization and stability. Such transposon functions are
likely associated to their ability to bind specific transcription factors.
We have previously reported that a genome-wide SINE-B1 retrotransposon (B1-X35S) has
a potent insulation activity by binding of the transcription factors dioxin receptor (AhR) and
Slug (Snai2) to their consensus elements present in the transposon sequence. In the human
genome, three Alu elements have been identified that are heterologous to the B1-X35S
mouse transposon and that contains a conserved AhR binding site. Interestingly, these Alus
(hereafter X14S, X36S and X45S) are present in most stemness-relevant genes including
Oct4, Nanog, Shh, Sox2 and Notch1.
In the undifferentiated embryonic carcinoma Ntera-2 cell line, treatment with retinoic acid
(RA) induces differentiation by decreasing Oct4, Nanog and Shh expression levels. Remarkably,
RA-induced differentiation promotes a parallel increase in AhR protein. Silencing
of AhR by RNA interference (siRNA of shRNA) blocks the decrease in Oct4, Nanog and Shh
induced by RA thus maintaining an undifferentiated state. Chromatin inmunoprecipitation
(ChIP) experiments were performed to confirm AhR binding to the X14S, X36S and X45S
Alu elements present in the promoter of such target genes. In addition, enhancer blocking
assays (EBAs) will be used to address the insulator activity of X14S, X36S and X45S Alu
elements. Their insulator activity will be confirmed in vivo in zebrafish.
We suggest that X14S, X36S and X45S Alu elements can represent evolutionary conserved
genome-wide insulators activated by the transcription factor AhR to control developmental,
oncogenic or toxicological-dependent processes.
This work was supported by grants from Ministerio de Economía y Competitividad
(BFU2009-07219 and ISCIII-RTICC: RD06/0020/0097) and Generalitat de Catalunya
(2009SGR312). EC is recipient of a FPU fellowship from Ministerio de Educación, Cultura
y Deporte
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