Untitled - Red Temática de investigación cooperativa en cáncer

P-20
THE POLARITY GENE PARD3 IS INACTIVATED IN SQUAMOUS CELL
CARCINOMAS OF THE LUNG
BONASTRE E 1 , ZONDERVAN I 2 , FACCHINETTI F 3 , CONDOM E 4 , SIDRANSKY D 5 , ROZ L 3 , SAVOLA
S 2 , SÁNCHEZ-CÉSPEDES M 1
1
IDIBELL, BARCELONA (RD06/0020/0062)
2
MRC-HOLLAND, NETHERLANDS
3
GINECOLOGÍA HOSPITAL VALL HEBRÓN BARCELONA (RD06/0020/0058)
4
HOSPITAL DE BELLVITGE, BARCELONA
5
JOHNS HOPKINS UNIVERSITY SCHOOL
In spite of the recent advances in cancer genomics, the genetics underlying the development
of lung squamous cell carcinomas (SCC) is still poorly understood. To get insights
into the genes that contribute to lung SCCs we tested for alterations at the cell polarity
related gene, PARD3, in a panel of 58 lung cancer cell lines from various histopathologocial
subtypes. The analysis confirmed an intragenic deletion in a SCC cell line, this and other
informations circumscribed PARD3 alterations to this lung cancer type. Next, we extended
the genetic screening, which also included the determination of intragenic deletions by
multiplex ligation-dependent probe amplification assay, to 107 primary lung SCCs. Six per
cent of the tumors endured biallelic and tumor-specific alterations at PARD3. The absence
of PAR3 protein was evident in those tumor carrying frameshift mutations or intragenic
deletions, while high levels and abnormal immunostaining of PAR3 were observed in some
PARD3 wild type tumors. Since some PARD3 alterations lead to aminoacid changes or predicted
for shorter proteins, we decided to evaluate how these affect the normal function
of the PAR3 protein. Our results clearly evidenced that, in contrast to mutants, only the
wild type PAR3 proteins restituted the formation of tight junctions, clearly evidencing that
these mutations affect the correct function of the protein. In conclusion, the presence of
deleterious mutations at PARD3 attest to its relevance in the development of lung SSCs.
P-21
FUNCTIONAL ANALYSIS OF THE GATA2 PROMOTER SHOWS THAT MUTA-
TIONS OF GATA2 IMPAIR ITS OWN TRANSCRIPTIONAL REGULATION
CORTÉS LAVAUD X 1 , MAICAS M 1 , VÁZQUEZ I 1 , VICENTE C 1 , URQUIZA L 1 , GARCÍA SÁNCHEZ
MA 1 , ODERO MD 1
1
CENTRO DE INVESTIGACIÓN MÉDICA APLICADA (CIMA), PAMPLONA (RD06/0020/0078)
GATA2 encodes a transcription factor with essential functions in hematopoiesis. Recent
studies report mutations in GATA2 in familial syndromes characterized by predisposition to
myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).
It has been showed in murine models that GATA2 activates its own transcription by binding
to regions located at -2.8 and -1.8 kb from the transcription start site (TSS). Thus, we hypothesized
that these GATA2 mutations could alter the GATA2 autoregulatory loop, affecting
the transcription of GATA2. Bioinformatic analysis showed two regions in the human GATA2
promoter, with two putative GATA2 binding sites. ChIP-qPCR assays showed that GATA2
binds to these sites.
To assess the ability of both wild-type and GATA2 mutations to regulate its own transcription,
we transfected these GATA2 gene variants along with different GATA2 promoter
constructs into HEK293T cells, and performed luciferase reporter assays. Wild-type GATA2
activated its transcription through the -2.4 kb site; however, it was not able to activate the
full length promoter construct containing both the -3.4 and -2.4 sites. CEBPA binding sites
near the -3.4 site could explain these results.
The T354M mutant activated GATA2 transcription in a similar manner than the GATA2 wildtype,
raising the question about the complex function of T354M. On the contrary, del355T
was unable of sustain any activation of GATA2.
Finally, the L359V mutation, present in 10% of CML cases with blast crisis, was able to activate
the GATA2 promoter, even the full length promoter construct that contains both -3.4
and -2.4 sites, supporting that L359V is a gain-of-function mutation.
In summary, GATA2 mutations had different effects on the GATA2 promoter that could
affect the dose of GATA2 and therefore, of other GATA2 targets. Studies to further clarify
these questions are in progress.
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