Untitled - Red Temática de investigación cooperativa en cáncer

P-14
AICAR INDUCES BAK/BAK-DEPENDENT APOPTOSIS THROUGH UP-REGU-
LATION OF THE BH3-ONLY PROTEINS BIM AND NOXA IN MOUSE EMBRYO
FIBROBLASTS
MONCUNILL-MASSAGUER C 1 , GONZÁLEZ-GIRONÈS D 1 ,IGLESIAS-SERRET D 1 , COSIALLS AM 1
PÉREZ-PERARNAU A 1 , MARIELA-PALMERI C 1 , RUBIO-PATIÑO C 1 , VIOLLET B 2 , PONS G 1 , VILLUNGER
A 3 , GIL J 1
1
DEPARTAMENT DE CIÈNCIES FISIOLÒGIQUES II, INSTITUT D’INVESTIGACIÓ BIOMÈDICA DE
BELLVITGE (IDIBELL)–UNIVERSITAT DE BARCELONA, L’HOSPITALET DE LLOBREGAT, CATALUNYA,
(RD06/0020/0097)
2
INSTITUT COCHIN, UNIVERSITÉ PARIS DESCARTES, CENTRE NATIONAL DE LA RECHERCHE SCIENTI-
FIQUE (CNRS), PARIS, FRANCE
3
DIVISION OF DEVELOPMENTAL IMMUNOLOGY, BIOCENTER, INNSBRUCK MEDICAL UNIVERSITY,
INNBRUCK, AUSTRIA
AICAR triggers apoptosis independently of AMPK and p53 through the upregulation of the
BH3-only proteins BIM and NOXA in chronic lymphocytic leukemia (CLL) cells. However,
it remains unknown how AICAR treatment modulates the expression of these BH3-only
proteins.
Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the
mechanism of AICAR-induced apoptosis. ZMP formation is required for AICAR-induced
apoptosis and it activates its main target Ampk. Nevertheless, direct Ampk activation with
A-769662 failed to induce apoptosis in MEFs, and AICAR also potently induced apoptosis
in Ampkα1-/-/α2-/- MEFs, demonstrating an Ampk-independent mechanism of cell death.
Notably, MEFs double knockouot for Bax and Bak were completely resistant to the apoptosis
triggered by AICAR, confirming the involvement of the mitochondrial pathway in its mechanism
of action. Apoptosis was preceeded by ZMP-dependent but Ampk-independent modulation
of mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2.
Bim protein levels are accumulated by AICAR treatment of MEF cells, suggesting its role in
the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa display high resistance to
AICAR, in concordance with their requirement for AICAR-induced cell death B lymphocytes.
Overall, we demonstrate that AICAR mechanism of apoptosis is highly similar in MEFs,
mouse B lymphocytes and CLL cells and thus MEFs will be used for the complete elucidation
of AICAR-induced apoptosis mechanism.
P-15
DETERMINATION OF SNAIL1 PARACRINE FUNCTIONS: IMPLICATION
IN PRO-TUMOROGENIC ABILITIES ON COLORECTAL EPITHELIAL CELLS
LINES
HERRERA-TEJERO A 1 , HERRERA-TORRES M 1 ,GARCÍA DE HERREROS A 1 , BONILLA-VELASCO
F 1 , PEÑA-MAROTO 1
1
HOSPITAL UNIV. PUERTA HIERRO-MAJADAHONDA (RD06/0020/0020)
Experimental data support the idea that the active recruitment of stromal cells by tumor
cells is essential for the generation of a microenvironment that actively fosters tumor
growth. Snail1 is a transcriptional factor that plays an important role in epithelial-mesenchymal
transition (EMT)-mediated metastasis through the regulation of E-Cadherin expression.
E-Cadherin is downregulated in human colon epithelial cells when co-cultured with Snail1
expressing cells. Moreover, expression of Snail1 in the tumor stroma correlates with lower
specific survival of cancer patients. The aim of this study was to determine the possible
pro-tumorogenic paracrine functions of Snail1 expressing cells.
Cancer-Associated Fibroblasts (CAFs) and Normal Fibroblasts (NFs) were isolated from 15
primary human colon tumors and 5 normal mucosa respectively. Colon cancer cell lines
SW480-ADH or LIM1215 were co-cultured with a panel of Snail1 expressing colon and
fibroblasts cell lines, or primary CAFs, to study migration and proliferation changes. Coculture
analyses were developed using trans-well culture system. Proliferation assays were
also analyzed with conditioned medium from fibroblasts and CAFs. The Snail1 expressing
cell lines induced higher cell migration of SW480-ADH or LIM1215 compared with Mock
cells. Moreover, increase of proliferation index in SW480-ADH cells were observed when
co-cultured with Snail1 expressing cells. In both migration and proliferation, the effect was
higher when Snail1 was expressed by fibroblasts, regarding tumor colon cells. Analysis of
primary CAFs showed higher Snail expression levels in CAFs compared with NFs. Interestingly,
co-culture of CAFs with LIM1215 or SW480-ADH cells induced a significant increase in
epithelial cell migration, as well as in proliferation, associated with Snail expression.
Together, these experiments demonstrate the Snail1-dependent paracrine pro-tumorogenic
effects of fibroblasts on colon cancer cells. Future analysis will include cytokine profile study
of Snail1 expressing cells, as well as primary CAFs and NFs, to characterize the cross-talk
between colorectal cancer cells and fibroblasts.
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