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143PD MET ACTIVATION BY AUTOCRINE LOOP RESCUES COLON
CANCER CELLS FROM SENSITIVITY TO EGFR INHIBITION
T. Troiani, D. Vitagliano, S. Napolitano, F. Morgillo, A. Capasso, V. Sforza,
A. Nappi, L. Berrino, F. Ciardiello, E. Martinelli
Division of Medical Oncology, Department of Experimental and Clinical Medicine
and Surgery “F. Magrassi and A. Lanzara”, Second University of Naples, Naples,
ITALY
Background: Cetuximab, a monoclonal antibody targeting the epidermal growth
factor receptor (EGFR), is active in K-RAS wild type colorectal cancer (CRC).
However, initially in responding patients cancer cells would become resistant to
EGFR inhibition by the activation of alternative growth controlling pathways
including the hepatocyte growth factor (HGF)/MET-dependent signal.
Methods: mRNA expression profiles of cetuximab-sensitive human GEO colon
cancer cells and of their cetuximab-resistant derived GEO-CR cells were analyzed by
microarrays. Protein expression levels were evaluated by western blot (WB). Growth
factors were measure in the culture medium by Luminex technology. The in vitro
antitumor activity of MET inhibitor (METi), was tested in a panel of ten human
colon cancer cell lines by MTT assay.
Results: Evaluation of gene expression profile identified a series of genes that were
up-regulated in GEO-CR cells possibly involved in acquired resistance to EGFR
inhibition. Among these we found several genes involved in the MET pathway. WB
analysis detected activated, phosphorylated MET in GEO-CR but not in GEO and in
the other colon cancer cell lines tested. We also found in GEO-CR cells up-regulation
of EGFR ligands such as transforming growth factor – α (TGFa) and Heparin
Binding- Epidermal Growth Factor (HB-EGF). We further observed expression of
HGF in GEO-CR cells, supporting HGF/MET autocrine activation following acquired
resistance to cetuximab treatment. In fact, the RAS/RAF/MEK/MAPK pathway was
constitutively active despite of EGFR inhibition by cetuximab in GEO-CR cells
possibly due to HGF-induced MET activation. Treatment with a potent and selective
METi was able to overcome cetuximab resistance in GEO-CR cells and causes cell
growth inhibition.
Conclusion: These results suggest that autocrine activation of HGF/MET could be a
relevant therapeutic target in colorectal cancer patients that become resistant to
anti-EGFR treatment.
Disclosure: All authors have declared no conflicts of interest.
144PD OPTIMIZING GEMCITABINE EFFICACY THROUGH
DEGRADATION OF RRM1
G. Bepler, Y. Zhang, X. Li
Karmanos Cancer Institute and Wayne State University, Detroit, MI, UNITED
STATES OF AMERICA
Objectives: RRM1 (ribonucleotide reductase M1) is the molecular target and key
efficacy determinant of gemcitabine. Gemcitabine binds directly to active sites
resulting in irreversible inactivation. Mechanisms that control RRM1 abundance are
largely unknown, but may provide an opportunity for optimization of gemcitabine
efficacy.
Methods and results: We have identified that the E3 ubiquitin-protein ligases RNF2
(RING finger protein 2, Ring1B) and Bmi1 (B cell-specific moloney murine leukemia
virus insertion site 1) interact with RRM1 using yeast two-hybrid screening. We
confirmed that RNF2and Bmi1 interact with RRM1 in vivo by immunoprecipitation.
Confocal immunofluorescence microscopy revealed that RNF2 and Bmi1 completely
co-localized with RRM1 in nucleus. RRM1 undergoes proteasome-mediated
polyubiquitination and degradation. The proteasome inhibitor MG132 blocked the
turnover of RRM1. We found that RNF2 and Bmi1 can induce polyubiquitination of
RRM1 in vitro and in vivo. Lysine (K) 548 and 224 are major polyubiquitination
sites of RRM1. Substitution of the lysine residues 548 and 224, either alone or
together, significantly reduced RRM1 polyubiquitination. Depletion of RNF2 and
Bmi1 by siRNA increased RRM1 protein level and promoted chemoresistance to
gemcitabine in vitro.
Conclusions: These results establish that RNF2 and Bmi1 are E3 ubiquitin ligases of
RRM1 that regulate RRM1 ubiquitination, degradation, and cellular response to
Annals of Oncology 23 (Supplement 9): ix67–ix72, 2012
doi:10.1093/annonc/mds390
gemcitabine. This suggests that RNF2 and Bmi1 might be attractive therapeutic
targets to overcome gemcitabine resistance in malignancies.
Disclosure: All authors have declared no conflicts of interest.
145P ZEB1 AND ZEB2 MEDIATE PANCREATIC FIBROBLAST
INDUCED EPITHELIAL-TO MESENCHYMAL TRANSITION
(EMT) IN PANCREATIC CANCER
L. Visa 1 , E. Samper 2 , M. Rickmann 2 , A. Postigo 3 , E. Sanchez-Tilo 3 ,
L. Fernandez-Cruz 4 , J. Maurel 5 , X. Molero 6 , E.C. Vaquero 2
1 Department Medical Oncology, Hospital Clinic Barcelona, Barcelona, SPAIN,
2 Gastroenterology Department, Hospital Clinic, Barcelona, SPAIN, 3 Oncology
and Hematology, IDIBAPS, Barcelona, SPAIN, 4 Surgical Department, Hospital
Clinic, Barcelona, SPAIN, 5 Medical Oncology, Hospital Clinic i Provincial,
Barcelona, SPAIN, 6 Gastroenterologist, Hospital Vall Hebron, Barcelona, SPAIN
EMT renders neoplastic cancer cells the ability to migrate and to invade distant
organs. The hallmark of EMT is the loss of E-cadherin, which is a prerequisite for
epithelial tumor cell invasion. In pancreatic cancer, loss of tumor E-cadherin is an
independent predictor of poor outcome.AIMS: To analyze the effect of pancreatic
fibroblasts (PF) on inducing EMT in pancreatic cancer cells and to identify the
transcription factors (Snail, Slug, ZEB1, ZEB2) that mediate EMT process.
Methods: Human PFs were isolated from human pancreatic specimens obtained
from chronic pancreatitis and from unaffected margins of pancreatic adenocarcinoma
and serous cistoadenoma. PF were cultured until complete cellular activation, as
assessed by expression of a-smooth muscle actin, vimentin and fibronectin.
Human pancreatic cancer cells Panc-1 were exposed to PF conditioned medium
(PF-CM) and EMT analyzed by cell morphology, migration, and E-cadherin
expression (quantitative RT-PCR and immunoblot). Gene expression of Snail, Slug,
ZEB1, and ZEB2 was analyzed by quantitative RT-PCR, and their activity modulated
by siRNA.
Results: Conditioned media from all types of activated PFs induced EMT changes in
Panc-1 cells, as shown by 1) morphological transition from cobblestone shaped to
fibroblast-like cells, 2) stimulation of cell migration, and 3) E-cadherin down–
regulation; mRNA expression of Snail transiently increased at 30 min after exposure
to PF returning to basal levels afterwards; mRNA levels of ZEB1 were not
up-regulated upon exposure to PF-CM. However, ZEB1 protein greatly accumulated
after 48h incubation with PF-CM, suggesting that PF prevent ZEB1 degradation in
Panc-1 cells. Combined RNA downregulation of ZEB1 and ZEB2, but not of Snail
and/or Slug, suppressed E-cadherin repression induced by PF.
Conclusion: Activated PFs promote the invasive phenotype of pancreatic cancer cells
through ZEB1 and ZEB2 activation.
Disclosure: All authors have declared no conflicts of interest.
146P THE PROGNOSTIC VALUE OF MMP-9, MMP-2, TIMP-1 AND
TIMP-2 IN BREAST CANCER AFTER TWELVE YEARS OF
INITIAL INVESTIGATION
D.C. Jinga 1 , A. Blidaru 2 , I. Condrea 3 , C. Ardeleanu 4 , D. Median 5 ,
M. Stefanescu 6 , C. Matache 6
1 Medical Oncology, University Bucharest Hospital, Bucharest, ROMANIA,
2 Surgical, “Al.Trestioreanu” National Institute Of Oncology, Bucharest, ROMANIA,
3 Pathology, “Al.Trestioreanu” National Institute of Oncology, Bucharest,
ROMANIA, 4 Pathology, Victor Babes National Institute, Bucharest, ROMANIA,
5 Medical Oncology, Institute of Oncology Bucharest, Fundeni Clinical Hospital
Alexandru Treistoreanu, Bucharest, ROMANIA, 6 Immunology, Cantacuzino
National Research for Microbiology and Immunology, Bucharest, ROMANIA
Background: The aim of this study was to investigate the prognostic power of
matrixmetalloproteinases (MMP-9, MMP-2) expression and activity as well as their
natural inhibitors (TIMP-1, TIMP-2) expression in breast cancer tumours.
Patients and methods: All of the experimental parameters were evaluated in fresh
tumour tissue samples from thirty-two consecutive patients with primary invasive
breast carcinomas collected between 2001-2002. Gelatinase activity (MMP-9 and
MMP-2) was analysed by zymography while the expression level of MMP-9, MMP-2,
TIMP-1 and TIMP-2 was quantified by commercial ELISA kits in 1% Triton-X 100
tumour extracts. The Mann-Whitney U test and Spearman’s coefficient where
calculated in order to establish the correlation of the experimentally determined
parameters with relapse free survival (RFS), overall survival (OS) and the presence of
metastasis and death (D) at the end of study.
© European Society for Medical Oncology 2012. Published by Oxford University Press on behalf of the European Society for Medical Oncology.
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