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S200
ESTRO 36
_______________________________________________________________________________________________
Material and Methods
To predict the potential targets of miR-200c, a microRNA
database was used for bioinformatics analysis. Malignant
glioma (U251), breast cancer (SKBR3, MDAMB468) and lung
carcinoma (A549) cell lines were obtained from the
American Type Culture Collection (ATCC, Manassas, VA,
USA). Cells were transfected with pre-miR-200c or control
pre-miRNA using siPORTNeoFX™ transfection reagent
(Ambion, Austin, TX, USA). Anti-miR-200c was mixed with
Opti-MEM (Invitrogen, Grand Island, NY, USA), incubated
and added directly to cells. RT-PCR was performed using
the Taqman miRNA reverse transcription kit and the Fast
Real-Time PCR System (Applied Biosystems, Carlsbad, CA,
USA).
Clonogenic
assay,
immunoblotting
and
immunocytochemisty was perfomed.
Results
Ectopic overexpression of miR-200c led to down-
regulation of p-AKT, p-EGFR, and p-HER2 and increased
the radiosensitivity of U251, A549, SKBR3, and MDA-MB-
468 cells. In contrast, a miR-200c inhibitor led to up-
regulation of p-AKT, p-EGFR, and p-HER2 and decreased
radiation-induced cell killing. miR-200c led to
persistent γH2AX foci formation and down-regulated
pDNA-PKcs expression. Autophagy and apoptosis were
major modes of cell death. Bioinformatics analysis
predicted that miR-200c could have association with
EGFR, AKT, MAPK, VEGFA, HIF1AN. We also confirmed that
miR-200c downregulated expression of VEGF, HIF-1α, and
MMP2. Overexpression of miR-200c inhibited invasion,
migration, and vascular tube formation. These were
associated with downregulation of E-cadherin and EphA2,
and up-regulation of N-cadherin. miR-200c showed no
observable cytotoxic effect on normal human fibroblasts
and normal human astrocytes.
Conclusion
Taken together, our data suggest that miR-200c is an
attractive target for improving the efficacy of
radiotherapy via unique modulation of the complex
regulatory network controlling cancer pro-survival
signaling and EMT.
PV-0371 Novel molecular radiobiology for
personalised prostate cancer radiotherapy
N. McDermott
1
, A. Meunier
1
, T. Jameson
2
, A. Mansour
2
,
C. Haynes
2
, A. Flores
2
, A. O'Callaghan
1
, L. Marignol
1
1
Trinity Centre for Health Sciences Discipline of
Radiation Therapy, Radiation Therapy, Dublin, Ireland
2
Mount Sinai School of Medicine, International Health,
New York, USA
Purpose or Objective
The integration of tumour-specific biological parameters
to the decision-making process is anticipated to overcome
the recognised limitations of the current risk stratification
system for prostate cancer and transform the practice of
radiation oncology. Molecular imaging techniques are
rapidly advancing our ability to assess the extent and
aggressiveness of prostate cancer. Differential analyses of
extensive genetic profiles of specimens have progressed
the use of genetic signatures from tumour tissue in
providing additional prognostic information. Adopting an
hypothesis-based approach to the identification of novel
radiobiology that can assist the personalisation of prostate
radiotherapy, we propose that that the signalling
pathways that regulate several cancer hallmarks and are
responsive to hypoxia, such as Notch and YB-1 regulate the
molecular response of cells to radiation.
Material and Methods
The analysis of a panel of 22Rv1 prostate cancer cells was
used to support the identification of novel biomarkers of
radioresistance. First, an isogenic model of
radioresistance was generated in 22Rv1 prostate cancer
cells through exposure to 30 x 2-Gy dose fractions. Second,
radioresistance was induced in 22Rv1 cells through
exposure to hypoxic conditions (0.5% O2, 24hrs). miRNA
profiling of these samples was performed and validated by
RT-PCR. Novel signalling interactions were characterised
by western blotting, and a series of cell-based assays.
Results
Exposure to fractionated 2Gy-irradition to a cumulative
total dose of 60Gy selected for 22Rv1 cells increase in
clonogenic survival following irradiation (1.3 fold increase
in survival after 2Gy and 2.2 fold increase after 10Gy)
when compared to both parent 22Rv1 and aged-matched
controls. The cross-evaluation strategy of the molecular
modifications associated with a combination of
radiobiological factors has identified miR-4284 as down
regulated amongst radiation resistant models. Further
evaluation of this miRNA indicates interaction with RLIM,
RASGEF1, YB-1 and Notch-3. YB-1 inhibition with Fisetin
significantly reduced clonogenic survival following
irradiation, and modified Notch-3 receptor activation.
Analysis of RNA extracted from a series of pooled samples
from prostate cancer patients identified elevation of
notch-3 mRNA levels in higher grade and hypoxic tumours.
Validation in cell lines further identifies modification in
Notch-3 activation following 5-Gy irradiation.
Conclusion
This study identifies novel molecular radiobiology that
may explain the multiple effects of radiation on the
molecular biology of prostate cancer cells. This work has
the potential to influence future direction of suitability
and treatment of radiotherapy prostate patients.
PV-0372 Histology-specific quantitative mapping and
targeting of glucose and glutamine metabolism in
NSCLC
T.W.H. Meijer
1
, P.N. Span
1
, W.J.M. Peeters
1
, R.
Biemans
3
, L.F. De Geus-Oei
2
, D. Vriens
2
, L.J. Dubois
3
, J.
Bussink
1
1
UMC St Radboud Nijmegen, Radiation Oncology,
Nijmegen, The Netherlands
2
Leiden university medical center, Radiology, Leiden,
The Netherlands
3
Maastricht University Medical Centre, Radiation
Oncology, Maastricht, The Netherlands
Purpose or Objective
Increased glycolysis and glutamine use are related to
resistance to radiotherapy. Therefore, targeting tumor
cell metabolism may improve radiotherapy efficacy in
NSCLC. In this prospective cohort study, we describe
pharmacokinetic rate constants of
18
F-FDG metabolism
(K
1
-k
3
) and fractional blood volume (V
B
) in regions with
different levels of glucose metabolic rate (MR
glc
) and
compare these between the major NSCLC histological
subtypes (adeno- (AC) and squamous cell carcinomas
(SCC)). Furthermore, glycolytic rate and growth delay plus
apoptotic index by glucose and/or glutamine inhibition
were assessed in six NSCLC cell lines
in vitro.
Material and Methods
One-hour dynamic
18
F-FDG-PET/CTs were acquired in 38
NSCLC patients (tumor size at least 30 mm in diameter).
Parametric images of Patlak MR
glc
values were obtained.
Lesions were delineated using the fuzzy locally adapted
Bayesian (FLAB) algorithm. Tumors were divided into
three equal volumes of increasing MR
glc
, in which K
1
-k
3
and
V
B
were computed.
For
in vitro
experiments, AC (H522, HCC827, H1975) and
SCC (H520, H226, SW900) NSCLC cell lines were used.
Glycolytic rate of cell lines was assessed by the
percentage extracellular acidification rate (% ECAR) under
normoxia and physiologic amount of glucose (i.e. 1.5 mM)
using Seahorse. Growth delay and apoptosis analyses were
performed under normoxia and 1.5 mM glucose using
IncuCyte. To examine the effect of metabolic inhibition
on growth delay and apoptotic index, the glycolysis
inhibitor lonidamine and/or glutaminase inhibitor 968
were used.