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theorized that the PI3K/AKT pathway and mTOR, one of its main effectors, play a role
in CSC maintenance. Moreover, hypoxia was known to be involved in CSC
maintenance and to have a regulating effect on mTOR signaling.
In our study, we therefore evaluated the effects of mTOR inhibitors on proliferation,
assessed the activity of the mTOR pathway and measured the mitochondrial activity
as well as levels of radical oxygen species (ROS) of human as well as murine CSCs
in vitro. Further, we examined basal levels of mediators of hypoxic signaling such
as HIF1α and its effectors.We isolated CSC like cells from the human PCa cell line
DU145 and the murine PCa cell line TRAMPC1 using FACS according to their
expression of the stem cell markers CD44, CD49f and Sca-1. We used sphere
formation assays to confirm stem and progenitor cell properties. Next, we treated
the CSC and non-CSC cell populations with mTOR inhibitors under normoxic and
hypoxic conditions in order to determine their cell viability at time points up to
72h. Mitochondrial activity was measured using extracellular flux (XF) analysis and
levels of ROS were obtained using ROS-specific fluorescent dyes and flow cytometry.
Our results suggest that CSC like PCa cells are more resistant to mTOR inhibitors
when compared to their non-CSC counterparts. Most interestingly, while the
non-CSC population displays higher sensitivity to mTOR inhibitors under hypoxic
conditions, the viability of CSCs remains unaffected. Immunoblotting uncovers
generally lower mTOR activity in CSCs compared to non-CSCs. Hypoxia leads to a
decrease in mTOR activity in both subpopulations, though this effect is stronger in
CSC like subpopulations through higher HIF1α-mediated negative feedback in both
cell lines studied. At the same time we do not observe alterations in signaling
through AR or PI3K downstream effectors besides mTOR, such as AKT. We
propose that prostate CSCs might be resistant against mTOR inhibition and that
inhibitors targeting multiple kinases along the PI3K/AKT/mTOR axis would be
more effective. Our findings could be helpful to assess the impact of mTOR
targeted therapy in prostate cancer in light of ongoing clinical trials.
Disclosure: All authors have declared no conflicts of interest.
1686P SRC INHIBITION AS A METHOD TO OVERCOME
TEMOZOLOMIDE RESISTANCE IN MELANOMA
E.J. Jordan 1 , A. Eustace 2 , D.M. Collins 2 , N. O Donovan 2 , J.P. Crown 3
1 Medical Oncology, Mater University Hospital, Dublin, IRELAND, 2 Dublin City
University, N.I.C.B, Dublin, IRELAND, 3 Medical Oncology, St Vincents University
Hospital, Dublin, IRELAND
Background: Malignant melanoma is resistant to chemotherapeutic agents. Src
signalling plays a role in many malignancies including melanoma. Src is important in
cell adhesion regulation, invasiveness and activating other downstream cellular
pathways. Src activity is elevated in melanoma and has potential to provide a
treatment target for melanoma. Temozolomide is an oral alkylating prodrug.
Dasatinib is a dual Src/Abl kinase inhibitor with antiproliferative activity. AZD0530
is a selective oral Src kinase inhibitor.
Methods: We examined the anti-proliferative effects of dasatinib and AZD0530 alone
and in combination with temozolomide (TMZ) in melanoma cell lines - MALME,
HT144 parent and temozolomide resistant cell lines MALME-TMZ and
HT144-TMZ. MALME-TMZ and HT144-TMZ were produced by the pulse method.
IC50 and combination proliferation assays were performed using an acid
phosphatase-based colourimetric assay.
Results: IC50 (50% inhibitory concentrations ± SD) values for TMZ were 338 ±25
µM in HTI44 and 490 ±19 µM in HT144-TMZ. IC50 values for TMZ were 306 ±29
µM in MALME and 515 ±45 µM in MALME-TMZ. Dasatinib was more effective as
a single agent in HT144-TMZ (71 ± 8% growth inhibition at 1 µM) and
Annals of Oncology
MALME-TMZ (60 ± 6% growth inhibition at 1 µM) as compared to MALME (14 ±
10% inhibition at 1 µM) and HTI44 (0 ± 3% inhibition at 1 µM). The combination of
dasatinib/TMZ was more effective than TMZ alone in HT144 and HT144-TMZ.
AZD0530 increased proliferation in HT144 (25 ± 8% at 1 µM) and HT144-TMZ
(35 ± 9% at 1 µM) as a single agent. AZD0530 caused growth inhibition as a single
agent in MALME-TMZ (18 ± 6% inhibition at 1 µM) and MALME (4 ± 2% at 1 µM).
The combination of AZD0530 and TMZ had no effect in HT144 or HT144-TMZ
while AZD0530 combined with TMZ was more effective than TMZ alone in
MALME and MALME-TMZ.
Conclusion: Dasatinib improved response to TMZ in both the chemotherapy naive
and resistant cell line models tested (HT144, HT144-TMZ, MALME,
MALME-TMZ). AZD0530 improved response to TMZ in one of the models only–
MALME and MALME-TMZ. These in vitro results support further preclinical
evaluation of Src Kinase inhibitors in combination with TMZ, and other
DNA-targeting agents, particularly in TMZ-refractory melanoma.
Disclosure: All authors have declared no conflicts of interest.
1687 VEGF AND VEGF RECEPTORS IN THYMIC EPITHELIAL
TUMORS (TET): PATHOLOGICAL FEATURES AND CLINICAL
IMPLICATIONS
L. Nappi 1 , V. Damiano 1 , M. Marino 2 , T. Gelardi 1 , L. Formisano 1 , P. Federico 1 ,
E. Matano 1 , L. Puglia 1 , S. De Placido 1 , G. Palmieri 1
1 Medical Oncology, University Federico II, Naples, ITALY, 2 Department of
Pathology, National Cancer Institute “Regina Elena”, Rome, ITALY
Background: Thymic Epithelial Tumors (TET) are very rare cancers. They derives
from Thymic gland and are associated with different clinical syndromes, such as
miastenic and Good syndrome and other immunological and paraneoplastic
disorders. The principal treatment of these patients is chemotherapy with
schedule containing cisplatin characterized by high response rates (60%).
Unfortunately some patients do not respond at all and most patients do not
achieve long-lasting remission. For these patients have not other therapeutic
chances at the moment. There are few data of the efficacy of anti-angiogenetic
drugs in TET but new knowledges are emerging on the potential use of this class
of drugs in patients with TET on the basis of laboratory and preclinical
translational findings.
Materials and methods: we investigated the expression of Vascular Endothelial
Growth Factor (VEGF) and its receptors (VEGFR1, 2 and 3) in 200 TET arranged in
Tissue Micro Array (TMA). We observed both histotype distribution and modulation
of angiogenetic factors (both VEGFRs and VEGFs) in TET.
Results: we observed that, when compared with the low-grade counterpart, high
grade TET (B2, B3 and Carcinomas) contained significantly higher numbers of
VEGFA, VEGFC, VEGFD, VEGFR1 and VEGFR2 expressing tumor cells. In
addition, in high grade TET, strong positive correlations between staining for
VEGFA and those for all other markers were found. In the same tumors, VEGFC
staining was not associated with VEGFR1, VEGFR3 and PDGFRB staining and no
correlation was obtained between VEGFD and VEGFR1 or VEGFR3 staining. The
co-expression in epithelial cells of growth factors and their receptors suggests the
presence of autocrine mechanisms in TET.
Conclusions: according to our data VEGFs and VEGFRs in TET are potential targets
of anti-angiogenesis drugs. Further studies are needed to investigate the potential role
of anti-angiogenetic agents.
Disclosure: All authors have declared no conflicts of interest.
ix540 | Abstracts Volume 23 | Supplement 9 | September 2012