Annals of Oncology

abstracts
were checked on the mRNA and protein level by qPCR and Western blot; possible
alterations in the EGFR signaling in the established cell line were checked by EGF
stimulation followed by the measurement of the EGFR and kinases phosphorylation.
Proteasomal, lysosomal degradation of EGFR and half-life of the receptor were assessed
by treatment of cells with corresponding inhibitors. Functional assays, examining
cellular proliferation and migration complemented the above-mentioned experiments.
Results: Our study is the first to describe the relations between PHD2 and EGFR in
both preclinical and clinical models of breast cancer. We identify PHD2 as a novel
contributor to EGFR signaling in breast cancer by showing its direct participation in
the regulation of EGFR stability.
Conclusions: Since the search for novel signaling interplays is a prerequisite for the
development of better treatment options of breast cancer, we believe that this novel
finding is of potential value for clinicians and medical researchers.
Legal entity responsible for the study: University of Oulu
Funding: FEBS, Finnish Center of International Mobility (CIMO), European
Association for Cancer Research (EACR), Biocenter Oulu, University of Oulu, Jane and
Aatos Erkko Foundation, Finnish Academy of Sciences, the Sigrid Juselius Foundation.
Disclosure: All authors have declared no conflicts of interest.
28P
The resistance mechanism of FGFR2 amplified gastric cancer
cells against AZD4547, a fibroblast growth factor receptor
inhibitor
S.Y. Lee, Y. Jeong, Y. Na, J.L. Kim, D.H. Lee, S.C. Oh
Oncology/Hematology, Korea University Guro Hospital, Seoul, Republic of Korea
Background: The fibroblast growth factor- fibroblast growth factor receptor
(FGF-FGFR) signaling pathway plays a role in cell proliferation, migration, survival,
and angiogenesis. Because approximately 10% of gastric cancers show amplification of
FGFR2, inhibition of FGFR2 activation has been regarded as one of therapeutic targets.
AZD4547, a selective inhibitor of the FGFR1-3 tyrosine kinases, was developed to
inhibit FGFR signaling, however, its efficacy is limited by emergency of the acquired
resistance. We tried to clarify a resistance mechanism against the FGFR inhibitor.
Methods: The cell line resistant against the AZD4547 was established using SNU-16
(SNU16R), a FGFR2 amplified gastric cancer cell line, by culturing with increasing
concentration of the AZD4547. The expression level of FGFR or phosphorylated FGFR
(pFGFR) and downstream signaling molecules was determined by Western blot. Cell
viability was measured with MTT assay. Relative level of tyrosine phosphorylation of
receptor tyrosine kinases (RTKs) was evaluated with proteome profiler TM array by
human phosphor-RTK kit (R&D Systems).
Results: Loss of expression of FGFR2 and pFGFR2 was confirmed in the SNU16R cell
line by Western blotting. The viability of SNU16R cell line was shown to be increased
than that of the parent cell line after incubation with AZD 4547. Change of the FGFR2
downstream signaling pathways was addressed, and upregulated expression level of
phosphorylated mammalian target of rapamycin (mTOR) was found. Overexpression
of downstream targets of mTOR, such as phosphorylated 4E-BP1 and S6K was also
confirmed. The SNU15R and parent SNU16 cell lines were incubated with everolimus
or AZD 4547, and inhibition of activated mTOR was observed with everolimus but not
with AZD 4547 by Western blotting and MTT assay. Because PI3K and Akt were not
activated, an alternative signaling pathway was sought and the phosphorylated level of
EphB3 was found to be elevated.
Conclusions: Activation of mTOR is one of mechanisms of acquiring resistance
against AZD 4547 in FGFR2 amplified gastric cancer cells. Targeting mTOR could
overcome the resistance by inhibition of activation of mTOR.
Legal entity responsible for the study: Korea University Guro Hospital
Funding: AstraZeneca
Disclosure: S.Y. Lee, Y. Jeong, Y. Na, J.L. Kim, D.H. Lee, S.C. Oh: This research was
funded by AstraZeneca. All experiments were performed independently from AstraZeneca.
29P
Anticancer activity of the mTOR inhibitor (everolimus) and dual
mTORC1/mTORC2 Inhibitor (AZD2014) on mouse lymphocytic
leukemia both in vitro and in vivo
Y-C. Su 1 , H-F. Liao 2 , C-C. Yu 3 , Y-C. Lin 2
1 Division of Hematology-Oncology, Department of Internal Medicine, E-DA
Hospital, Kaohsiung, Taiwan, 2 Department of Biochemical Science and
Technology, National Chiayi University, Chiayi, Taiwan, 3 Department of Medical
Research, Dalin Buddhist Tzu Chi General Hospital, Chiayi, Taiwan
Background: The mammalian target of rapamycin (mTOR) controls cell growth and
enlargement and has been found to be aberrant in a wide variety of malignancies.
Allosteric mTOR inhibitors, which inhibit mTORC1 but not mTORC2, result in
feedback activation of AKT signaling, which can attenuate their antitumor activity.
AZD2014 is a second generation mTOR inhibitor that blocks activation of both
mTORC1 and mTORC2, and activates apoptosis in cancer cells without activation of
AKT signaling. Here, we investigated the therapeutic efficacy of everolimus and
AZD2014 in lymphocytic leukemia cell line (L1210) and mouse xenograft model.
Methods: Cytotoxicity effect of AZD2014 and everolimus in L1210 cells was assessed
after 24 and 48 h by using cell viability assays, clonogenic survival assays, and cell cycle
analyses. Cell cycle, mTOR signal transduction pathway and the relative regulatory
molecules were examined in mTOR inhibitor-treated L1210 cells by western blotting to
detect protein expression. Then, the in vivo anti-leukemic effect of AZD2014 was
assessed in L1210 cell-transplanted DBA/2 mice.
Results: AZD2014 significantly inhibited L1210 cell proliferation with an IC50 of
100nM. In contrast, everolimus was a poor growth inhibitor of L1210 cells
(IC50 > 200nM). Treatment with AZD2014 was more effective than RAD001 to
down-regulate the levels of mTORC1 downstream effectors, including S6K1, 4EBP1,
eIF4E and a significant decrease in protein levels of rictor, a component of mTORC2
protein.We also observed inhibition of mTORincreased G1 arrest by reducing cyclin
D1 and CDK4 levels, which augmented growth-inhibitory effect of L1210 cells. In vivo,
AZD2014 oral administration significantly inhibited the growth of L1210 cell xenograft
in DBA/2 mice, and the mice survival was dramatically improved.
Conclusions: These data indicate that AZD2014 may be a better therapeutic agent
than mTORC1 inhibitors to enhance the antitumor activity of lymphocytic leukemia
both in vitro and under xenograft model in vivo conditions. Antitumor activity of
AZD2014 was inhibited of both mTORC1 and mTORC2 activity and cell cycle arrest,
leading to lymphocytic leukemia growth inhibition.
Legal entity responsible for the study: N/A
Funding: Without funding from a pharma, biotech, or other commercial company.
Disclosure: All authors have declared no conflicts of interest.
30P
Transforming growth factor beta receptor (TGFβR) pathway is
involved in ligand independent transactivation of AXL receptor
in colorectal cancer (CRC) cell lines
E. Franzese, G. Martini, C. Cardone, T. Troiani, V. Sforza, M.L. Ferrara, V. Belli, P.
P. Vitiello, S. Napolitano, N. Zanaletti, P. Vitale, F. De Vita, M. Orditura, F. Morgillo,
F. Ciardiello, E. Martinelli
Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale, AOU
Seconda Università degli Studi di Napoli (AOU-SUN), Naples, Italy
Background: It has been described that AXL,a tyrosine kinase receptor, can be
activated through different mechanisms: GAS6-ligand dependent dimerization and
ligand-independent activation. As we previously demonstrated the absence of GAS6 in
a panel of CRC cell lines, we tried to investigate the possible mechanisms of AXL
activation in our in vitro CRC model.
Methods: We used a panel of CRC cell lines (HCT116, SW480, SW620 and LOVO).
Expression and activation of AXL and its ligand GAS6 were analyzed by Western Blot
(WB) and real time-PCR (RTPCR). We generated, in our laboratory, stable
(shRNA)-sh-AXL LOVO cells clones (shAXL clone 1, shAXL clone3 and shAXL clone
5) to evaluate the effect of AXL specific knockdown. GAS6, TGF-β1 levels were
measured by ELISA assay.
Results: TGF-β1 was detected at variable levels in AXL expressing CRC cell lines
(HCT116, SW480, SW620 and LOVO). We stimulated CRC cells with TGF-β1finding
increased levels of pAXL, pAKT and pMAPK by WB. To reveal how TGF-β1 actives AXL
we measured the mRNA levels of AXL or GAS6 by RT-PCR after TGF-β1 24h stimuli. No
fold mRNA increased was observed, suggesting a different interaction between AXL and
TGFβ pathway. To further correlate TGFβR pathway with AXL transactivation, we
stimulated LOVO cell line, shAXL clone1, 3 and 5 with TGF-β1 for 24 hrs. An increased
activation of p38MAPK in LOVO cells was demonstrated, accompanied by no change in
shAXL clones, suggesting that, at least in our model, the downstream protein p38 MAPK is
strictly correlated with AXL transactivation stimulated by TGF-β1. Further experiments
with p38MAPK inhibitor are currently ongoing.
Conclusions: Our findings suggested a relationship between TGF-b1, p38 MAPK and
AXL as a possible mechanism of AXL independent ligand activation. Further
investigations are ongoing.
Legal entity responsible for the study: Second University of Naples
Funding: Second University of Naples
Disclosure: All authors have declared no conflicts of interest.
31P
Annals of Oncology
Taselisib enhances effects of anti-microtubule chemotherapic
agents in phosphatidylinositol 3-kinase (PI3Kα) mutant breast
cancer cell lines
A. Diana, F. Morgillo, C.M. Della Corte, C. Di Mauro, V. Ciaramella, F. De Vita,
F. Ciardiello, M. Orditura
Medicina Clinica Sperimentale Magrassi Lanzara, AOU Seconda Università degli
Studi di Napoli (AOU-SUN), Naples, Italy
Background: 30-50% of breast cancer are characterized by activating mutations of
PI3Kα, and higher activity of this pathway is often associated with resistance to
conventional therapies. Thus, selective PI3K inhibitors could represent a novel option
to prevent treatment resistance, including chemotherapy. Several trials are evaluating
the efficacy of Taselisib, a novel selective inhibitor of mutant PI3Kα, in combination
with hormonal therapy (NCT02340221, NCT02273973, NCT01296555) or taxanes
vi8 | abstracts Volume 27 | Supplement 6 | October 2016