Annals of Oncology

abstracts
proteins, and classified into multiple classes. One of the MT species, MT1H, is down
regulated in HCC. However, the roles of MT1H in HCC are not fully understood.
Methods: The expression of MT1H in large datasets derived from TCGA databases.
Hepatoma HepG2 and Hep3B cells were transfected with full length Flag-tagged
MT1H. The cell growth curved was obtained through MTT assay. Invasiveness and
migration ability of hepatoma cells was studied through the matrigel-coated transwell
assay. HepG2-Vector and HepG2-MT1H cells were subcutaneously injected into nude
mice for assessing in vivo tumorigenicity of MT1H over-expression.
Results: The results from TCGA RNASeq2 data showed that MT1H is significantly
down regulated in most HCC analyzed. MT1H expression level was found to be
markedly decreased in all HCC tumors. Hepatoma HepG2 and Hep3B cells were
transfected with full length Flag-tagged MT1H. Enforced expression of MT1H in
HepG2 and Hep3B cells led to a more than 50% decrease in colony numbers as
compared with control cells. The DNA synthesis capability was significantly decreased
in MT1H-overexpressed hepatoma cells. Ectopic overexpression of MT1H significantly
impaired the migration capability of hepatoma cells. We found that Wnt/β-catenin
pathway related genes were significantly enriched in the HCC patients with low MT1H
expression. Consistently, ectopic over-expression of MT1H in HCC cells significantly
suppressed the mRNA level of β-catenin signaling downstream targets (c-Myc, MMP7,
LEF1, and TCF4) . As expected, Wnt/β-catenin nuclear translocation was significantly
attenuated in MT1H over-expressed HCC cells. To assess the effect of MT1H
over-expression on tumorigenicity in vivo, we subcutaneously injected nude mice with
HepG2-Vector and HepG2-MT1H cells. The tumors formed by HepG2-MT1H cells
were significantly smaller than that of control cells. The average tumor weight of the
MT1H- transfected cells inoculated mice was significantly decreased at day 30.
Conclusions: Our findings suggest that MT1H functions as tumor suppressor in HCC
mediated by Wnt/β-catenin signaling pathway.
Legal entity responsible for the study: N/A
Funding: Zhejiang Provincial Natural Science Fund (grant no. LY13H160007) and the
Zhejiang Medicines and Health Science and Technology Project (grant no. 201348801).
Disclosure: All authors have declared no conflicts of interest.
5P
AMPKalpha1 restrains fibroblasts transformation and
tumorigenesis in vivo
P. Song, Y. Ding, Q. Lu, M-H. Zou
Center for Molecular and Translational Medicine, Georgia State University, Atlanta,
GA, USA
Background: Adenosine monophosphate-activated protein kinase (AMPK) is a critical
metabolic sensor and redox modulator. Despite evidence linking AMPK tumor
suppressor functions, the role of AMPK in chromosome instability and tumorigenesis
is obscure.
Methods: Murine embryo fibroblasts (MEF) were isolated from AMPK alpha1 knock
out (AMPKα1-KO), AMPKα2-KO, and wild type (WT) C57BL/6J mouse embryos,
and immortalized by employing standard 3T3 protocol to investigate the mechanisms
of chromosomal instability and fibroblast-mediated angiogenesis. Chromatin proteins
and aneuploidy were monitored in cultured MEFs by Western blot and metaphase
spread. Athymic nude mice were employed to test tumorigenesis of fibroblasts in vivo.
Results: Deletion of AMPKα1, but not AMPKα2, exhibited a significant reduction in
centromere-specific binding proteins C (CENP-C) and elevation of Polo-like kinase 4
(PLK4), a trigger of centriole biogenesis. Both CENP-C and PLK4 may be associated
with the increased aneuploid (34%-66%) and micronucleus. Allograft model data
demonstrated that knock out of AMPKα1 enhanced the cellular proliferation of
immortalized MEFs, vascularization, and tumor development in athymic nude mice in
vivo. Mechanistically, it was shown that the protein level of noncanonical nuclear factor
kappa B2 (NF-κB2)/p52 was elevated in AMPKα1-KO MEFs and was responsible for
induction of erythropoietin (Epo) expression. Lastly, the most conclusive evidence for
AMPKα1-dependent inhibition of fibroblast-mediated angiogenesis as well as tumor
progression was that the allograft growth of the inoculated AMPKα1-KO MEFs was
attenuated by treatment with Epo neutralization antibody.
Conclusions: Taken together, these data indicate that AMPKα1 activation opposes
tumor development and its loss fosters tumor progression in part by controlling
chromosome stability and angiogenesis that support tumor growth.
Legal entity responsible for the study: Georgia State University
Funding: National Institutes of Health, American Heart Association
Disclosure: All authors have declared no conflicts of interest.
receptors (VEGFR)1–3, fibroblast growth factor receptors (FGFR) 1–4, platelet-derived
growth factor receptor–α, and KIT and RET proto-oncogenes. We previously reported
that LEN + EVE showed significantly greater antitumor activity in human RCC
xenograft models compared with each monotherapy. LEN + EVE inhibited in vitro
angiogenesis driven by VEGF and FGF in an additive and a synergistic manner,
respectively. Here, we examined FGFR and VEGFR signaling pathway participation in
tumor growth and angiogenesis in human RCC xenografts treated with LEN + EVE.
Methods: Antitumor activity was evaluated in 2 human RCC xenograft models (A–498
and Caki–1). To assess dependence of in vivo growth of RCC xenografts on FGFR
signaling, nude mice with RCC xenografts were treated daily with the selective FGFR
inhibitor PD173074 (50 mg/kg, orally [p.o.]). To evaluate antiangiogenic activity, mice
were treated daily with LEN (10 mg/kg, p.o.), EVE (30 mg/kg, p.o.) or LEN + EVE
(10 + 30 mg/kg p.o.). Microvessel density (MVD) was evaluated by
immunohistochemistry using an α-CD31 antibody.
Results: PD173074 inhibited in vivo growth of RCC xenografts. In the Caki-1 model,
administration of LEN or EVE decreased MVD in tumor tissues, with significantly
reduced MVD observed with LEN + EVE (P < 0.001 and P < 0.05), compared with EVE
and LEN respectively (mean ± standard deviation [/mm 2 ]; control, 135.8 ± 26.0; LEN,
65.3 ± 14.8; EVE, 89.4 ± 24.0; LEN + EVE, 38.1 ± 6.0). In the A–498 model, LEN and
EVE monotherapies showed antiangiogenic and antiproliferative activity, respectively.
These results demonstrate FGFR signaling pathway participation in in vivo growth of
RCC xenografts.
Conclusions: The combined activity of LEN + EVE was therefore based on 1)
enhanced inhibition of VEGF- and FGF-driven angiogenesis and 2) the combination of
the antiangiogenic activity of LEN and antiproliferative activity of EVE. These data
demonstrate that both FGFR and VEGFR pathways are necessary for the LEN + EVE
combined activity in RCC xenograft models.
Legal entity responsible for the study: Eisai Co., Ltd
Funding: This study was funded by Eisai Co., Ltd
Disclosure: All authors are employees of Eisai Co., Ltd, Tsukuba, Ibaraki, Japan.
7P
Identification of novel CRC pathway genes in familial CRC
M.S. Lung 1 , A. Trainer 2 , I. Campbell 1
1 Research, Peter MacCallum Cancer Centre, East Melbourne, Australia, 2 Familial
Cancer Centre, Peter MacCallum Cancer Centre, East Melbourne, Australia
Background: The Wnt, DNA repair and bone morphogenetic protein (BMP) pathways
are implicated in development of familial colorectal cancer (CRC) through inherited
mutations in genes such as APC, MLH1, MSH2, MSH6, PMS2, POLE, POLD1,
MUTYH, SMAD4 and BMPR1A. We hypothesised that mutations in other genes
within these pathways may predispose to unexplained familial colorectal cancer, and
sought rare potentially deleterious variants in genes within these pathways in a cohort
of familial and/or young-onset CRC cases.
Methods: We performed whole exome sequencing on constitutional DNA from 51
individuals with unexplained familial or young-onset (