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Annals of Oncology
the STAT signalling pathway and NF-κB. This study sought to determine the
expression and regulation of IL-23A in NSCLC.
Methods: IL-23A expression levels were determined in a series of 34 tumour
specimens with matched normal tissue taken from patients presenting with NSCLC.
Basal expression levels were also examined in a panel of normal and NSCLC cell
lines at the mRNA level. Using histone deacetylase (HDi), DNA methyltransferase
(DNMTi) inhibitors and a ChIP assay it was determined if IL-23A was subject to
epigenetic regulation in NSCLC cell lines (adenocarcinoma and squamous cell
carcinoma). The effect of Gemcitabine on IL-23A expression was also studied. In
addition, the effect of recombinant IL-23 treatment on NSCLC cell line proliferation
was examined.
Results: In a cohort of NSCLC patients, IL-23A expression levels were significantly
elevated in tumour tissue compared with normal (p < 0.05). This elevation was also
evident within the NSCLC sub types. Treatment with TSA (p < 0.05) and SAHA
(p < 0.05) induced the expression of IL-23A in the A549 and SK-MES-1 cell lines. A
ChIP assay confirmed dynamic remodelling of the IL-23A promoter region.
Significant induction of IL-23A was also evident post treatment with a DNMTi
(5-Aza-2’-Deoxycytidine) and Gemcitabine. Furthermore, treatment with
recombinant IL-23 increased cellular proliferation in NSCLC cell lines that express
functional IL-23R receptors.
Conclusion: IL-23A was significantly elevated in a cohort of NSCLC patient tissues
and is epigenetically regulated through histone post-translational modifications and
DNA CpG residue methylation. The chemotherapy agent, Gemcitabine, also induced
IL-23A expression. Additionally, IL-23 promoted NSCLC cell line proliferation.
These results may have important implications for treating NSCLC patients with
epigenetic targeted therapies or Gemcitabine.
Disclosure: All authors have declared no conflicts of interest.
151P INTRATUMOR HETEROGENEITY OF ER STATUS IS A
FEATURE OF INTERACTION BETWEEN MAMMARY
CARCINOMA CELLS AND MICROENVIRONMENT
I. Boichuk 1 , D. Burlaka 2
1 Department of Cancer Experimental Therapeutics, R.E. Kavetsky Institute of
Experimental Pathology, Oncology and Radiobiology, Kyiv, UKRAINE,
2 Biotechnical Problems of Diagnostic, Institute Problems of Cryobiology and
Cryomedicine of the National Academy of Sciences (IPCC), Kiev, UKRAINE
The growth and survival of mammary carcinoma cells often depend on their
estrogen sensitivity proving a rationale for targeted therapy. The relationship between
tumor growth and changing hormonal environment is demonstrated both in
experiment and in clinical setting (age-related difference in breast cancer prognosis,
changing estrogen and progesterone receptor patterns in breast carcinoma during the
menstrual cycle and menopause, etc.). The aim of this study was to analyze the
effects of the changing hormonal environment in the setting of the oestrus cycles on
tumor growth and levels of estrogen receptors (ER) in murine mammary tumors.
Methods: The dextran-coated charcoal radioactive ligand-binding ER assay method
was used followed by Scatchard analysis.
Results: The increment of tumor volume in mouse with mammary carcinoma is not
a linear one but followed the pattern of changing hormonal levels within 4-day
oestrus cycle in a wave-shaped manner. Upon the plateau of tumor growth, receptors
are lost in half of cells. Thus, the population becomes heterogeneous in ER content.
The widely accepted hypothesis that the interaction of estrogen with cellular ER
determines the hormone dependency of mammary tumors should now be challenged
on the basis of the following observations: a) tumors arising in experimental animals
with permanent high estrogen levels are receptor-positive and that with low estrogen
levels are receptor-negative; b) tumors regrowing after complete or partial regression
as a result of endocrine ablation or hormone administration are considered to be
environment dependent rather than autonomous or hormone dependent; c)
mammary tumors growing in the setting of cyclically changing hormonal level
become heterogeneous.
Conclusion: Tumor cells are highly sensitive to alterations in hormonal background.
The adaptation to the altered microenvironment could promote metastases, tumor
heterogeneity, and, oddly enough, transform the tumors into a nonmalignant state.
Disclosure: All authors have declared no conflicts of interest.
152P BONE MARROW TRANSPLANTATION RESCUES INTESTINAL
MUCOSA AFTER WHOLE BODY RADIATION VIA PARACRINE
MECHANISMS
H. Chang 1 , Y.H. Chang 1 , L. Lin 1 , C. Lou 1 , C. Chou 2
1 National Institute of Cancer Research, National Health Research Institutes,
Tainan, TAIWAN, 2 National Laboratory Animal Center, National Applied Research
Laboratories, Taipei, TAIWAN
Background: Our previous study reveals BM transplantation (BMT) triggers
trafficking of host CD11b(+) myelomonocytic cells from the host marrow to the
radiation-injured intestinal mucosa, enhancing the proliferation of intestinal stroma
cells, leading secondarily to epithelial regeneration. In this study, we propose that
BMT ameliorates intestinal damage via paracrine mechanisms.
Materials and methods: Mouse survival, intestine microcolony assay, angiogenic
cytokine array, immunonhistochemical studies of both intestine and BM were
evaluated in mice after WBI and BMT. Epithelial and stromal components within
intestine mucosa were measured by immunoblotting and flowcytometry. BM
conditioned medium (BMCM) with or without treatment with neutralizing
antibodies to angiogenic cytokines were continuously infused to mice after WBI or
whole abdomen irradiation. Carrageenan was used to deplete myelomonocytic cells
of recipient mice.
Results: BMT increases VEGF, bFGF and other angiogenic cytokines within intestine
mucosa within 24 hrs after WBI. Continuous infusion of BMCM alone ameliorated
radiation-induced intestine damage and improved survival of mice. Proliferation
index, endothelial cells, myofibroblasts as well as myelomonocytic cells within
intestine were increased by BMCM within one week after radiation. Neutralization of
bFGF, PDGF and other angiogenic cytokines within BMCM abolished the mitigating
effect to intestine. Pretreatment of carrageenan to recipient mice depleted CD11b(+)
myelomonocytic cells and reversed some of the angiogenic cytokine levels, including
VEGF, within intestine mucosa after BMT from healthy donors.
Conclusions: Our results suggest that BMT recruits host CD11b(+) myelomonocytic
cells and enhances intestine stroma proliferation by secreting angiogenic cytokines
including PDGF, bFGF. Host CD11b(+) myelomonocytic cells further uplift the
angiogenic effect via cytokines including VEGF.
Disclosure: All authors have declared no conflicts of interest.
153P TOCILIZUMAB CAN ENHANCE THE ANTI-TUMOR EFFECT OF
IFN-α IN RENAL CELL CARCINOMA
T. Oguro 1 , K. Ishibashi 1 , T. Sugino 2 , S. Kumagai 1 , N. Takahashi 1 , N. Haga 1 ,
T. Yanagida 1 , K. Aikawa 1 , O. Yamaguchi 3 , Y. Kojima 1
1 Urology, Fukushima Medical University, Fukushima, JAPAN, 2 Pathology,
Fukushima Medical University, Fukushima, JAPAN, 3 Bioenginnering, Nihon
University, Koriyama, JAPAN
Background: Interleukin 6 (IL-6), one of the proinflammatory cytokines, contributes
to the onset and maintenance of various types of cancer. IL-6 signaling induces
up-regulation of SOCS3, which leads to interferon (IFN) –α resistance in renal cell
carcinoma (RCC) cells. To propose a new treatment for mRCC, we investigated the
effect of combination therapy with IFN-α and humanized antihuman IL-6R
antibody, tocilizumab.
Material and methods: Real time PCR (RT-PCR) was used to analyze gene
expression of IL-6 and SOCS3 after IFN stimulation and ELISA were used for IL-6
secretion in RCC cell lines. These expression levels were compared among the RCC
cell lines, i.e., ACHN, Caki-1, TUHR3TKB, TUHR4TKB and 786-O.
Phosphorylation of STAT-1, STAT-3 and ERK were investigated by Western blotting
(WB) analysis. We investigated alteration of IL-6 signaling by tocilizumab, and
analyzed its impacts on the susceptibility to IFN-α in RCC cells by MTT assay. The
effects of tocilizumab on in vivo growth on 786-O xenografts were determined by
SOCS3 expression, morphological observation and tumor volume.
Results: Among the RCC cell lines, the expression levels of IL-6, SOCS3 in RT-PCR
and the production level of IL-6 were highest in 786-O. These results revealed a
correlation between the expression levels IL-6 and SOCS3 (P = 0.0001, r = 0.99974).
Although the 786-O cells had IFN-α resistance, MTT assay showed that the
tocilizumab induced a growth inhibitory effect of IFN-α. WB analysis showed that
phosphorylation level of STAT-1 was increased and the levels of STAT-3 and ERK
were decreased with the simultaneous use of tocilizumab in 786-O cells. An in vivo
study demonstrated that tocilizumab promoted IFN-α-induced cell death and growth
suppression in 786-O cell xenograft in nude mice.
Conclusions: IL-6 could be a key component in the resistance to IFN-α treatment of
renal cell carcinoma. Antihuman IL-6R antibody can be an effective strategy to
enhance the anti-tumor effect of IFN-α, and probably, the effect of angiogenic
inhibitors, in human RCC cells.
Disclosure: All authors have declared no conflicts of interest.
154P CO-TARGETING THE PI3K-MTOR & MEK PATHWAYS IN
NSCLC
S. Heavey, M. Barr, A. Ahmad, A. Davies, K.J. O’Byrne, K.A. Gately
Clinical Medicine, Trinity College Dublin, Dublin, IRELAND
The PI3K/Akt/mTOR and MAP/ERK kinase (MEK) pathways regulate cell growth
and proliferation and are often dysregulated in cancer due to mutation, amplification,
deletion, methylation and post-translational modifications. We and others have
shown that activation of the PI3K pathway in Non Small Cell Lung Cancer (NSCLC)
leads to a more aggressive, drug-resistant disease which correlates to poor prognosis
for patients. Combinations of PI3K and MEK and inhibitors have shown promise in
preclinical cancer models, leading to the initiation of clinical trials cotargeting these
two key cancer signalling pathways. The selection of appropriate patient cohorts who
Volume 23 | Supplement 9 | September 2012 doi:10.1093/annonc/mds390 | ix69