Journal Thoracic Oncology

Abstracts Journal of Thoracic Oncology • Volume 12 Issue S1 January 2017
POSTER SESSION 3 – P3.01: BIOLOGY/PATHOLOGY
MODELS OF LUNG CANCER –
WEDNESDAY, DECEMBER 7, 2016
P3.01-054 ANTAGONISM OF A NOVEL KINASE, MAP3K19, BY
SPECIFIC SMALL MOLECULE INHIBITORS BLOCKS HUMAN
PRIMARY NSCLC TUMOR GROWTH IN MURINE XENOGRAFT
MODELS
Stefen Boehme, John Ludka, Danielle Ditirro, Tai Wei Ly, Kevin Bacon
Biology, Axikin Pharmaceuticals, Inc., San Diego/CA/United States of America
Background: We have identified a novel serine/threonine protein kinase,
MAP3K19, whose expression in normal lung was predominantly localized
to alveolar and interstitial macrophages, bronchial epithelial cells and
type II pneumocytes of the epithelium. We have also found MAP3K19 to be
expressed in multiple, primary NSCLC tumor samples, as well as human lung
cancer cell lines, including A549. The kinase is transcriptionally upregulated
in cells upon various types of cell stress, including oxidative, endoplasmic
reticulum and osmotic stress. Methods: Using two different murine xenograft
models, we assayed the role of MAP3K19 to inhibit the growth of either
primary human NSCLC tumors and A549 cells using small molecule inhibitors.
Results: The ability of i.v. injected A549 cells to colonize and grow in the lung
was significantly reduced in mice that received orally administered, selective
MAP3K19 inhibitors. Similar results were observed in a subcutaneous
xenograft model, as A549 tumor cell growth was inhibited by both MAP3K19
antagonists and other standard of care kinase inhibitors. These studies also
showed an additive anti-proliferative effect when gefitinib or sorafenib
and MAP3K19 inhibitors were co-administered. Importantly, xenograft
models using primary human NSCLC tumors implanted subcutaneously in
immunodeficient mice showed a statistically significant inhibition of tumor
growth when the mice were treated with the orally administered MAP3K19
antagonists. IHC analysis of the tumors showed that mice treated with
the MAP3K19 inhibitors also had decreased levels of Ki-67, c-myc, p27 and
phospho-Bim staining and increased caspase-3 staining. Conclusion: These
results suggest a molecular mechanism by which MAP3K19 may inhibit tumor
cell growth, and further suggest that inhibition of MAP3K19 either by itself
or in combination with other therapies may represent a new avenue for the
treatment of NSCLC. The clinical development of the MAP3K19 inhibitor is
expected to initiate Phase I clinical trials in early 2017.
Keywords: NSCLC, MAP3K19, novel kinase
POSTER SESSION 3 – P3.01: BIOLOGY/PATHOLOGY
Stem Cells in Lung Cancer –
WEDNESDAY, DECEMBER 7, 2016
P3.01-055 IN VITRO CONSTRUCTION OF LUNG CANCER ORGANOIDS
FROM INDUCED LUNG CANCER STEM LIKE CELLS
Hiroyuki Ogawa 1 , Takashi Aoi 2 , Nahoko Shimizu 1 , Daisuke Hokka 1 , Yugo
Tanaka 1 , Michiyo Koyanagi-Aoi 2 , Yoshimasa Maniwa 1
1 Thoracic Surgery, Kobe University Hospital, Kobe City/Japan, 2 Department of Ips
Cell Applicationse, Kobe University Hospital, Kobe City/Japan
Background: Lung cancer stem cells are considered to be responsible for lung
cancer progression. However, little is known about how they actually promote
lung cancer progression and metastasis. Methods: We retrovirally introduced
three defined factors (OCT3/4, SOX2, and KLF4) into lung cancer cell line,
A549. We evaluated cancer stem cell properties in the A549 cells transduced
with the three factors (OSK-A549) in terms of their chemo resistance, and
sphere formation ability. We also assessed lung cancer organoid constructing
ability by co-culturing with mesenchymal stem cells (MSC) and human
umbilical vein epithelial cells (HUVEC). Results: OSK-A549 cells formed
dome-shaped colonies in 10 to 15 days after transfection. These colonies were
picked up for further expansion in DMEM/10%FBS medium, and we named
these cells OSK-A549-Colony cells. Induced OSK-A549-colony cells were more
resistant to cisplatin than parental A549 cells. Cell cycle analysis revealed that
the rate of G0/G1 cells was significantly increased in OSK-A549-colony cells.
Sphere forming ability was enhanced in OSK-A549-colony cells. These results
suggested that OSK-A549-colony cells acquired the properties of lung cancer
stem like cells. Co-culture with MSC and HUVEC showed that A549 and OSK-
A549-colony cells could form large spheres equally, however, HE staining of
spheres revealed that OSK-A549-colony cells could form much denser spheres
than those of parental cells (Figure). As the morphology was similar to real
lung cancer tissue, we named this spheres “lung cancer organoids”.
Conclusion: By introducing defined factors, A549 cells acquired lung cancer
stem cell like properties, and these cells could form lung cancer organoids by
co-culturing with MSC and HUVEC. Analysis of these organoids might enable
us to elucidate the molecular mechanism of lung cancer progression and
metastasis.
Keywords: cancer organoid, lung cancer, Cancer stem cell
POSTER SESSION 3 – P3.01: BIOLOGY/PATHOLOGY
STEM CELLS IN LUNG CANCER –
WEDNESDAY, DECEMBER 7, 2016
P3.01-056 ASSOCIATION OF ANGIOGENESIS, EMT AND STEM CELL
CHARACTERISTICS USING HYPOXIC STRESS IN LUNG CANCER
Chankwon Park 1 , Nahyeon Kang 2 , Hyonsoo Joo 1 , Young Kyoon Kim 1 , Seung
Joon Kim 1
1 Department of Internal Medicine, The Catholic University of Korea, Seoul/Korea,
Republic of, 2 The Cancer Research Institute, College of Medicine, the Catholic
University of Korea, Seoul/Korea, Republic of
Background: Hypoxia, a major phenomenon in solid tumors, can promote the
metastatic potential of tumor cells which is associated with chemoresistance
and poor prognosis. It was reported that various angiogenesis factors
including VEGF and HIF, were associated in cancer development and
progression by hypoxia. In addition, both epithelial-mesenchymal transition
(EMT) and cancer stem cells play an important role in malignant progression
in many human tumors. We investigated the effect of hypoxic stress on
the angiogenesis, EMT and stemness acquisition in lung cancer. Methods:
Normal lung cell (BEAS-2B) and lung cancer cell lines (A549, H292, H226
and H460) were incubated in either normoxic or hypoxic (below 1% O 2
)
conditions. For transcriptome analysis, mRNA of BEAS-2B and A549 cell lines
were analyzed using next-generation sequencing (HiSeq 2500 system). For
further validation, angiogenesis markers were analyzed by western blotting.
EMT was assessed with western blotting, wound healing assay and Matrigel
invasion assay, and stem cell characteristics were assessed with RT-PCR,
immunostaining, soft agar colony formation assay, sphere formation assay
and in vivo mice tumor model. Results: Next-generation sequencing revealed
significant changes in the expression of angiogenesis, EMT and stem cell
markers after hypoxic stress. Among the angiogenesis markers, VEGF and
HIF-2α were increased. EMT markers related in hypoxia showed decrease in
E-cadherin and increase in fibronectin, vimentin, N-cadherin, α-SMA, Snail,
Slug, ZEB1 and ZEB2. Stem cell markers such as CXCR4, Oct4 and Nanog were
increased at least one lung cancer cell line in hypoxic condition compared with
in normoxic condition. Functional assays for EMT and stemness acquisition
indicated that hypoxic stress increased wound healing, Matrigel invasion,
sphere formation and in vivo mice tumor formation. Conclusion: These results
suggest that hypoxia induces angiogenesis markers expression which is
associated with EMT and stemness acquisition in lung cancer.
Keywords: hypoxic stress, angiogenesis, lung cancer
POSTER SESSION 3 – P3.01: BIOLOGY/PATHOLOGY
STEM CELLS IN LUNG CANCER –
WEDNESDAY, DECEMBER 7, 2016
P3.01-057 TGF-Β INDUCED EMT AND STEMNESS CHARACTERISTICS
IN LUNG CANCER
Seung Joon Kim 1 , Nahyeon Kang 2 , Hyonsoo Joo 1 , Chankwon Park 1 , Young
Kyoon Kim 1
1 Department of Internal Medicine, The Catholic University of Korea, Seoul/Korea,
Republic of, 2 The Cancer Research Institute, College of Medicine, the Catholic
University of Korea, Seoul/Korea, Republic of
Copyright © 2016 by the International Association for the Study of Lung Cancer
S605