Page 15 - D. Cancer biology

Caveolin-1 supplies energy necessary for migration and metastasis of gastric cancer cells.

Nahee Hwang1,2, Bo Kyung Yoon1, Jae-woo Kim1,2
1. Department of Biochemistry and Molecular Biology, Chronic Intractable Disease Systems Medicine Research Center, Yonsei University
College of Medicine, Seoul 120-752, Korea
2. Brain Korea 21 Project for Medical Science, Yonsei University, Seoul 120-752, Korea
BACKGROUND AIM
Gastric cancer (GC) is the third most fatal cancer in the world, which requires
thorough understanding of the disease. To conquer GC, finding new targets By understanding the roles of caveolin-1 in GC cells
and appropriate therapeutic strategies are essential. Stem-like GC has worst
prognostic value as well as predictive value accounting for the resistance to associated with their migration, we expect it would
the chemotherapy. [1] Based on the gene expression of stem-like GC, stem- bring a new point of view in studies on cancer
like GC cell lines are selected and studied to prove that they have similar metabolism and ultimately in development of anti-
characteristics to stem-like type of tumor. [1][2] Caveolin-1, a member of the
caveolin complex, has been studied in various cellular behaviors over the cancer drug targeting cancer metastasis
years. However, the functions of caveolin-1 in GC have been controversial
and still remained unclear. In this study, we found that high expression of
caveolin-1 correlates with poor prognosis of GC patients in TCGA STAD. In
addition, we found that stem-like GC cell lines characterized caveolin-1 as one
of their phenotypes.
METHODS
Electroporation
5
2×10 MKN1 or HS746Tcells were electroporated with 1 µg Cav1_mCherry and ATP_biosensor or GAPDH_GFP by Neon™ Transfection
System 10μL Kit according to the manufacturer’s instructions.
Confocal microscopy
GC cells were grown on coverslips (VWR) in 12-well plates and treated as indicated. Cells were fixed with 4% (w/v) paraformaldehyde (PFA,
Santa Cruz) for 20 min at RT, permeabilized with 0.5% (v/v) Triton-X-100 (Sigma-Aldrich) in PBS and then blocked with 5% (v/v) fetal bovine
serum (Gibco) in PBS for 1 h at RT. Adherent cells were mounted on microscope slides (VWR) in DAPI-containing Mowiol mounting medium
(Mowiol 4–88 10% (w/v, Carl Roth), glycerol 25% (w/v, Sigma-Aldrich), H 2 O 25% (v/v), 0.2 M Tris HCl pH 8.5 50% (v/v, AppliChem GmbH, VWR),
DABCO 2.5% (w/v, Carl Roth)) to co-stain nuclei. All laser scanning images were acquired on a Zeiss LSM 780 confocal microscope.
RESULTS

a. b. a. MKN1 Ctrl MKN1 CAV1 OE b.
TCGA STAD GC patients
CAV1 KD CAV1 KD
Cav1-high (n=113) 0hr
Cav1-low (n=113) *** Gastric Intestinal
Inflammatory Mixed MKN1
Survival probability Log intensity 10 72hr
12.5
Stem-like
7.5 c.

c. GC cell lines Cav1 Cav2 MKN1

CAV1 DAPI mCherry ATP Merge
CAV2
Caveolin complex CAVIN1 d.
CAVIN2
CAVIN3 HS746T
DAPI GAPDH mCherry Merge
MKN1
SNU16
MKN74
HS746T
NCIN87
KATOIII
SNU668
SNU484
SNU601
SNU719
SNU638
SNU620
Fig 1. Cav1-high GC tumor is aggressive.
a) Tumor mRNA expression levels were obtained from TCGA for 226 patients with Cav1 b) Cav1 gene expression levels elevated in tumors of stem-like GC patients
compared to those of intestinal-type GC patients. c) mRNA expression levels of caveolin complex elevated in stem-like cell lines compared to those of intestinal-type
GC cell lines. Pink : Stem-like GC cell lines; Sky blue: Intestinal-type GC cell lines.
Fig 2. Cav1 facilitates cell movement in stem-like GC cells by enabling efficient energy supply .
a) Migration assay using wound healing test. Cav1 overexpression cells are fast in migration and proliferation b) Representative images of the invasion assay in
MKN1 cells after 10hrs. Cav1 overexpression cells are fast in invasion. c) Confocal visualization of transiently co-expressed ATP-biosensor (green) and Cav1-
mCherry (red), which exhibit localization to the plasma membrane and peri-nuclear site in MKN1 cells. Scale bar, 20μm. d) Confocal visualization of co-expressed
GAPDH-GFP(green) and cav1-mCherry (red), which exhibit localization to the plasma membrane and peri-nuclear site in MKN1 cells. Scale bar, 20μm.
CONCLUSION REFERENCES
We confirmed that caveolin-1 was [1] Jae-Ho Cheong*, Han-Kwang Yang, Hyunki Kim, Woo Ho
Kim, Young-Woo Kim, Myeong-Cherl Kook, Young-Kyu Park,
localized at the tip and of highly Yan Kim, Jinae Lee, Seung Ho Choi, Soonwon Hong, Jong Contact information
Hyung-Ho Kim, Hye Seung Lee, Kyung Hee Lee, Mi Jin Gu, Ha
stem-like GC cells. Live imaging of Won Kim, Yoon Young Choi, resectable gastric cancer: a multi-
cohort, retrospective analysis.” Lancet Woo Jin Hyung, Eunji
cells indicates that the role of Jang, Hyeseon Kim, Yong-Min Huh, Sung Hoon Noh. naheechel@naver.com
caveolin-1 is thought to localize “Predictive test for chemotherapy response in Oncol 2018; 19:
629–38
ATP at the tip of the cells, enabling [2] Jooyoung Lee, Hyosil Kim, Jae Eun Lee, Su-Jin Shin, Sejin
efficient energy supply necessary Oh, Sungjin Kwon, Hakhyun Kim, Yoon Young Choi, Michael A.
White, Soonmyung Paik, Jae-Ho Cheong,* and Hyun Seok
for the movement in actin- Kim*. “Selective Cytotoxicity of the NAMPT Inhibitor FK866
Toward Gastric Cancer Cells with Markers of the Epithelial–
dependent manner Mesenchymal Transition, Due to Loss of NAPRT.”
Gastroenterology, 2018 May