Page 47 - D. Cancer biology

Dexras1 plays pivotal roles in immune escape in mesenchymal GC cells

Kyu-Hye Chun1,2, Bo Kyung Yoon1, Jae-Woo Kim1,2
1Department of Biochemistry and Molecular Biology, Chronic Intractable Disease Systems Medicine Research Center, Yonsei University College of Medicine, Seoul 120-752, Korea
2Brain Korea 21 Project for Medical Science, Yonsei University, Seoul 120-752, Korea

BACKGROUND AIM

tGastric cancer is a heterogeneous cancer, and has two distinct Dexamethasone-induced Ras-related protein1, called
molecular subtypes which is, mesenchymal phenotype and epithelial Dexras1, is a protein which regulates Ras signal
phenotype. Mesenchymal subtype is associated with markedly poor transduction pathways. In TCGA and GSEA Database,
survival and resistance to standard chemotherapy. Among Gastric Dexras1 low group was highly enriched in Interferon gamma
cancer cell lines, Dexras1 shows high expression in mesenchymal signaling pathway and NF-ΚB signaling pathway. Dexras1
phenotypes. Here we show ablation of Dexras1 can approach to could be novel therapeutic targets.
progression of chemotherapy
ransduction pathways.
METHODS

1. The whole-cell lysate was made in lysis buffer [1% Triton X-100, 50 mM KCl, 25 mM HEPES (pH 7.8), 10 g/ml leupeptin, 20
g/ml aprotinin, 125 M dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM sodium orthovanadate] with sonication
to break both cytoplasmic and nuclear membrane. The protein(15 ug) in 25 ul of reducing sample buffer was boiled for 5 min and
resolved in 6% SDS-PAGE for 80 min at 130 V. Then, the protein was transferred onto polyvinylidene difuoride membrane at 290
A for 100min. The membrane was blotted with first antibody for 24h and secondary antibody for 60min in milk buffer.
2. The luciferase assay was conducted using a 96-well luminometer with the dual luciferase substrate system(Promega, Madison,
WI). Relative luciferase activity was normalized to the internal control renilla luciferase activity, and the relative luciferase activity
was presented as mean value plus sd of the quadruplicates.

RESULTS

To evaluate whether Dexras1 functions Fig.1 GO_IFNγ mediated signaling pathway GO_Response_to_interferon_gamma Fig.2
0
as a immune related genes in GC, we NES: -2.08 0 Nom p-val : 0.17 IFN-γ (10ng/ml) shctrl HS746T shDexras1
NES: -1.14
Nom p-val: 0
firstly analyzed Enrichment Score - 0.5 -0.35 Time [hr] 0 1 4 0 1 4
p-STAT1
(Y701)
according to the TCGA database. high GO_NIK_NF-Κb signaling low KEGG_JAK STAT_signaling pathway NF-κB
shCtrl
shDexras1
Dexras1
Analysis of theses data revealed that the 0.10 0 NES: -1.11 0 c-Jun
Nom p-val: 0.27
Dexras1 low group was highly enriched - 0.25 -0.4 GAPDH
PD-L1
in Immune related pathway. Also, in high Dexras1 low shCtrl shDexras1
shDexras1 group was highly enriched in
same pathways. Fig.3 ISMARA 1600 Luciferase activity assay CTRL
Dexras1 O/E
10
**
We next analyzed ISMARA motif shCtrl_rep1 shCtrl_rep2 shRasD1l_rep1 shRasD1l_rep2 * **
analysis assay using shDexras1 group. STAT1-STAT3-BCL6 Firefly/renilla 800 5
IRF2-STAT2-IRF8-IRF1
Consistent with our GSEA database, Motif activity REL-A
shDexras1 group was related with JAK- CREB1 0
NRF1
STAT and NF-KB motif activity. 0 TNF-α IFN-γ LPS
CEBPB
pGL3-p65REX3 - + + + + pGL3-p65REX3 + + + + + + + +
p65 - - + + +
-1 0 1 - - - + +
BRD4
Dexras1 - - - - +
We also assessed Luciferase reporter gene assay for the effect of NF-Kb and Dexras1 on the activity of p65
promoter. No significant change in luciferase activity was observed upon p65 promoter. However, substitution
of the NF-Kb binding site increased the promoter activity. In addition, overexpression of Dexras1 in cells
reduced the promoter activity, suggesting the Dexras1 may reduce NF-Kb activity.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
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