The 57th Annual Meeting of Japan Society of Clinical Oncology 2019/10/24
Roles of DNAJC12 In Gastric Cancer Progression
Department of Gastroenterological Surgery Nagoya University Graduate School of Medicine
Yasuo Uno, Mitsuro Kanda, Dai Shimizu, Chie Tanaka, Daisuke Kobayashi Fuminori Sonohara, Norifumi Hattori, Hideki Takami, Masamichi Hayashi Suguru Yamada, Goro Nakayama, Masahiko Koike, Michitaka Fujiwara, Yasuhiro Kodera The 57th Annual Meeting of the Japan Society of Clinical Oncology Disclosure of Conflict of Interest
Name of first author： Yasuo Uno
I have no COI with regard to our presentation. Background
• Identification of gastric cancer-related molecules is necessary to elucidate the pathological mechanisms of this heterogeneous disease.
Purpose
• The aim of this study was to identify novel genes associated with aggressive phenotypes of gastric cancer. Methods
Transcriptome analysis Metastatic gastric cancer (n = 4) Gastric cancer tissues vs normal tissues in primary gastric cancer
overexpressed in gastric cancer expressed at comparable expression levels in primary gastric cancer and metastatic tissues
25 candidate genes
highly expressed genes in primary gastric cancer tissues encoded a trafficking protein Roles in gastric cancer are unknown
DNAJC12 Methods
DNAJC12
14 GC cell lines evaluated using siRNA 262 clinical samples mRNA expression Cell proliferation mRNA expression analysis Cell invasion analysis Cell adhesion Correlation analysis of Cell migration clinicopathological factors Results List of candidate genes
GC/Normal Meta/GC Function Symbol Name Log2 P Log2 P DNAJC12 4.15 <0.0001 dnaJ heat shock protein family member C12 -1.16 0.1038 Trafficking protein RBP4 4.25 <0.0001 retinol binding protein 4 1.51 0.0515 highly expressed genes in SYT7 4.29 <0.0001 synaptotagmin 7 0.30 0.6281 FNDC1 4.50 <0.0001 fibronectin type III domain containing 1 -0.89 0.1592 primary gastric cancer tissues GNG4 4.84 <0.0001 G protein subunit gamma 4 0.29 0.7296 Transcription factor ELF5 5.00 0.0001 E74 like ETS transcription factor 5 -0.85 0.3319 encoded a trafficking protein HOXC10 6.49 0.0001 homeobox C10 1.68 0.0752 that may be involved in growth GRB7 3.98 <0.0001 growth factor receptor bound protein 7 -0.03 0.9716 factor signaling and Cell membrane receptor UTS2R 4.50 <0.0001 urotensin 2 receptor 0.50 0.5675 transportation of anticancer TNFRSF11B 4.57 <0.0001 TNF receptor superfamily member 11b 0.53 0.4265 drugs through the membrane. Cell-surface glycoprotein MELTF 3.27 <0.0001 melanotransferrin -0.19 0.7380 COMP 3.15 0.0003 cartilage oligomeric matrix protein 0.91 0.1072 Published data on the CLDN1 3.27 <0.0001 claudin 1 0.71 0.1568 oncological roles in gastric Cellular adhesin THBS2 3.76 <0.0001 thrombospondin 2 0.20 0.7759 cancer are lacking THBS4 4.01 <0.0001 thrombospondin 4 0.95 0.2787 Growth factor INHBA 3.76 <0.0001 inhibin beta A subunit -0.37 0.5028 VSNL1 4.04 <0.0001 visinin like 1 1.09 0.1528 Mediator of neural CPLX2 4.36 0.0007 complexin 2 1.88 0.2436 transmission NPY 4.86 <0.0001 neuropeptide Y 0.09 0.9008 PADI2 3.01 <0.0001 peptidyl arginine deiminase 2 -1.29 0.0758 KLK10 3.26 0.0003 kallikrein related peptidase 10 -0.76 0.2984 Metabolic enzyme AKR1C4 3.28 0.0009 aldo-keto reductase family 1 member C4 0.59 0.4064 PLA2G2A 3.70 <0.0001 phospholipase A2 group IIA -0.43 0.4529 CDC25B 3.17 0.0006 cell division cycle 25B -0.66 0.3947 Regulator of cell cycle CCNE1 3.41 <0.0001 cyclin E1 -1.06 0.0709 DNAJC12 expression levels of cell lines Confirmation of siRNA-mediated knockdown of DNAJC12 qPCR-PCR assay
MKN1 AGS Confirmation of siRNA-mediated knockdown of DNAJC12 western blotting analysis
MKN1 AGS Effects of siRNA-mediated knockdown of DNAJC12 Cell proliferation assay
MKN1 AGS Effects of siRNA-mediated knockdown of DNAJC12 Cell invasion assay
MKN1
Untransfected
siControl
siDNAJC12 Effects of siRNA-mediated knockdown of DNAJC12 Cell adhesion assay
MKN1 Effects of siRNA-mediated knockdown of DNAJC12 Cell wound-healing assay
MKN1
) 600 μm 500
400
300
200
100
Mean Mean length of migration( 0 0h 4h 8h 12h 16h
Untransfected siControl siDNAJC12 DNAJC12 expression levels of clinical samples
The AUC value of DNAJC12 levels was 0.606 for detection of cancer-related mortality within 3 Normal Gastric years after surgery. We categorized patients into tissues cancer high (above the cutoff value) or low DNAJC12 tissues (below the cutoff value) groups. DNAJC12 expression and clinical characteristics
Low DNAJC12 High DNAJC12 Variables P (n = 139) (n = 123) Age < 70 year 78 70 0.897 ≥ 70 year 61 53
Sex Male 106 83 0.114 Female 33 40
CEA (ng/ml) ≤ 5 112 96 0.614 > 5 27 27
CA19-9 (IU/ml) ≤ 37 106 95 0.935 > 37 32 28
Tumor location Entire 7 12 0.067 Upper third 42 25 Middle third 47 35 Lower third 43 51
Tumor size (mm) < 50 60 44 0.222 ≥ 50 79 79
Macroscopic type Borrmann type 4/5 16 21 0.197 Others 123 102
Multifocal lesions Absent 130 108 0.109 Present 9 15 DNAJC12 expression and clinical characteristics
Low DNAJC12 High DNAJC12 Variables P (n = 139) (n = 123) Tumor depth (UICC) pT1-3 73 55 0.207 pT4 66 68
Differentiation Differentiated 63 38 0.016 Undifferentiated 76 85
Lymphatic involvement Absent 18 7 0.042 Present 121 116
Vascular invasion Absent 45 31 0.201 Present 94 92
Infiltrative growth type Invasive growth 44 56 0.021 Expansive growth 95 67
Lymph node metastasis Absent 40 22 0.037 Present 99 101
Peritoneal lavage cytology Negative 119 97 0.152 Positive 20 26
UICC stage I 16 4 0.011 II 33 36 III 63 46 IV 27 37 DNAJC12 expression and overall survival (Stage I - IV)
Institutional dataset Extra-validation dataset Prognostic factors for overall survival
n = 262, Stage I - IV
Univariate Multivariate Variables n Hazard ratio 95% CI P Hazard ratio 95% CI P Age (≥ 70) 114 1.02 0.65–1.57 0.932 Gender (female) 73 1.06 0.64–1.67 0.825 CEA (> 5 ng/ml) 54 1.87 1.15–2.96 0.012 1.35 0.79–2.25 0.272 CA 19-9 (> 37 IU/ml) 60 1.99 1.23–3.14 0.006 1.36 0.81–2.23 0.239 Tumor location (lower third) 94 0.90 0.57–1.40 0.658 0.57 0.34–0.93 0.024 Tumor size (≥ 50 mm) 158 3.76 2.19–6.96 <0.001 2.32 1.30–4.41 0.004 Macroscopic type (Borrmann type 4/5) 37 2.14 1.26–3.47 0.006 1.19 0.64–2.15 0.578 Multifocal lesions 24 1.16 0.54–2.19 0.683 Tumor depth (pT4, UICC) 134 3.04 1.90–5.02 <0.001 1.53 0.87–2.73 0.140 Tumor differentiation (undifferentiated) 161 1.59 1.01–2.57 0.044 1.03 0.61–1.78 0.901 Lymphatic involvement 237 3.03 1.25–9.96 0.011 1.49 0.37–4.94 0.548 Vascular invasion 186 2.15 1.30–3.75 0.002 1.59 0.91–2.93 0.107 Invasive growth 100 2.20 1.43–3.40 <0.001 1.32 0.73–2.38 0.353 Lymph node metastasis 200 4.44 2.25–10.1 <0.001 2.66 1.14–7.34 0.022 Peritoneal lavage cytology (positive) 46 4.28 2.71–6.67 <0.001 1.82 1.07–3.07 0.028 High DNAJC12 123 2.08 1.34–3.28 0.001 1.70 1.07–2.72 0.024 Subgroup Analysis Patient with Stage I - III
n = 198, Stage I - III, R0 resection
Recurrent patterns DnaJ Heat Shock Protein Family Member C12 (DNAJC12)
• DNAJC12 is a member of the heat shock proteins (HSP), an evolutionary protein family that act as molecular chaperones. DNAJC12 belongs to DnaJ heat shock protein family (HPS40), which acts as a cofactor of HSP70.
Cell Stress Chaperones. 2014;19:439-446.
• HSPs can exhibit anti-apoptotic properties and function in various cancer- related processes, such as in tumor cell proliferation, invasion, metastases and death. Cell Mol Immunol. 2004;1:274-279. Mol Ecol. 2018;27:3040-3054.
Gene Cards: The Human Gene Database http://www.genecards.org/cgi- bin/carddisp.pl?gene=DNAJC12 DnaJ Heat Shock Protein Family Member C12 (DNAJC12)
• DNAJC12 is ubiquitously expressed in various organs and is reported as responsible for hyperphenylalaninemia. Mol Genet Metab. 2018;123:1-5.
• High levels of DNAJC12 expression in breast cancer tissues were associated with estrogen receptor transactivation activity. Int J Mol Med. 2006;17:363-367.
• DNAJC12 overexpression acts as a negative predictive factor for the response to neoadjuvant concurrent chemoradiotherapy. Exp Mol Pathol. 2015;98:338-345. Discussion
• We found that tissue DNAJC12 mRNA levels were higher in GC tissues compared with normal adjacent tissues, and elevated expression of DNAJC12 was found in 85% GC cell lines, supporting our hypothesis that DNAJC12 might play oncogenic roles in GC based on the transcriptome analysis.
• Dysregulated proliferation and invasion are one of the major contributors to cancer-related growth, progression, and metastasis. Our findings indicated that DNAJC12 plays an important role in the malignant behavior of GC.
• Further studies, including pathway analyses manly focusing on HSP70 families and apoptotic analyses, are needed to elucidate the underlying molecular mechanisms of DNAJC12 contributing to these phenotypes. Conclusion
• Our findings support DNAJC12 as a candidate gene associated with aggressive phenotypes of gastric cancer.