Supplementary Information - chimeric fusion transcript in human gastric cancer promotes tumorigenesis through activation of PI3K/AKT signaling Sun Mi Yun, Kwiyeom Yoon, Sunghoon Lee, Eunjeong Kim, Seong-Ho Kong, Jinny Choe, Jin Muk Kang, Tae-Su Han, Pyounggang Kim, Yojun Choi, Sungwoong Jho, Hana Yoo, Jong Bhak, Han-Kwang Yang, and Seong-Jin Kim 1 1 Correspondence should be addressed to S-J.K. (kimsj@cha.ac.kr) These authors equally contributed to this work. This PDF file includes Supplementary Figures S1 to S7 Supplementary Tables S1 to S Supplementary Note Supplementary Reference
Index of Supplementary Figures and Tables Supplementary Figure Legends Supplementary Figure S1. (a) Displaying the different sequence reads across the fusion junction among 56 spanning reads. (b) - fusion gene sequences. Black and blue indicate exon 6-7 and exon, respectively. (c) Schematic illustration of predicted fusion protein and each wild-type proteins. (d) Read distributions of and estimated by RNA sequencing are displayed as a custom track using the UCSC genome browser. Supplementary Figure S. - expression of gastric cancer cell lines. (a-c) The expressions of - fusion with 18 gastric cancer cell lines at transcriptional level (a) or genomic DNA level (b), and human gastric patient tissues at transcriptional level (c) were evaluated by RT-PCR. (d) Sequencing chromatograms show fusion junction in SNU-5, MNK-5 and KATOⅢ gastric cancer cell lines, and 119T human gastric cancer tissue. (e) RNA transcriptional levels of - and each wild-type in 7 primary human gastric cancer tissues were measured by Q- PCR Supplementary Figure S3. (a) Schematic representation of genes co-amplified with ERBB locus at 17q1-q1. (b) The levels of RNA expression of genes in the ERBB amplicon in 18 gastric cancer cell lines were estimated by RNA sequencing. (c) Copy number variations of chromosomal region containing and (chromosome 17:37,78,~38,3,). The calculated copy numbers of each sample were plotted as a bold line. The border lines of white and grey regions represent the thresholds of copy gain (upper border) and copy loss (lower border). Calculated copy numbers located in grey regions represent copy gains (upper grey region) or copy losses (bottom grey region).
Supplementary Figure S. - expression of several cancer cell lines. (a-c) The expression of - were assessed by RT-PCR in eleven breast cancer cell lines (a), nine endometrial cancer cell lines (b) and eight ovarian cancer cell lines (c). (d) The fusion junction of - in several different cancer cell lines was confirmed by Sanger sequencing. Supplementary Figure S5. Cell proliferation of - expression in AGS stable cells. (a) Western blot analysis showing the levels of and - in AGS cells stably expressing empty vector, or -. (b) Proliferation assay with AGS stable cells was performed using cell counting. (*, P =.9; **, P =.17; ***, P =.3) (c) Quantitative result of focus-forming assay demonstrated significant increase in relative cell survival in AGS cells stably expressing -. (*, P <.1; **, P =.5; ***, P =.1) Supplementary Figure S6. The expression of and - in recurrent tumors of xenografts was confirmed by RT-PCR. Supplementary Figure S7. Induction of the PI3K-AKT activation by - in gastric cancer cells. (a-b) Western blot analysis of AKT and phospho-akt (S73) in empty vector, or - expressing AGS stable cells (a) and in empty vector,, - and truncated form of ( truncated) expressing MKN-8 stable cells (b). (c) Proliferation assay with empty vector,, - and truncated form of expressing MKN-8 stable cells was assessed using cell counting (*, P <.1; **, P <.1; N.S., not significant). (d) Western blot analysis of AKT activation in empty vector, alone, - alone, or and - together stably expressing MKN-8 cells. (e-f) AKT phosphorylation in AGS stable cell lines upon PI3K inhibitor, LY9, treatment (5 M) (e) and in MKN-8 stable cell lines upon AKT inhibitor, AKT-IV, treatment ( M) (f) was examined by western
blotting. Supplementary Table Legends Supplementary Table S1. The clinical information of the primary gastric cancer patient tissues in this study. Supplementary Table S. The list of Q-PCR and RT-PCR primers for validation of the expression of fusion transcript and wild-type genes. Because the fusion transcript possessed shared sequences of their parental genes, we designed primer sets capable of amplifying the breakpoint junction of the genes. Wildtype specific primers were designed in exon 6 and exon 7 of, and exon 1 and exon of. The primer sequences against exon 6 of and exon of were used for the amplification of fusion transcript fragment. Supplementary Notes Whole-genome sequencing Illumina platform was used for whole-genome sequencing. The whole-genome sequencing has been previously described in detail. 5 Calculation of copy number variations (CNVs) Copy number variations of cell line genomes were detected using ReadDepth v..9.8.. 1. Supplementary Reference 1. Miller CA, Hampton O, Coarfa C, Milosavljevic A. ReadDepth: a parallel R package for detecting copy number alterations from short sequencing reads. PLoS One 11; 6: e1637.
Supplementary Figure S1. a exon 6 exon 5 AGGACCAAGUGGAAGACCCAGCACUAAGUG CCCUGCUGCUGAGGCCGCGCCCUCCCCGCC 3 AGGGTCTGGAAGGGCCCTGGGAGCGCCCACCCCCTCTGGATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCAAGCTGCCCAGGGAGCTGACCCGAGACTTGGAGC AGGGCCCTGGGAGCGCCCACCCCCTCTGGATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCT GGGCCCTGGGAGCGCCCACCCCCTCTGGATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTG GGCCCTGGGAGCGCCCACCCCCTCTGGATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGC GCCCTGGGAACGCCCACCCCCTCTGGATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCT GCCCACCCCCTCTGGATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGC CCCACCCCCTCTGGATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCG CACCCCCTCTGGATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCC CTGGATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGC TGGATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCC GGATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCC GATGAGTCCGAGAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCT GAGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGC GAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCC GATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCAC ATGGAGCCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACC TGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCA GGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAG GAGGCTCTGAGGACCAAGTGGAAGCCCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGG GCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATG ACTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATG CTGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGC TGAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCA GAGGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGGCCACCAGGATGAGCAA GGACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCAAGC ACCAAGTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCAAGCTG CCAAGTGGAAGCCCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCAAGCTGC GTGGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCAAGCTGCCCAG GGAAGACCCAGCACTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCAAGCTGCCCAGGG CAGCCCTATGTC CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCAAGCTGCCCAGGGAGCTGACC AGCACTAGGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCAAGCTGCCCAGGGAGCTGACCC CTAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCAAGCTGCCCAGGGAGCTGACCCGAGA TAAGTG CCCTGCTGCTGAGGCCGCGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCAAGCTGCCCAGGGAGCTGACCCGCGAA Black : exon 6 Blue : exon b c AGGAAGAAGAAGAGGACAGCCAGGCTGAAGTCCTGAAGGTCATCAGGCAGTCTGCTGGGCAAAA GACAACCTGTGGCCAGGGTCTGGAAGGGCCCTGGAGCGCCCACCCCCTCTGGATGAGTCCGAG AGAGATGGAGGCTCTGAGGACCAAGTGGAAGACCCAGCACTAAGTG/CCCTGCTGCTGAGGCCG CGCCCTCCCCGCCCTGAGGTGGGGGCCCACCAGGATGAGCAAGCTGCCCAGGGAGCTGACCCG AGACTTGGAGCGCAGCTGCCTGCCGTGGCCTCCCTGGGCTCCTCACTGTCCCACAGCCAGAGCC TCTCCTCGCACCTCCTTCCGCCGCCTG (5 a.a ) ( a.a) MENTAL DARPP-3 START Black : exon 6 Blue : exon - (188 a.a + 57 a.a) DARPP-3 d chr17 37,785, 37,79, Read depth 5 variants chr17 37,795, 37,8, 37,85, 37,81, 37,815, 37,8, Read depth 1 variants
Supplementary Figure S. a d - - 18S rrna 1 3 6 3 1 15 mrna expression (FPKM) 5- - 75-5- 5-15- - ATG 5 a.a Stop SNU-5 b gdna cdna SNU-1 SNU-5 SNU-16 SNU-16 MKN-5 Cell Tissue KATOⅢ MKN-7 SNU-8 NCI-N87 SNU-5 119N SNU-61 119T SNU-6 SNU-638 SNU-668 SNU-719 MKN-1 MKN-8 MKN-5 MKN-7 KATOIII AGS NCI-N87 NCI-N87 - c - 18S rrna 119T mrna expression (FPKM) 3 5 15 1 5 Exon6 Exon e 1 1 8 6 - Relative mrna expression (Normalized to 18S) 16 1 1 1 8 6 5 15 WT WT 1 5 87 T 91 T 9 T 95 T 99 T 1 T 13 T 11 T 119 T 13 T 13 T 18 T 18 T 195 T 11 T T 36 T 38 T T 55 T 6 T 79 T 337 T 9 T 3 T 36 T 77 T 9 T 56 T 516 T 57 T 57 T 55 T 551 T 557 T 565 T 58 T 718 T 76 T 773 T 777 T 783 T 89 T 859 T 88 T 889 T 917 T
Supplementary Figure S3 a Chromosome17q1 TCAP PNMT PGAP3 ERBB GRB7 Amplicons 37,78, 37,8, 37,86, 37,9, b 3 mrna expression (FPKM) 5 15 1 5 NCI-N87 SNU-5 SNU-16 SNU-5 MKN-5 KATO III SNU-1 SNU-16 SNU-8 SNU-61 SNU-6 SNU-638 SNU-668 SNU-719 MKN-1 MKN-8 MKN-7 AGS - Fusion positive - Fusion negative c chr17:37,7,-38,, - Fusion positive - Fusion negative SNU-5 SNU-16 SNU-5 MKN-5 SNU-1 SNU-638 SNU-668
Supplementary Figure S BT7 ACI-5 SNU8 HS578T ACI-61 TOV11D M1 ACI-98 OVCA338 M ACI-16 OVCAR3 M3 ECC1 OVCA9 M RL95- DOV13 MDA-MB31 NC1EC1 SKOV3 MDA-MB35 HHUA SKBR3 HELA Ishikawa T7D ZR75B a Breast Cancer cell lines d - 1 3 6 3 1 15 ATG 5 a.a Stop - SKBR3 b Endometrial Cancer cell lines Ishikawa - c SKOV-3 Ovarian Cancer cell lines Exon6 Exon -
Supplementary Figure S5 a AGS WT - Anti- Anti-a-Tubulin b Number of cells (x1,) 1 1 8 6 WT - 1 3 Time (d) * * * *** c Relative foci formation 3 1 * *** WT ** -
#1 # #5 #6 - #3 - # Supplementary Figure S6 MKN8-18S rrna
Number of cells (x5,) WT - WT + - WT - WT - truncated Supplementary Figure S7 a b AGS MKN-8 Anti-p-AKT Anti-AKT Anti- Anti-a-Tubulin Anti-p-AKT Anti-AKT Anti-HA Anti-a-Tubulin c d MKN-8 1 1 8 WT - truncated * N.S. ** 6 Anti-p-AKT Anti-AKT 1 3 Anti- Time (d) Anti-a-Tubulin e f AGS MKN-8 WT - WT - - + - + - + LY9 Anti-p-AKT Anti-AKT Anti- Anti-a-Tubulin - + - + - + AKT-IV Anti-p-AKT Anti-AKT Anti- Anti-a-Tubulin
Supplementary Table S1. Serial Sex Age Location Size (cm) Histologic subtype (WHO classification) Lauren Stage 3 M 51 U 7. WD Gastric IIIb O 8 M 56 L 5. PD Diffuse IIIb O RNA- qpcr - Sequencing Validation 87 M 53 U 8. MD Diffuse IIIb O O 95 M 65 L 6. WD Intestinal IIIa O O 119 M 8 L 1. WD Intestinal IIIa O O O 13 M 57 L 6.5 MD Intestinal IIb O O 13 M 57 L 8.5 WD Intestinal IIIb O O 135 M 37 L 6. Mucinous Intestinal IIb O 136 M 55 L.5 MD Diffuse IIIa O 195 M 67 U 6. Papillary Intestinal IIIa O O O 36 F 7 L 6.5 MD Mixed IIa O O 777 M 86 L 19 MD Diffuse IIIc O O 783 M 7 L 1 PD Intestinal Iia O O 89 M 6 L 5. PD Intestinal Ib O O 859 F 6 L 5. MD Intestinal Ib O O 88 F 75 L 3.5 MD Intestinal Ia O O 889 F 66 L 8.7 MD Intestinal Iia O O O 917 M 73 L 7.9 MD Intestinal IIa O O 91 M 81 L.5 SRCC N/A IIIa O 9 M 61 L 13. MD with mucin N/A IIIb O O 99 M 7 M 7. SRCC N/A IIa O 1 M 5 M 13. PD N/A IIIb O 13 M 78 L 6. Mucinous N/A IIIb O 11 M 5 U 11. MD N/A IIb O 18 M 76 L 6. MD Intestinal IIIb O 18 F 6 L 1. MD Intestinal IIa O 11 M 6 U 1. Mucinous N/A IIa O M L 13. PD N/A IIb O 38 M 67 L 6.5 PD Mixed IIIa O M 67 L 7.5 PD Diffuse IIIb O 55 M 65 M 7. Mucinous Diffuse IIb O 6 M 6 L 9. MD Intestinal IIa O O 79 F 59 L 1. PD Diffuse IIIb O 337 F 51 M 13. PD with mucin Diffuse IIa O 9 F M 5. PD Mixed IIIc O 3 F 66 M 1.5 Undifferentiated Intestinal IIb O O 36 M M/L 8. PD Mixed IV O 77 M 8 M 1.7 Undifferentiated Intestinal IIIc O 9 M 68 N/A N/A N/A N/A N/A O 56 M 6 L 6. MD Intestinal IIa O O 516 M 59 L 6. MD Intestinal IIIb O 57 F 3 M 5.5 PD Diffuse IV O 57 F 7 L 8. MD with mucin Mixed IIIc O O 55 M 9 L 5. PD Mixed IIIc O 551 M 8 U/M 6. PD Diffuse IIb O 557 M 63 Whole 1. SRCC Diffuse IIIb O 565 F 73 M/L 1. PD Diffuse IIIa O 58 M 66 L Unmeasurable Papillary Intestinal IIIa O 718 F 67 U/M 17. PD Diffuse IV O O 76 M 7 M 13. Mucinous Intestinal IIIc O 773 M 68 U 3.5 WD Intestinal Ia O O U, upper third; M, middle third; L, lower third WD, tubularadenocarcinoma and well differentiated; MD, tubularadenocarcinoma and moderately differentiated; PD, tubularadenocarcinoma and poorly differentiated N/A, not applicable
RT-PCR Q-PCR Supplementary Table S Accession # Gene Symbol Primer sequences NM_319.3 exon6 qf; GAAGACCCAGCACTAAGTGAG exon7 qr; AAAACAGGGTGAGGATAGAGTG NM_68.3 exon1 qf; GTGGCTGACATGGAGCAG exon qr; ACAGTGAGGAGCCCAGG - PS fusion ( exon6) qf; AGATGGAGGCTCTGAGGAC PS fusion ( exon) qr; CAAGTCTCGGGTCAGCTC NM_319.3 exon6 F; GGCTGAAGTCCTGAAGGTCA exon7 R; CCCAGGTTCTCTGGGTATCA NM_68.3 exon1 F; GATCTTCTTCCGCTCTGAGG exon R; GATGAAGAGCAGGTCGAAGG - exon6 F; GGCTGAAGTCCTGAAGGTCA exon R; GATGAAGAGCAGGTCGAAGG