Tong M et al.

Rab25 is a tumor suppressor with anti-angiogenic and anti-invasive activities in esophageal squamous cell carcinoma

SUPPLEMENTAL INFORMATION Supplemental Materials and Methods 5-aza-2’-deoxycytidine (5-aza-dC), trichostatin A (TSA) and valporic acid (VPA) treatment ESCC cell lines with absent / low Rab25 expression (EC109 and KYSE520) were treated with 0.1- 100 µmol/L 5-aza-dC, 1-5 mM VPA or 100-400 nM TSA (Sigma-Aldrich, St. Louis, MO) for 72 hrs with a medium change every 24 hrs.

Lentiviral transduction Rab25 shRNA lentiviral knockdowns (Sigma-Aldrich NM_020387) or shRNA non-target control (NTC) were packaged using MISSION Lentiviral Packaging Mix (Sigma-Aldrich) and used to infect KYSE30 cells to establish cells constitutively repressing Rab25. Rab25 lentiviral overexpression plasmid (EX-T8368-Lv105, GeneCopoeia, Rockville, MD) or empty vector (EV) control plasmid were packaged using HIV-based packaging mix (GeneCopoeia) and used to infect EC109 cells to establish cells constitutively expressing Rab25. Stable clones were selected using puromycin. Cells were infected with lentiviral media at a multiplicity of infection of 10, in the presence of 8 mg/mL polybrene (Sigma-Aldrich) overnight in a 37°C incubator.

Capillary tube formation assay Rab25 knockdown or overexpressing cells and their respective controls were cultured in serum-free medium for 24 hrs. Conditioned media were collected and filtered for subsequent treatment of human umbilical vein endothelial cells (HUVECs) (Invitrogen). A total of 5x103 HUVECs were seeded into 96-well plate pre-coated with 50 μl Matrigel (BD Biosciences, San Jose, CA) and co- cultured with conditioned medium for 4 hrs. The formation of tube-like structures was examined and photographed using an inverted microscope.

Invasion and migration assay Invasion and migration assays were performed in 24-well BioCoat Matrigel Invasion Chambers (BD Biosciences) according to manufacturer’s instructions or a 24-well millicell hanging insert (Millipore). In brief, 1x105 cells were added to the top chamber, and 10% FBS in DMEM was added to the bottom chamber as a chemoattractant. After 48 hrs incubation at 37°C, the number of cells that invaded through the Matrigel (invasion) or membrane (migration) was counted in 10 fields under a 20x objective lens and imaged using SPOT imaging software (Nikon, Japan).

Western blot Cells were harvested and directly lysed with RIPA buffer supplemented with 1x protease inhibitor. lysates were resolved on SDS-PAGE, transferred onto a PVDF membrane (Millipore) and probed with rabbit anti-human Rab25 (1:800), rabbit anti-human phospho-FAK (Abcam, Cambridge), rabbit anti-human β1 integrin, rabbit anti-human phospho-ERK1/2, rabbit anti-human phospho-c-Raf, rabbit anti-mouse phospho-MEK1/2 (Cell Signaling), rabbit anti-human c-Raf, rabbit anti-human FAK, rabbit anti-human ERK, rabbit anti-human Sp1 or mouse anti-human β-actin (Santa Cruz), or mouse anti-human α-tubulin (Sigma-Aldrich), followed by incubation with secondary HRP-conjugated antibodies. Antibody signal was detected using an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ). Band intensities were quantified by densitometry software ImageJ (http://rsb.info.nih.gov/ij/).

Xenograft transplantation in nude mice The study protocol was approved by and performed in accordance with the Committee of the Use of Live Animals in Teaching and Research at The University of Hong Kong. A total of 1x106 cells were resuspended in 100 μl culture medium and injected subcutaneously into the left or right flanks of four-week old male nude mice. Each group contained 5-6 animals. Cryosections were stained with H&E and used for IHC. Animals that were injected with tumor cells but showed no sign of tumor burden were generally terminated three months after tumor cell inoculation, and animals were opened up at the injection sites to confirm that there was no tumor development.

Statistical analysis All statistical analyses were performed using PASW Statistics 18.0 (SPSS Inc., Chicago, IL), with the exception of the significance in bar graphs, in which case analyses were performed by applying the independent t-test using Microsoft Office Excel software (Microsoft Corp., Redmond, WA). Differences in Rab25 expression among different clinico-pathological stages were analyzed by Paired Sample t-test and Chi-squared test (χ2 test). The overall survival time, which was defined as the time from surgery to death (living patients were censored at the time of their last follow-up), of ESCC patients with different Rab25 expression levels was estimated by Kaplan-Meier analysis and long-rank test. A p-value of less than 0.05 was considered significant.

Fluorescence in situ hybridization (FISH) Dual-color FISH was undertaken using BAC clone at 1q22 covering the Rab25 gene (RP11-336K24) labeled in Spectrum Red (Vysis, Illinois, IL) and a reference BAC clone localized to the centromere of 1, labeled in Spectrum Green (Vysis). BACs were obtained from the BACPAC Resource Center (BPRC) at the Children’s Hospital Oakland Research Institute (Oakland, CA). FISH reactions were performed as previously described (1). Stained sections were counterstained with 40-6-diamidino-2-phenylindole in an anti-fade solution and were examined under a Zeiss Axiophot microscope equipped with a triple-band pass filter. Supplemental Table 1. Clinico-pathological features of patients used for RNA-Seq.

Patient Sex Age LN metastasis* Differentiation TNM stage 1 Male 60 Yes Moderate T3N1M0 2 Male 71 No Well T2N0M0 3 Male 62 No Well T2N0M0 4 Male 48 Yes Poor T3N1M0 5 Male 57 No Well T2N0M0 6 Male 43 No Moderate T2N0M0 7 Male 68 Yes Moderate T2N1M0 8 Female 60 No Moderate T2N0M0 9 Male 54 Yes Moderate T3N1M0

*LN, lymph node Supplemental Table 2. Primer sequences used for qPCR, MSP and BGS analyses.

Analysis CpG Island Name Sequence Rab25 q-F 5’-CCCTCCTGGTGTTTGACCTA-3’ qPCR - Rab25 q-R 5’-TGGTAGAGTCCAGGGCTGAG-3’ 1a MSP-F 5’-TTTTTTCGTAGGGCGTTTTTTATC-3’ MSP CpG I1a 1a MSP-R 5’-TTATAATCTACGATCGAACCTCGAT-3’ 1b MSP-F 5’-GCGGATTGTTTCGGGAGATC-3’ MSP CpG I1b 1b MSP-R 5’-CAAAAAACGAAAATAAAACGACGAC-3’ 2 BGS-F 5’-GTTGGGATTATAGGTGGGAGTTAT-3’ BGS CpG I2 2 BGS-R 5’-AACAACAAAAACTACTTCTCAAAAA-3’ 3 BGS-F 5’-AGGTTGGTTTTGAATTTTTGATT-3’ BGS CpG I3 3 BGS-R 5’-TACCTATAATTCCAACACTTTAA-3’ 4 BGS-F 5’-TGTTATTTAGGTTGGAGTGTAAGGG-3’ BGS CpG I4 4 BGS-R 5’-CACCAAAAAAATTAAAAACCAACTCA-3’

Supplemental Table 3. Summary of sequencing statistics and reads mapping for 12 ESCC / non- tumor samples.

Total Uniquely % Sample Filtered Exon Intron mapped mapped mapped ID reads reads reads reads1 reads reads 8N 7,925,657 5,342,049 3,038,623 2,925,007 2,417,042 67.4 9N 10,091,169 5,247,439 2,672,637 2,618,200 2,629,239 52.0 5N 6,864,933 5,970,338 3,624,371 3,516,441 2,453,897 87.0 4N 2,447,035 2,031,544 1,178,741 1,093,440 938,104 83.0 6N 4,206,226 3,692,441 1,728,571 1,579,231 2,113,210 87.8 6T 11,751,995 7,210,123 3,834,804 2,916,489 4,293,634 61.4 9T 13,369,142 7,921,953 3,829,541 2,992,335 4,929,618 59.3 1T 6,999,000 6,000,639 3,310,382 2,632,229 3,368,410 85.7 7T 7,625,761 5,642,895 3,521,506 2,696,034 2,946,861 74.0 8T 10,006,376 7,992,134 4,810,168 3,861,815 4,130,319 79.9 2T 5,772,653 4,899,559 3,298,388 2,595,159 2,304,400 84.9 3T 4,148,077 3,600,125 2,387,613 1,925,250 1,674,875 86.8 Average 7,600,669 5,462,603 3,102,945 2,612,636 2,849,967 71.9

1Solexa sequencing reads were mapped to NCBI Build 37.1 (GRCh37) with at most 2 mismatches. Supplemental Table 4. List of differentially expressed involved in integrin signaling (p < 0.001).

Full Name Fold Δ p-value# Up-regulated genes COL4A1 Collagen, type IV, alpha 1 69.757 8.26E-14 COL4A5 Collagen, type IV, alpha 5 64.85 3.70E-06 COL4A2 Collagen, type IV, alpha 2 59.125 0 LAMC1 Laminin, gamma 1 (formerly LAMB2) 43.909 6.72E-07 ITGA5 Integrin, alpha 5 (fibronectin receptor, alpha polypeptide) 42.039 0 ITGA1 Integrin, alpha 1 35.033 1.73E-05 CAV1 Caveolin 1, caveolae protein, 22kDa 26.171 1.05E-04 ITGA6 Integrin, alpha 6 25.673 7.60E-04 LAMB1 Laminin, beta 1 24.616 1.29E-05 ZYX Zyxin 16.727 3.33E-08 ITGB4 Integrin, beta 4 16.054 5.10E-06 Integrin, alpha V (vitronectin receptor, alpha polypeptide, ITGAV 15.868 0 antigen CD51) ROCK1 Rho-associated, coiled-coil containing protein kinase 1 11.931 3.15E-05 Integrin, beta 1 (fibronectin receptor, beta polypeptide, ITGB1 10.207 1.24E-05 antigen CD29 includes MDF2, MSK12) ICAM1 Intercellular adhesion molecule 1 8.988 4.48E-09 RAPGEF1 Rap guanine nucleotide exchange factor (GEF) 1 8.747 6.29E-04 Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of ITGA3 8.22 1.38E-07 VLA-3 receptor) RAP1B RAP1B, member of RAS oncogene family 8.046 0 LAMB2 Laminin, beta 2 (laminin S) 7.977 9.63E-07 PTK2 Protein tyrosine kinase 2 6.616 2.23E-07 VCL Vinculin 5.139 3.19E-11 PXN Paxillin 4.819 7.84E-05 SHC (Src homology 2 domain containing) transforming SHC1 4.307 0 protein 1 ITGB8 Integrin, beta 8 3.623 6.79E-07 GRB2 Growth factor receptor-bound protein 2 3.23 0 CRK V-crk sarcoma virus CT10 oncogene homolog (avian) 3.086 2.45E-04 MAPK1 Mitogen-activated protein kinase 1 2.626 2.05E-06 Down-regulated genes RAB25 RAB25, member of RAS oncogene family -3.533 4.85E-31

#p-value, Bonferroni-corrected; fold Δ, fold change

Supplemental Figure Legends Supplemental Figure 1. Expression tiling of Rab25 in 6 unpaired ESCC and non-tumor clinical samples.

Supplementary Figure 2. Detection of DNA copy number change of Rab25 by dual-color fluorescent in situ hybridization (FISH) in ESCC cell lines KYSE520, EC109 and formalin-fixed paraffin-embedded ESCC clinical tissue specimens (#1 and #2) with no or low Rab25 expression. BAC probe to Rab25 and the control reference probe to the centromere of are represented by red and green signals, respectively. Magnification at 1000x. Reference 1. Guan XY, Sham JST, Tang TCM, Fang Y, Huo KK et al. Isolation of a novel candidate oncogene within a frequently amplified region at 3q26 in ovarian cancer. Cancer Res 2001;61:3806-9.