Volume 143, Issue 3, Pages 675-686.e12 (September 2012) Identification of PTK6, via RNA Sequencing Analysis, as a Suppressor of Esophageal Squamous Cell Carcinoma  Stephanie Ma, Jessie Y.J. Bao, Pak Shing Kwan, Yuen Piu Chan, Carol M. Tong, Li Fu, Na Zhang, Amy H.Y. Tong, Yan–Ru Qin, Sai Wah Tsao, Kwok Wah Chan, Si Lok, Xin–Yuan Guan  Gastroenterology  Volume 143, Issue 3, Pages 675-686.e12 (September 2012) DOI: 10.1053/j.gastro.2012.06.007 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 RNA-Seq expression profiling data. (A) Clustering tree diagram based on the gene expression values for 3 pairs of matched normal/tumor samples. (B) Venn diagram of differentially regulated genes based on gene fold change >1.2 between matched tumor and control 16N/16T, 18N/18T, and 19N/19T. A total of 10,250 and 721 genes were up-regulated and down-regulated, respectively. (C) Heat map of expression profiles for genes with a P value ≦.005 (1425 genes). (D) Validation study to determine the correlations between real-time qPCR and RNA-Seq data. Results indicate a high level of concordance of the differential expression measurements between both platforms at varying abundances. (E and F) A selection of the top enriched GO biological process, GO molecular function, and pathway for genes with a P value ≦.005. The numbers for each pathway indicate the fold enrichment based on Fisher exact test. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Down-regulation of PTK6 in ESCC. (A) Validation of altered PTK6 expression by qPCR in the original 3 ESCC (T) and their adjacent nontumor (N) samples. (B) PTK6 expression in matched NT and primary ESCC (n = 73) as detected by qPCR. The boxes contain the values between the 25th and 75th percentiles, the lines across the boxes indicate the median, and the whiskers extend to the highest values excluding outliers and extremes. (C) PTK6 protein levels in nontumor (N) and primary ESCC (T) as detected by Western blotting. (D) Representative IHC staining of PTK6 in matched nonneoplastic squamous epithelium (NT) and poorly differentiated ESCC (T) tissue from 3 patient samples. (E) Summary of IHC staining for PTK6 in NT and ESCC specimens. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Re-expression of PTK6 following treatment with 5-aza-dC, VPA, or TSA. (A and B) Measurement of PTK6 expression and protein levels in a panel of esophageal cell lines by semiquantitative reverse-transcription PCR, real-time qPCR, and Western blotting. (C) Genomic map of the PTK6 5′ region with +1 as the transcription start site. A 589–base pair CpG island including 45 CpG sites were identified at location −3684 to −3096 base pairs (with a small part overlapping with the SRMS gene that is in close proximity upstream of PTK6). PCR regions for clonal bisulfite sequencing (BGS), methylation-specific PCR (MSP), and ChIP PCR assays are also shown. (D) qPCR analysis of PTK6 expression after demethylation and/or histone acetylation treatment with 5-aza-dC (blue bars), VPA (green bars), and/or TSA (red bars) in ESCC cell lines that are absent for PTK6. The gray bars represent combined 10 μmol/L 5-aza-dC and 200 nmol/L TSA treatment. *P < .05. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 The PTK6 promoter region is frequently hypermethylated and deacetylated in ESCC. (A) High-resolution mapping of the methylation status of individual CpG sites in the PTK6 promoter by bisulfite sequencing in ESCC cells KYSE140, KYSE180, and HKESC1, immortalized normal esophageal cell NE1, and one representative matched NT/ESCC clinical sample. A 459–base pair region spanning the CpG island with 31 CpG sites was analyzed. Open circles represent unmethylated CpG dinucleotide, and closed circles represent methylated CpG dinucleotide. (B) Methylation-specific PCR (M, methylated allele; U, unmethylated allele) was performed to investigate the methylation status in the promoter region of PTK6 in ESCC cell lines and clinical samples. (C) Expression of acetylated histones H3 and H4 in KYSE140, KYSE180, EC18, and HKESC1 ESCC cells after 72 hours of TSA or VPA treatment, as detected by Western blotting. (D) Representative quantification of ChIP-PCR analyses on NE1 and KYSE180 cells. A fragment covering the majority of the CpG island 5′ upstream of PTK6 was immunoprecipitated with anti-acetylated H3, anti-acetylated H4, or anti-trimethylated histone H3K9 antibody and amplified by PCR. *P < .05. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 PTK6 is an important tumor-suppressor gene in ESCC. (A) Stable repression or overexpression of PTK6 in KYSE30 and KYSE510 cells, respectively, by lentiviral transduction was confirmed by Western blotting. (B) Representative foci formation assays in PTK6 repressed or overexpressed clones as compared with their controls. (C) Representative image of the tumors formed in nude mice following injection of NTC cells (left dorsal flank) and PTK6 shRNA clone 552 (right dorsal flank) or empty vector control (EV CTRL) cells (right dorsal flank) and PTK6 overexpression (O/E) clone (left dorsal flank). (D) H&E staining of tumors injected confirmed a malignant phenotype. IHC staining for PTK6 expression in the resected xenografts. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 PTK6 regulates invasion, migration, and cell cycle progression of ESCC cells. (A and B) Representative images of invasion and migration assays in PTK6 repressed (KYSE30) or overexpressed (KYSE510) clones as compared with their controls. (C) Comparison of DNA content between NTC and PTK6 repressed KYSE30 cells or empty vector (EV) and PTK6 overexpressed KYSE510 cells by flow cytometry. Summary of cell proportions in different phases of cell cycle. The results are expressed as means ± SD of 3 independent experiments. *P < .05. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 (A) Comparison of expression of phospho Akt (Ser473), total Akt, phospho GSK3β, total GSK3β, and active β-catenin (ABC) by Western blot analysis in PTK6-overexpressed (KYSE510) or -repressed (KYSE30) cells. (B) Representative IHC staining of active β-catenin and PTK6 in 2 ESCC patient samples. Pictures taken at 100× original magnification followed by a higher magnification at 400×. (C) Representative IHC staining of active β-catenin in resected tumor xenografts. (D) Proposed model illustrating the tumor-suppressive effect of PTK6 in the pathogenesis of ESCC. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 RNA-Seq expression profiling data. (A) Distribution of number of exon reads and RPKM for genes expressed with at least 10 reads in at least one of the samples. (B) Scatter plot of RPKM for genes in paired samples 16N/16T, 18N/18T, and 19N/19T. Black indicates genes with a P value >.05 (11,352 genes), blue indicates genes with .005 < P ≦ .05 (1962 genes), and red indicates genes with a P value ≦.005 (1425 genes). Spearman rank correlation between paired samples was ∼0.82. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 SNP detection. (A) Percentage distributions of the SNPs in each functional category. (B) Chromatograms show sequences from NT and T for 3 representative genes that were validated (ALDH3A1, SLC25A5, and WDR1). A total of 20 mutations were validated. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 PTK6 expression levels do not correlate with the number of epithelial cells in NT and ESCC samples. (A) PTK6 expression in matched NT and primary ESCC (n = 60) as detected by real-time qPCR. PTK6 levels were normalized with cytokeratin 18 (KRT18), an epithelial-specific marker. The boxes contain the values between 25th and 75th percentiles, the lines across the boxes indicate the median, and the whiskers extend to the highest values excluding outliers and extremes. (B and C) Measurement of PTK6 expression and protein levels in a panel of esophageal cell lines by quantitative real-time qPCR and Western blot, normalized to KRT18. (D) PTK6 protein levels in nontumor (N) and primary ESCC (T) as detected by Western blotting. KRT18 used as normalization control. (E) Western blot analysis of PTK6 and epithelial specific markers cytokeratin 5/6 and cytokeratin AE1/3 in a panel of esophageal cell lines and nontumor (N) and primary ESCC (T) clinical samples. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 Real-time qPCR analysis of PTK6 expression after demethylation and/or histone acetylation treatment with 5-aza-dC (blue bars), VPA (green bars), and/or TSA (red bars) in ESCC cell lines that express PTK6. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 Western blot analysis for expression of PTK6 following nuclear and cytoplasmic fractionation of KYSE30 control (NTC) and PTK6-repressed clones (552 and 912) or KYSE510 control (EV) and PTK6 overexpressed clones (O/E). Sp1 and α-tubulin served as markers for nuclear and cytoplasmic fractions, respectively. Gastroenterology 2012 143, 675-686.e12DOI: (10.1053/j.gastro.2012.06.007) Copyright © 2012 AGA Institute Terms and Conditions