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Proteome Atlas of Human Chromosome 8 and Its Multiple 8p Deﬁciencies in Tumorigenesis of the Stomach, Colon, and Liver † ○ † ○ ‡ § ○ ∥ ○ † † † Yang Zhang, , Guoquan Yan, , Linhui Zhai, , , Shaohang Xu, , Huali Shen, Jun Yao, Feifei Wu, † # † † † ‡ § ‡ § Liqi Xie, Hailin Tang, Hongxiu Yu, Mingqi Liu, Pengyuan Yang, Ping Xu, , Chengpu Zhang, , ‡ § ‡ § ‡ § ‡ § ‡ § ∥ ⊥ ∥ Liwei Li, , Cheng Chang, , Ning Li, , Songfeng Wu, , Yunping Zhu, , Quanhui Wang, , Bo Wen, ∥ ∥ ∥ □ † ∥ ⊥ ‡ § Liang Lin, Yinzhu Wang, Guiyan Zheng, Lanping Zhou, Haojie Lu,*, Siqi Liu,*, , Fuchu He,*, , † and Fan Zhong*, † Institutes of Biomedical Sciences and Department of Chemistry, Fudan University, Shanghai 200032, China ‡ State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China § National Engineering Research Center for Protein Drugs, Beijing 102206, China ∥ BGI-Shenzhen, Shenzhen 518083, China ⊥ Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China # College of Mechanical & Electronic Engineering and Automatization, National University of Defense Technology, Changsha 410073, China □ State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China

*S Supporting Information

ABSTRACT: Chromosome 8, a medium-length euchromatic unit in humans that has an extraordinarily high mutation rate, can be detected not only in evolution but also in multiple mutant diseases, such as tumorigenesis, and further invasion/ metastasis. The Chromosome-Centric Human Proteome Project of China systematically profiles the proteomes of three digestive organs (i.e., stomach, colon, and liver) and their corresponding carcinoma tissues/cell lines according to a chromosome organizational roadmap. By rigorous standards, we have identified 271 (38.7%), 330 (47.1%), and 325 (46.4%) of 701 chromosome 8-coded proteins from stomach, colon, and liver samples, respectively, in Swiss-Prot and observed a total coverage rate of up to 58.9% by 413 identified proteins. Using large-scale label-free proteome quantitation, we also found some 8p deficiencies, such as the presence of 8p21−p23 in tumorigenesis of the above-described digestive organs, which is in good agreement with previous reports. To our best knowledge, this is the first study to have verified these 8p deficiencies at the proteome level, complementing genome and transcriptome data. KEYWORDS: chromosome 8, proteome, 8p deletion, tumorigenesis, liver, colon, stomach

■ INTRODUCTION Chromosome 8 is a medium-length euchromatic unit in humans that has an extraordinarily high mutation rate by The Chromosome-Centric Human Proteome Project (C-HPP) 3,4 5 6,7 ﬁ positive selection. DEF and MCPH1 are widely known has received considerable attention for its signi cance in rapidly evolving gene clusters in 8p. Telomere shortening, understanding gene function and structure in terms of especially in chromosome 8, appears as a mechanism fostering chromosome proteins.1,2 The International Human Genome the development of chromosomal instability during aging and Sequencing Consortium recently completed a sequence of the chronic disease.8 This relatively high genomic instability of human genome including chromosome 8. It has reported a chromosome 8 is found not only in evolution but also in manually curated gene catalog, containing 793 gene loci and 301 pseudogene loci, including all previously known genes on Special Issue: Chromosome-centric Human Proteome Project 3 chromosome 8 and giving 701 nonredundant proteins in Received: August 31, 2012 Swiss-Prot (Version 2012-7-11).

Figure 1. Chromosome 8 proteome atlas with data on the quantification (A) and identification (B) of gastric (green text and frame), colorectal (blue text and frame), and hepatic (red text and frame) systems. (A) The colors denote the abundance of the protein expression data. The median − normalized protein data were transformed by log2 and rescaled into a [ 1,1] region. The protein data above the median are shown in red, whereas those below the median are shown in blue. The color legend is provided at the top portion. (B) The protein coding genes are shown in blue. multiple mutant diseases, such as tumorigenesis, and further information, is more robust for LTQ data. More details on invasion/metastasis. Chromosome 8 and its 8p deletion have proteome quantification are provided in the related article. The been studied and determined to be associated with mass spectrum results detected rather high incidences of hepatocellular carcinoma (HCC) metastasis for many years.9 missing values (without identification in some replicates). Use As part of the Chinese C-HPP consortium, chromosomes 1, of each proteomic experimental replicate as input column 8, and 20 have been selected to systematically profile the would disrupt further Gene Set Enrichment Analysis (GSEA) proteomes of three digestive organs, namely, stomach, colon, processing. To solve this problem, we calculated the average of and liver, as well as their corresponding carcinoma tissues/cell SIN or LFQuant values for each sample as representation, rather lines. From the perspective of pathophysiological significance in than deal with each fluctuant experimental replicate in further China and worldwide, digestive cancers, such as gastric cancer analysis. For comparative analysis of liver samples, we further (GC), colorectal cancer (CRC), and HCC, are among the most normalized SIN and LFQuant values by dividing their medians. frequently reported cancers and are characterized by metastatic Biological Category Statistics and Enrichment Analysis potential and poor outcomes. This digestive group includes some of the most critical cancers (among them are those “Data set Files” and “Analyses” under “Human Genes ranked second to fourth in cancer-related mortality) and, Chromosomal Location” in Ingenuity Pathway Analysis (IPA, despite all sustained efforts, maintains a profile of low survival Ingenuity Systems; www.ingenuity.com) Version 14197757 rates and lacks successful therapies.10 Herein, we report the were used for cell, tissue, and organ (CTO) and cell line proteome of chromosome 8 based on measured proteins from distribution statistics as well as biological category enrichment samples of selected digestive tissues/cells and summarize the analysis. total nonredundant proteins reported so far for the said Enrichment analysis of 288 missing chromosome 8-coded proteome. We also discuss the connections of measured 8p proteins was carried out using the web-accessible Database for deletions with these three digestive cancers. Annotation, Visualization, and Integrated Discovery (DAVID) tool.14 DAVID can recognize the UniProt AC from data sets. ■ MATERIALS AND METHODS Medium classification stringency and default items were chosen for enrichment calculation. Proteome Identification and Quantification GSEA Proteomes from 18 samples, namely, liver tissue11 Hep3B, SNU398, SNU449, SNU475, MHCC97L (97L), MHCC97H GSEA15 was performed to find down-regulated enriched (97H), HCCLM3 (LM3), HCCLM6 (LM6), colon tissue, cytobands in chromosome 8. The data set was loaded from CRC tissue, SW480, HCT116, stomach tissue, GC tissue, AGS, HUGO Gene Nomenclature Committee-approved gene BGC823, and SGC7901, were identified or researched as symbols16 and analyzed using the Java GSEA package.17 We described in a companion article (DOI: 10.1021/pr3008286). scanned (Signal2Noise metric, weighted scoring, 1,000 Protein abundance in the human liver data set and that in 17 phenotype permutations) the cytoband-organized c1 gene fi 12 13 other samples were quanti ed by SIN and by LFQuant, sets of Molecular Signature Database v3.0. As GSEA requires respectively. Both are label-free proteome quantification at least three (sample) columns for each phenotype group, we methods, and the SIN method, which does not use XIC replicated stomach, colon, and liver proteome data to appear

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Figure 2. Overlapping status of samples in total and chromosome 8-coded protein identifications. (A−C) Unique (diagonal, red text) and overlapped identification scales within gastric (A), colorectal (B), and hepatic (C) systems. Numbers of chromosome 8-coded proteins are given in brackets. (D−K) Venn diagrams comparing total proteins (D−F) and chromosome 8-coded proteins (G−I) identified within the three digestive systems and among three normal digestive organs (J, K). (L, M) Venn diagrams comparing total proteins (L) and chromosome 8-coded proteins (M) from the present study, PeptideAtlas, and MaxQB. thrice as well as HCT116 and SW480 proteome data to appear respectively, using rigorous standards. The proteomes from twice for comparison. The gene sets with normalized these samples provided 413 identified proteins in total and gave enrichment scores lower than −1.00 were enriched in a a total coverage of up to 58.9%. The total identification down-regulated direction. coverage of chromosome 8-coded proteins in other 5 databases were Ensembl (gene level, v.69) 402/696 = 57.8%, neXtProt ■ RESULTS (v.2012-10-16) 413/700 = 59.0% (Table S2 in Supporting Information), GPMdb (green, v.2012-10-18) 355/472 = 75.2%, Proteomes of the Three Digestive Organs Peptide Atlas (1% FDR at protein level, v.2012-07) 333/385 = All proteome data sets for liver, stomach, and colon samples 86.5%, and Human Protein Atla (v.10.0) 314/467 = 67.2%. have been thoroughly discussed elsewhere for the proteome of These findings suggest that selections of any CTOs (Table S3 chromosome 1 (see the companion article, DOI: 10.1021/ in Supporting Information) and cell lines (Table S4 in pr3008286). In brief, we identified 271 (38.7%), 330 (47.1%), Supporting Information) seldom cover more than 50% of and 325 (46.4%) of 701 chromosome 8-coded proteins in genes or those with expression bias in each chromosome. When Swiss-Prot (Version 2012-7-11) from stomach, colon, and liver the sample was limited to merely normal or paracancer tissues, samples (Figure 1 and Table S1 in Supporting Information), these identification scales decreased to 112 (16.0%, stomach),

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Figure 3. 8p deficiencies in the three digestive organs by cancer versus normal pairing. (A) GSEA normalized enrichment score (NES)of chromosome 8 cytobands in cancer versus normal comparisons. The negative value indicates down-regulation, and |NES| > 1.00 can be significant. (B) Heatmaps of protein abundance located on 8p21−p23 in each sample are displayed based on the protein quantification values, leaving those unidentified as blank blocks. The color legend is shown on the right side of the heatmaps.

303 (43.2%, colon), and 238 (34.0%, liver). The 288 missing detected in the colon (Figure 2K). We also compared our data proteins of chromosome 8-coded proteins were found to be set with published data sets for the whole proteome (Figure mostly enriched in the “disulfide bond, signal peptide, secreted, 2L) and the chromosome 8-coded proteome (Figure 2M). and glycoprotein” cluster (enrichment score = 3.45, p = 1.3 × Aebersold et al.18 created a PeptideAtlas data set including 406 − − 10 5, Benjamini = 7.7 × 10 3) and “defensin, antimicrobial, and proteins of chromosome 8 among 12,173 proteins collected defense response” cluster (enrichment score = 2.68, p = 9.0 × from the literature. In addition, a MaxQB data set with 313 − 10 4, Benjamini = 0.57) according to DAVID. proteins of chromosome 8 among 10,463 proteins measured IPA preanalysis showed no major expression bias of from 11 cell lines was established by Mann et al.19 Altogether, chromosomes among different CTOs (Table S3 in Supporting these three data sets provide 487 nonredundant proteins and an Information) and cell lines (Table S4 in Supporting increased coverage of 69.5% for the chromosome 8-coded Information) at the transcriptome level, thus answering the proteome. intriguing question of whether any expression bias exists as we 8p Deficiencies in Tumorigenesis select a sample for C-HPP profiling. These results also confirm that chromosome 8 is distinctly typical in character, being very We performed GSEA based on quantified proteome data and close to the genome median for each characteristic in terms of found that multiple significantly down-regulated enriched length, gene content, repeat content, and segmentally cytobands existed in chromosome 8, especially 8p21−p23 of duplicated fraction,3 although some chromosomes apparently all three digestive organs (Figure 3A). Detailed heatmaps with exhibited extreme characteristics. There were 2 insufficient cancer versus normal pairing showed that several 8p21−p23- protein identified cytobands that 8p23.1 (52/288) and 8q24.3 coded proteins (Figure 3B) were clearly down-regulated or (39/288), and the proteome quantification distribution among absent and occurred in other cytobands of chromosome 8 the 18 samples and their chromosome 8-coded subsets did not (Figure S2 in Supporting Information). We determined that significantly differ (Figure S1 in Supporting Information). chromosome 8 exhibits the highest enrichment of breast cancer − We observed that the overlap of proteomes was high genes (p = 1.68 × 10 10) and orthologs to (mouse) mammary − between cancer and normal/paracancer tissues of the same tumor genes (p = 2.23 × 10 10) (Table S5 in Supporting organ and among cancer cell lines from the same organ but Information), as well as the highest coverage (32.0%) of relatively low between tissues and cell lines (Figure 2A−F). A tumorigenesis genes among all chromosomes (Table S6 in similar status was observed in chromosome 8-coded proteomes Supporting Information). Our proteome results for chromo- (Figure 2G−I). Not surprisingly, the proteome of the stomach some 8 seem to be in good agreement with the findings for the was almost inclusive of that of the colon (3,254/3,375), mainly genome. due to the similar compositions of these two organs from Particularly, the proteins of some reported HCC, CRC, and gastrointestinal epithelia (Figure 2J). Only two chromosome 8- GC suppressor genes in 8p, namely, CCDC25 (8p21.1),20 coded proteins were identified in the stomach, namely, IDO1 DLC1 (8p21.3−p22),20,21 EPHX2 (8p12−p21),20,22 LZTS1 (8p11−p12) and NACAP1 (8q22.3), but these were not (8p22),23 NRG1 (8p12−p21),24 PCM1 (8p22),25 PROSC

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Figure 4. Protein abundances located on 8p of HCC, CRC, and GC suppressor genes were down-regulated or deﬁcient in the three carcinomatous digestive organs.

(8p11.2),20 SCARA5 (8p21.1),26 SH2D4A (8p21.2),20,27 the missing 214 proteins in chromosome 8 could be cell- SORBS3 (8pter−p23.3),20,28 TUSC3 (8p22),29 WRN specific proteins in cells other than those we have explored. (8p11.2−p12),24 and ZNF703 (8p12),24 were significantly Although each cell type might express a few tens of cell-specific down-regulated or deficient in the three carcinomatous proteins only, the total number of cell-specific proteins could be digestive organs (Figure 4). Some proteins of HCC and CRC accumulated as the amount of measured cell type would suppressor genes located in other cytobands were also found to increase. As such, we strongly suggest that more types of CTOs be down-regulated/deficient, including BIRC2 (11q22);30 CA1 and cell lines be measured to observe all 701 possibly expressed (8q13-q22.1), CA2 (8q22), and CA13 (8q21.2);31 CDH1 proteins in chromosome 8, because the numbers of cell types in (16q22.1); TCF4 (18q21.2);32 TP53 (17p13); and YAP1 the present and previous studies are still considerably lower (11q13)30 (Figure S3 in Supporting Information). than the minimum possible of 230 for all cell types. We investigated some 8p deficiencies, such as the presence of − ■ DISCUSSION 8p21 p23 in tumorigenesis of the stomach, colon, and liver. 8p11.21, 8p21.3, and 8p22−p23 losses and 8q24 gain Using the latest instruments and methodology in this study, we frequently occurred in HCC tumorigenesis and its meta- fi 9,34−37 have determined that the identi ed proteins in each sample stasis. 8p deletions/LOH including 8p21, 8p22−23.1, from the stomach, colon, and liver are less than or close to only and 8q24 gains also promoted CRC and its meta- − half of the proteome (701) of chromosome 8. We thus stasis.24,32,38 41 The 8p deletion/LOH is also a frequent − speculated that the average of half of a chromosome proteome event in GC.42 45 8p contains many tumor suppressor genes might be the upper limit for expressed proteins. Aebersold et al. and is essential for cancer progression and metastasis.45,46 In provided a quantitative description of the proteome of a addition to HCC, CRC, and GC, 8p deletions can be found in commonly used human cell line and demonstrated that the lung cancer,47 larynx cancer,48 and renal cancer.49 8p LOH also − human cultured cells express at least approximately 10,000 occurs in bladder cancer,50,51 breast cancer,45 49,52 B cell 33 − proteins. This number is close to half of the total number of lymphoma,53 prostate cancer,54 56 and head and neck the protein-coded genome (∼20,344), which might explain why squamous cell carcinoma.57,58 Particularly, loss of chromosome only approximately half of or fewer proteins on average can be 8p associated with poor outcome (reduced survival time) and found in chromosome 8 for a cell or several cells. Mann et al. deletion of a cluster of six genes on 8p (CCDC25, DLC1, ELP3, identified 313 proteins belonging to chromosome 8 from PROSC, SH2D4A, and SORBS3) among the 10-gene signature 10,463 proteins in 11 cell lines, comparable with our 325 in HCC from patients with poor outcomes have been proteins from 8 cell lines and tissues from the liver (Figure 1I). reported.9,34 Thus, the deletion of chromosome 8p (e.g., PeptideAtlas has summarized 406 proteins of chromosome 8 8p23) might contribute to the development of HCC among 12,173 proteins from literature data sources (cells and metastasis.9 tissues) to date, and the present work study increases this The 8p deletion/LOH in chromosome 8 can be attributed to number to 487 from a total of 16,079 proteins for the proteome its genome instability. The chromosome has a vast region of of chromosome 8 (Figure 1L and M). Therefore, we feel that approximately 15 megabases on distal 8p with a strikingly high

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