Integrated Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Quantitative Proteomic Analysis Identifies Galectin-1 as a Potential Biomarker for Predicting Sorafenib Resistance in Liver Cancer MOL CELL PROTEOMICS 5.912 2017-05-25 IMI CONFIDENTIAL

Background 索拉非尼(Sorafenib)属于多激酶抑制剂，是一种新型多靶点抗肿瘤药物。具有抑制肿瘤细胞增殖和血管形成的双重作用，即一方面可以抑制信号传导系统Raf抑制肿瘤细胞增生；另一方面通过抑制血管内皮生长因子受体(vascular endothelial growth factor receptor, VEGFR)和血小板衍生生长因子受体(platelet-derived growth factor receptor, PDGFR)等酪氨酸激酶受体从而抑制肿瘤新生血管的形成和切断肿瘤细胞的营养供应而达到遏制肿瘤生长目的。 IMI CONFIDENTIAL

EMT的发生主要包括细胞形态学的改变、细胞间粘附结构的缺失和细胞免疫表型的变化： (1) 细胞形态学上：上皮细胞极性缺失，细胞变为梭形； (2) 细胞间超微结构发生变化：骨架结构由以角蛋白为主的转化为以波形蛋白(Vimentin)为主，同时细胞间的连接结构蛋白如闭锁小带蛋白1(ZO-1)和泡膜蛋白1(VMP1)下调或缺失，导致细胞间粘附能力降低； (3) 细胞的免疫表型：上皮细胞标记物E-cadherin表达降低，-catenin向细胞核内转移，间质细胞标记物N-cadherin、Vimentin等表达上调，这一过程使细胞失去上皮表型，表现出间质细胞特性。 IMI CONFIDENTIAL

2. 物理缺氧法：应用三气培养箱根据调控不同浓度的O2, CO2和N2使细胞出现不同程度的缺氧。 bla 细胞缺氧模型建立： 1. 化学缺氧法：细胞培养基中加入化学诱导剂如二氯化钴（Cocl2）、氰化物、亚硫酸钠等进行化学诱导，这些化学试剂可造成细胞内呼吸链功能障碍或者导致培养基内氧浓度降低，从而使细胞出现缺氧。 2. 物理缺氧法：应用三气培养箱根据调控不同浓度的O2, CO2和N2使细胞出现不同程度的缺氧。 用三气培养箱、亚硫酸钠和 Cocl2进行肿瘤细胞缺氧研究发现，三种方法都能导致细胞缺氧，引起细胞中HIF-1表达的升高，其中三气培养箱法和Cocl2诱导法还能使细胞发生EMT的改变，通过低氧浓度或Cocl2干预后，细胞中的EMT相关蛋白的表达发生了改变，细胞中HIF-1和Vimentin表达显著上调，而E-cadherin的表达下调，由此说明这两种方法均能引起细胞缺氧，并能致使细胞发生EMT。 Cocl2已被广泛用于细胞缺氧致EMT模型，该方法造模稳定。 IMI CONFIDENTIAL bla

Methods HuH-7：HCC cell HuH-7R：sorafenib-resistant HCC cell line Tumor xenograft model： HuH-7/HuH-7R：subcutaneously injecting (Tumor dimensions and tumor volume) OR tail vein inoculation (H&E) 91 advanced HCC patients/17 healthy volunteers SILAC (for in vitro labeling) iTRAQ (for in vivo labeling) Off-line 2D-LC-MS/MS Protein Identification and Quantification Mascot search engine (http://www.matrixscience.com) Ingenuity Pathway Analysis Tool (IPA) (http://www.ingenuity.com) Immunoblotting and Immunohistochemistry (IHC) Chromatin Immunoprecipitation (ChIP) Assays IMI CONFIDENTIAL

1. Functional Analyses of HuH-7 and HuH-7R Cells HuH-7R cells possess a more aggressive phenotype than HuH-7 cells IMI CONFIDENTIAL

The sorafenib resistant cells showed an activation of the EMT process with enhanced invasive and metastatic potentials. IMI CONFIDENTIAL

2. Identification and Quantification of Differentially Expressed Proteins in HuH-7 and HuH 7R Cells and Cell-Derived Tumors IMI CONFIDENTIAL

4616 2836 A total of 2,450 proteins common to both SILAC and iTRAQ experiments were reliably. 699 644 156 proteins were differentially expressed between HuH-7 and HuH-7R cells. 81 proteins was increased in HuH-7R cells (>2.0-fold) 75 proteins was decreased (<0.5-fold) 301 392 398 252 IMI CONFIDENTIAL

3. Biological Function, Pathway, and Network Analysis bla 3. Biological Function, Pathway, and Network Analysis Red ：EMT-related proteins 9 increased; 1 decreased 详见Table 1 和Table 2 IMI CONFIDENTIAL bla

81 up-regulated proteins 75 down-regulated proteins IMI CONFIDENTIAL

Supplemental Table S2 IMI CONFIDENTIAL Cellular Movement The enriched molecular and cellular biological functions of up-regulated proteins (>2-fold) Category p-value Molecules # Molecules Cellular Movement 9.16E-06 ANXA1, ANXA2, CCDC88A, CTGF, EPHA2, EZR, IQGAP1, LGALS1, VIM 9 Cellular Growth and Proliferation 1.03E-04 AKAP12, ANXA1, ANXA2, CCDC88A, CDC37, CTGF, EPHA2, EZR, HEXIM1, IGF2R, IQGAP1, KPNA2, LGALS1, MNAT1, PFKP, SDCBP, TMSB10/TMSB4X, TPM1, WBP2 19 Cellular Development Cellular Assembly and Organization 3.79E-04 ALDOA, ANXA1, ARL2, CCDC88A, CKAP5, CNN2, EPHA2, EZR, IQGAP1, TPM1, VIM 11 Cellular Function and Maintenance Inflammatory Response 1.78E-03 ANXA1, FKBP8, LGALS1, OPTN, P4HA2, TPM1 6 Tissue Development 2.00E-03 CDC73, CTGF, EIF2AK2, LGALS1, TPM1 5 Cell Death and Survival 2.85E-03 AKAP12, ANXA1, CDC37, CKAP5, CNN2, CTGF, EIF2AK2, EPHA2, EZR, FKBP8, FXR1, HSPB1, IGF2R, KPNA2, LGALS1, PRAF2, SH3BGRL3, TMSB10/TMSB4X 18 Protein Trafficking 4.20E-03 CDC37, MYO1C, OPTN 3 Amino Acid Metabolism 4.27E-03 PLOD1 1 DNA Replication, Recombination, and Repair VIM Energy Production ALDOA Organismal Development 4.78E-03 ANXA1, CTGF, EPHA2, PLOD1 4 Cell Morphology 5.70E-03 ANXA2, ATP6AP1, EPHA2, EZR, IQGAP1, TPM1, VIM 7 Cell-To-Cell Signaling and Interaction 8.13E-03 AKAP12, ANXA1, CCDC50, EPHA2, FKBP8, HSPB1, IGF2R, IQGAP1, LGALS1, OPTN, PVRL2 RNA Post-Transcriptional Modification 8.52E-03 EIF2AK2 Small Molecule Biochemistry 1.97E-02 AKAP12, ALDOA, CTPS1, MNAT1, TOR1B Nucleic Acid Metabolism Carbohydrate Metabolism 2.08E-02 ALDOA, ALDOC, CTGF, OPTN, PFKM Cell Cycle 2.56E-02 ANXA2, EIF2AK2, LGALS1, MNAT1, RRM2B Lipid Metabolism 2.63E-02 LGALS1, VIM 2 Molecular Transport Post-Translational Modification 3.11E-02 EIF2AK2, HEXIM1, IQGAP1 Protein Synthesis 3.26E-02 ANXA2, PFKM, TOR1B Cellular Compromise 3.33E-02 CDC37, TOR1B Supplemental Table S2 IMI CONFIDENTIAL

Supplemental Table S3 IMI CONFIDENTIAL Cellular Movement RALGAPA2 The enriched molecular and cellular biological functions of down-regulated proteins (<0.5-fold) Category p-value Molecules # Molecules Amino Acid Metabolism 9.39E-08 ALDH6A1, ASS1, GCLC, GLDC, GLUD1, PSPH, QDPR 7 Small Molecule Biochemistry Nucleic Acid Metabolism 3.06E-04 ABCD3, ACOT1, AK4, ALDH6A1, CTPS2, GLDC, MTAP, UGDH 8 Lipid Metabolism 4.00E-04 AKR1C3, EBP, RBP1, SULT1A1, SULT1E1 5 Vitamin and Mineral Metabolism Drug Metabolism 5.56E-04 MAOA, SULT1A1 2 Cell-To-Cell Signaling and Interaction Molecular Transport 1.60E-03 GCLC, MAOA Carbohydrate Metabolism 3.55E-03 ALDH2, GALM, GK, PRDX2, SORD, UGDH 6 Energy Production 3.75E-03 ABCD3, ALDH1A1, EHHADH 3 Cell Cycle 7.97E-03 IDH1 1 DNA Replication, Recombination, and Repair GLDC Organismal Development 1.19E-02 ITGA1 Cellular Movement 1.68E-02 RALGAPA2 Cell Death and Survival 2.26E-02 GLRX, MAOA, PRDX2 Cellular Assembly and Organization 2.55E-02 ANPEP, RPS6KA3 Cellular Function and Maintenance Cell Morphology Cellular Compromise 2.76E-02 RPS6KA3 Post-Translational Modification EHHADH Cellular Growth and Proliferation 3.15E-02 Cellular Development Cell Signaling 3.54E-02 ADH5 Protein Synthesis 3.98E-02 A1CF, HIST1H1B Inflammatory Response 4.35E-02 CES1, CSRP1 Supplemental Table S3 IMI CONFIDENTIAL

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10 significantly differentially expressed proteins mainly participated in cellular movement or invasion (ANXA1, ANXA2, CCDC88A, CTGF, EPHA2, EZR, LGALS1（galectin-1）, IQGAP1, RALGAPA2 and vimentin). IMI CONFIDENTIAL

4. Selected In Vitro- and In Vivo-Overexpressed Proteins Associated with Epithelial-Mesenchymal Transition (EMT) 6 proteins associated with EMT including vimentin, CTGF, IQGAP1, galectin-1, ezrin, and annexin A2 were selected. SILAC spectra are shown for sorafenib-regulated proteins IMI CONFIDENTIAL

iTRAQ spectra are shown for sorafenib-regulated proteins bla iTRAQ spectra are shown for sorafenib-regulated proteins IMI CONFIDENTIAL bla

Validation of comparative proteomic results by western blotting IMI CONFIDENTIAL

Proteins dysregulated in HuH-7R cells that used as HCC serum biomarkers for predicting sorafenib resistance？ A total of 22 proteins were putative secreted proteins (SignalP program). Two of these candidates—galectin-1 （半乳糖凝集素1）and CTGF（结缔组织生长因子）—were highly expressed in HuH-7R cells. IMI CONFIDENTIAL

5. Galectin-1 Knockdown Inhibits HuH-7R Cell Proliferation, Migration, and Invasion, and Restores Sorafenib Sensitivity IMI CONFIDENTIAL

IC50 value HuH-7 cells (4.13) Knockdown of galectin-1 not only attenuates cell proliferation and metastasis in HuH-7R cells, it also restores sorafenib sensitivity. IMI CONFIDENTIAL

6. High Expression of Galectin-1 in HuH-7R Cells Promotes Tumorigensis and Pulmonary Metastasis In Vivo Ki67作为标记细胞增殖状态的抗原，阳性说明癌细胞增殖活跃。 IMI CONFIDENTIAL

HuH-7R cells have greater tumorigenic and metastatic potential than HuH-7 cells in vivo. IMI CONFIDENTIAL

7. Galectin-1 Expression is Regulated by PI3K/AKT, mTOR and HIF-1 Pathways Bioinformatics analyses indicated that up-regulation of the mTOR signaling pathway involved in facilitating the sorafenib resistance of HuH-7R cells. IMI CONFIDENTIAL

AKT and mTOR pathways are involved in galectin-1 up-regulation. IMI CONFIDENTIAL

Galectin-1 is a direct target of HIF-1. HIF-1作为肿瘤在低氧条件下最主要的调控因子，在促进肿瘤的侵袭转移过程中起着关键作用。在肿瘤缺氧微环境中， HIF-1能诱导和（或）调控EMT的过程。 Galectin-1 is a direct target of HIF-1. Cocl2(二氯化钴)缺氧诱导剂建立体外细胞缺氧模型。 IMI CONFIDENTIAL

The expression of galectin-1 is mediated by the PI3K/AKT/mTOR/HIF-1 pathway. IMI CONFIDENTIAL

8. Prognostic Value of Galectin-1 in Advanced HCC Patients PFS（Progression-Free-Survival, 无进展生存期）：从治疗开始至肿瘤客观进展或任何原因的死亡的时间。 OS（Overall survival, 总生存期） ：从治疗开始至随访截至时间所观察到的存活时间。 IMI CONFIDENTIAL

52 39 High galectin-1 serum level is associated with poor treatment efficacy of sorafenib, and shorter survivals in advanced HCC patients treated with sorafenib. IMI CONFIDENTIAL

多因素分析显示表达galectin-1可作为肝癌无进展生存期和总生存期的独立预后因素。   PFS OS HR p High galectin-1 1.888 0.008 2.179 0.002 Male 2.379 0.054 1.100 0.830 Age 0.980 0.084 1.006 0.609 HBsAg (+) 1.269 0.449 1.426 0.273 Anti-HCV (+) 0.681 0.287 0.705 0.328 Macrovascular invasion 1.383 0.331 1.627 0.187 AFP > 400 ng/mL 1.169 0.636 1.088 0.794 Extrahepatic metastasis 1.761 0.042 1.486 0.146 BCLC C (vs. B) 0.867 0.783 3.710 CLIP ≥ 3 1.463 0.352 0.900 0.699 ECOG PS = 1 or 2 (vs. 0) 1.058 0.831 1.068 0.893 Abbreviations: PFS = progression-free survival; OS = overall survival; HR = hazard ratio; HBsAg = hepatitis B surface antigen; HCV = hepatitis C virus; AFP = α-fetoprotein; BCLC = Barcelona Clinic Liver Cancer; CLIP = Cancer of the Liver Italian Program; ECOG PS = Eastern Cooperative Oncology Group performance status 多因素分析显示表达galectin-1可作为肝癌无进展生存期和总生存期的独立预后因素。 综合表明过表达galectin-1导致sorafenib抵抗。 IMI CONFIDENTIAL

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  Galectin-1 N (%) Total High Low p 91 52 39 Complete response 0 (0) Partial response 5 (5) 1 (2) 4 (10) Stable disease 48 (53) 24 (46) 24 (62) Progressive disease 33 (36) 25 (48) 8 (21) Not evaluable 5 (6) 2 (4) 3 (8) Response rate* 0.160 Disease control rate# 53 (58) 28 (72) 0.023 *Defined as the percentage of patients with complete response or partial response #Defined as the percentage of patients with complete response, partial response or stable disease IMI CONFIDENTIAL