Identification of metastasis-associated exoDEPs in colorectal cancer using label-free proteomics

Exosomes are secreted nanovesicles consisting of biochemical molecules, including proteins, RNAs, lipids, and metabolites that play a prominent role in tumor progression. In this study, we performed a label-free proteomic analysis of exosomes from a pair of homologous human colorectal cancer cell line with different metastatic abilities. A total of 115 exoDEPs were identified, with 31 proteins upregulated and 84 proteins downregulated in SW620 exosome. We also detected 30 proteins expressed only in SW620 exosomes and 60 proteins expressed only in SW480 exosomes. Bioinformatics analysis enriched the components and pathways associated with the extracellular matrix, cytoskeleton-related pathways, and immune system changes of colorectal cancer (CRC). Cellular function experiments confirmed the role of SW620 exosomes in promoting the proliferation, migration, and invasion of SW480 cells. Further verifications were performed on six upregulated exoDEPs (FGFBP1, SIPA1, THBS1, TGFBI, COL6A1, and RPL10), three downregulated exoDEPs (SLC2A3, MYO1D, and RBP1), and three exoDEPs (SMOC2, GLG1, and CEMIP) expressed only in SW620 by WB and IHC. This study provides a complete and novel basis for exploring new drug targets to inhibit the invasion and metastasis of CRC.     

Proteomics identification of ITGB3 as a key regulator in reactive oxygen species-induced migration and invasion of colorectal cancer cells

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and second in females worldwide. Unfortunately 40-50% of patients already have metastatic disease at presentation when prognosis is poor with a 5-year survival of <10%. Reactive oxygen species (ROS) have been proposed to play a crucial role in tumor metastasis. We now show that higher levels of ROS accumulation are found in a colorectal cancer-derived metastatic cell line (SW620) compared with a cell line (SW480) derived from the primary lesion from the same patient. In addition, ROS accumulation can affect both the migratory and invasive capacity of SW480 and SW620 cells. To explore the molecular mechanism underlying ROS-induced migration and invasion in CRC, we have compared protein expression patterns between SW480 and SW620 cells using a two-dimensional electrophoresis-based proteomics strategy. A total of 63 altered proteins were identified from tandem MS analysis. Cluster analysis revealed dysregulated expression of multiple redox regulative or ROS responsive proteins, implicating their functional roles in colorectal cancer metastasis. Molecular and pathological validation demonstrated that altered expression of PGAM1, GRB2, DJ-1, ITGB3, SOD-1, and STMN1 was closely correlated with the metastatic potential of CRC. Functional studies showed that ROS markedly up-regulated expression of ITGB3, which in turn promoted an aggressive phenotype in SW480 cells, with concomitant up-regulated expression of STMN1. In contrast, knockdown of ITGB3 expression could mitigate the migratory and invasive potential of SW620 or H(2)O(2)-treated SW480 cells, accompanied by down-regulated expression of STMN1. The function of ITGB3 was dependent on the surface expression of integrin αvβ3 heterodimer. Furthermore, STMN1 expression and the PI3K-Akt-mTOR pathway were found to be involved in ROS-induced and ITGB3-mediated migration and invasion of colorectal cancer cells. Taken together, these studies suggest that ITGB3 plays an important role in ROS-induced migration and invasion in CRC.     

Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells

Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways.     

联合应用RNA-seq和ATAC-seq寻找FOXQ1转录因子的下游靶基因

结直肠癌(Colorectal cancer, CRC)是一种全球高发的恶性肿瘤,发病原因复杂且预后较差。近年来发现叉头框Q1(Forkhead box Q1,FOXQ1)基因作为一类核转录因子在结直肠癌中高表达,可控制下游基因转录活性。本实验拟探究CRC细胞中FOXQ1的转录调控功能并寻找其下游基因。方法:(1)构建低表达FOXQ1基因的稳定转染CRC细胞株;(2)应用RNA-seq检测FOXQ1敲低前后表达量显著差异的基因;(3)应用转座酶可接近性核染色质区域测序分析(Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq)检测FOXQ1敲低前后细胞染色质易接近性的变化;(4)进一步对FOXQ1敲低前后的RNA-seq和ATAC-seq数据进行一系列生物信息学分析,寻找CRC中FOXQ1转录调控的潜在下游基因。结果:应用RNA-seq筛选出了敲低FOXQ1后表达显著差异的基因EI24、TLR2、SMAD3,通过联合分析两细胞系的测序结果,发现FOXQ1基因敲低后,在DLD1和SW480两个细胞系中染色质易接近性均增强且表达量均上调的基因有61个,染色质易接近性均减弱且表达量均下调的基因有70个,且EI24、TLR2、SMAD3基因均位于重叠分析结果中,其中TLR2、SMAD3基因的染色质区域有明显变化,而EI24基因的染色质区域变化不明显。通过代谢通路分析找到了EI24、TLR2、SMAD3基因所富集的代谢通路。其中SMAD3、TLR2基因在炎症性肠病(Inflammatory bowel disease, IBD)通路中显著富集。EI24基因在p53信号通路(p53 signaling pathway)通路中显著富集。结论:基于染色质易接近性的变化和转录水平的研究发现:敲低FOXQ1基因对CRC细胞系中染色质的开放情况有较大的影响,且影响FOXQ1转录调控的下游基因的表达。找到了FOXQ1敲低后在SW480、DLD1中均发生变化的基因,为丰富FOXQ1转录因子的下游调控网络提供了研究基础。     

Lupulone, a hop bitter acid, activates different death pathways involving apoptotic TRAIL-receptors, in human colon tumor cells and in their derived metastatic cells

Our study aimed to compare death signalling pathways triggered by lupulone in TRAIL-sensitive human colon cancer cells (SW480) and in their derived TRAIL-resistant metastatic cells (SW620). Lupulone (40 μg/ml) up-regulated expression of TRAIL DR4/DR5 death receptors at the cell surface of both cell lines, even in the absence of exogenous TRAIL ligand. Cell death induced by lupulone was inhibited in SW480 and SW620 cells exposed to blocking anti-DR4/DR5 antibodies. In SW480 cells, lupulone triggered cell death through a cross-talk between TRAIL-DR4/DR5 and the mitochondrial (intrinsic) pathways involving caspase-8 activation and Bid protein cleavage. As a consequence mitochondrial cytochrome c was released into the cytosol and activation of caspases-9 and -3 was observed. In the metastatic SW620 cells, lupulone restored the sensibility of these cells to TRAIL ligand and activated the extrinsic apoptotic pathway via DR4/DR5 death receptors and the involvement of the caspase-8/caspase-3 cascade. The demonstration that lupulone is able to activate TRAIL-death signalling pathways even in TRAIL resistant cancer cells highlights the potential of this natural compound for cancer prevention and therapy.     

Human Voltage-Gated Proton Channel Hv1: A New Potential Biomarker for Diagnosis and Prognosis of Colorectal Cancer

Solid tumors exist in a hypoxic microenvironment, and possess high-glycolytic metabolites. To avoid the acidosis, tumor cells must exhibit a dynamic cytosolic pH regulation mechanism(s). The voltage-gated proton channel Hv1 mediates NADPH oxidase function by compensating cellular loss of electrons with protons. Here, we showed for the first time, that Hv1 expression is increased in colorectal tumor tissues and cell lines, associated with poor prognosis. Immunohistochemistry showed that Hv1 is strongly expressed in adenocarcinomas but not or lowly expressed in normal colorectal or hyperplastic polyps. Hv1 expression in colorectal cancer is significantly associated with the tumor size, tumor classification, lymph node status, clinical stage and p53 status. High Hv1 expression is associated significantly with shorter overall and recurrence-free survival. Furthermore, real-time RT-PCR and immunocytochemistry showed that Hv1 is highly expressed in colorectal cancer cell lines, SW620, HT29, LS174T and Colo205, but not in SW480. Inhibitions of Hv1 expression and activity in the highly metastatic SW620 cells by small interfering RNA (siRNA) and Zn²⁺ respectively, markedly decrease the cell invasion and migration, restraint proton extrusion and the intracellular pH recovery. Our results suggest that Hv1 may be used as a potential biomarker for diagnosis and prognosis of colorectal carcinoma, and a potential target for anticancer drugs in colorectal cancer therapy.     

Differential role of Fas/Fas ligand interactions in cytolysis of primary and metastatic colon carcinoma cell lines by human antigen-specific CD8+ CTL

We have previously identified mutated ras peptides reflecting the glycine to valine substitution at position 12 as HLA-A2-restricted, CD8+ CTL neo-epitopes. CTL lines produced against these peptide epitopes lysed the HLA-A2+ Ag-bearing SW480 primary colon adenocarcinoma cell line, although IFN-gamma treatment of the targets was necessary to achieve efficient cytotoxicity. Here, we compared the lytic phenotype of the SW480 cell line to its metastatic derivative, SW620, as an in vitro paradigm to further characterize the nature of a HLA class I-restricted, Ag-specific CTL response against neoplastic cell lines of primary and metastatic origin. Although both colon carcinoma cell lines were lysed by these Ag-specific CTL following IFN-gamma pretreatment, the mechanisms of lysis were distinct, which reflected differential levels of sensitivity to the Fas pathway. Whereas IFN-gamma pretreatment rendered SW480 cells sensitive to both Fas-dependent and -independent (perforin) pathways, SW620 cells displayed lytic susceptibility to Fas-independent mechanisms only. Moreover, pretreatment of SW480 cells with the anti-colon cancer agent, 5-fluorouracil (5-FU), led to enhanced Fas and ICAM-1 expression and triggered Ag-specific CTL-mediated lysis via Fas- and perforin-based pathways. In contrast, these phenotypic and functional responses were not observed with SW620 cells. Overall, these data suggested that 1) IFN-gamma and 5-FU may enhance the lytic sensitivity of responsive colon carcinoma cells to immune effector mechanisms, including Fas-induced lysis; 2) the malignant phenotype may associate with resistance to Fas-mediated lysis in response to Ag-specific T cell attack; and 3) if Ag-specific CTL possess diverse lytic capabilities, this may overcome, to some extent, the potential "escape" of Fas-resistant carcinoma cells.     

Knockdown of DNA‐binding protein A enhances the chemotherapy sensitivity of colorectal cancer via suppressing the Wnt/β‐catenin/Chk1 pathway

DNA‐binding protein A (dbpA) is reported to be upregulated in many cancers and associated with tumor progress. The present study aimed to investigate the role of dbpA in 5‐fluorouracil (5‐FU)‐resistant and oxaliplatin (L‐OHP)‐resistant colorectal cancer (CRC) cells. We found that 5‐FU and L‐OPH treatment promoted the expression of dbpA. Enhanced dbpA promoted the drug resistance of SW620 cells to 5‐FU and L‐OHP. DbpA knockdown inhibited cell proliferation, induced cell apoptosis, and cell cycle arrested in SW620/5‐FU and SW620/L‐OHP cells. Besides, dbpA short hairpin RNA (shRNA) enhanced the cytotoxicity of 5‐FU and L‐OHP to SW620/5‐FU and SW620/L‐OHP cells. Meanwhile, dbpA shRNA inhibited the activation of the Wnt/β‐catenin pathway that induced by 5‐FU stimulation in SW620/5‐FU cells. Activation of the Wnt/β‐catenin pathway or overexpression of checkpoint kinase 1 (Chk1) abrogated the promoting effect of dbpA downregulation on 5‐FU sensitivity of CRC cells. Importantly, downregulation of dbpA suppressed tumor growth and promoted CRC cells sensitivity to 5‐FU in vivo. Our study indicated that the knockdown of dbpA enhanced the sensitivity of CRC cells to 5‐FU via Wnt/β‐catenin/Chk1 pathway, and DbpA may be a potential therapeutic target to sensitize drug resistance CRC to 5‐FU and L‐OHP.     

Ursolic acid synergistically enhances the therapeutic effects of oxaliplatin in colorectal cancer

Oxaliplatin is a key drug in chemotherapy of colorectal cancer (CRC). However, its efficacy is unsatisfied due to drug resistance of cancer cells. In this study, we tested whether a natural agent, ursolic acid, was able to enhance the efficacy of oxaliplatin for CRC. Four CRC cell lines including SW480, SW620, LoVo, and RKO were used as in vitro models, and a SW620 xenograft mouse model was used in further in vivo study. We found that ursolic acid inhibited proliferation and induced apoptosis of all four cells and enhanced the cytotoxicity of oxaliplatin. This effect was associated with down-regulation of Bcl-xL, Bcl-2, survivin, activation of caspase-3, 8, 9, and inhibition of KRAS expression and BRAF, MEK1/2, ERK1/2, p-38, JNK, AKT, IKKα, IκBα, and p65 phosphorylation of the MAPK, PI3K/AKT, and NF-κB signaling pathways. The two agents also showed synergistic effects against tumor growth in vivo. In addition, ursolic acid restored liver function and body weight of the mice treated with oxaliplatin. Thus, we concluded that ursolic acid could enhance the therapeutic effects of oxaliplatin against CRC both in vitro and in vivo, which offers an effective strategy to minimize the burden of oxaliplatin-induced adverse events and provides the groundwork for a new clinical strategy to treat CRC.     

Proteomic profiling reveals key cancer progression modulators in shed microvesicles released from isogenic human primary and metastatic colorectal cancer cell lines

Extracellular vesicles comprise two main classes - exosomes and shed microvesicles (sMVs). Whilst much is known about exosome cargo content and functionality, sMVs are poorly understood. Here, we describe the large-scale purification of sMVs released from primary (SW480) and metastatic (SW620) human isogenic colorectal cancer (CRC) cell lines using a combination of differential ultracentrifugation and isopycnic iodixanol density centrifugation. The yield of SW480-sMVs and SW620-sMVs was 0.75 mg and 0.80 mg, respectively. Both SW480-/SW620-sMVs are heterogeneous in size (100–600 nm diameter) and exhibit identical buoyant densities (1.10 g/mL). In contrast to exosomes, sMVs are ALIX⁻, TSG101⁻, CD63⁻ and CD9⁻. Quantitative mass spectrometry identified 1295 and 1300 proteins in SW480-sMVs and SW620-sMVs, respectively. Gene Ontology enrichment analysis identified ‘cell adhesion’ (CDH1, OCLN, CTN families), ‘signalling pathway’ (KRAS, NRAS, MAPK1, MAP2K1), and ‘translation/RNA related’ processes (EIF, RPL, HNRNP families) in both sMV types. Strikingly, SW480- and SW620-sMVs exhibit distinct protein signatures - SW480-sMVs being enriched in ITGA/B, ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling networks, while SW620-sMVs are enriched in PRKCA, MACC1, FGFR4 and MTOR/MARCKS signalling networks. Both SW480- and SW620-sMVs are taken up by NIH3T3 fibroblasts resulting in similar cell invasion capability. This study provides, for the first time, molecular insights into sMVs and CRC biology.     

Identification of Novel Biomarkers for Metastatic Colorectal Cancer Using Angiogenesis-Antibody Array and Intracellular Signaling Array

Colorectal cancer (CRC) is one of the three leading causes for cancer mortality. CRC kills over 600,000 people annually worldwide. The most common cause of death from CRC is the metastasis to distant organs. However, biomarkers for CRC metastasis remain ill-defined. We compared primary and metastatic CRC cell lines for their angiogenesis-protein profiles and intracellular signaling profiles to identify novel biomarkers for CRC metastasis. To this end, we used primary and metastatic CRC cell lines as a model system and normal human colon cell line as a control. The angiogenesis profiles two isogenic CRC cell lines, SW480 and SW620, and HT-29 and T84 revealed that VEGF was upregulated in both SW620 and T84 whereas coagulation factor III, IGFBP-3, DPP IV, PDGF AA/AB, endothelin I and CXCL16 were downregulated specifically in metastatic cell lines. Furthermore, we found that TIMP-1, amphiregulin, endostatin, angiogenin were upregulated in SW620 whereas downregulated in T84. Angiogenin was downregulated in T84 and GM-CSF was also downregulated in SW620. To induce CRC cell metastasis, we treated cells with pro-inflammatory cytokine IL-6. Upon IL-6 treatment, epithelial-mesenchymal transition was induced in CRC cells. When DLD-1 and HT-29 cells were treated with IL-6; Akt, STAT3, AMPKα and Bad phosphorylation levels were increased. Interestingly, SW620 showed the same signal activation pattern with IL-6 treatment of HT-29 and DLD-1. Our data suggest that Akt, STAT3, AMPKα and Bad activation can be biomarkers for metastatic colorectal cancer. IL-6 treatment specifically reduced phosphorylation levels of EGFR, HER2 receptor, Insulin R and IGF-1R in receptor tyrosine kinase array study with HT-29. Taken together, we have identified novel biomarkers for metastatic CRC through the angiogenesis-antibody array and intracellular signaling array studies. Present study suggests that those novel biomarkers can be used as CRC prognosis biomarkers, and as potential targets for the metastatic CRC therapy.     

人野生型p53基因增强5-FU对大肠癌化疗敏感性的分子生物学机制

为了探讨人野生型p53（wt-p53）基因增强大肠癌细胞化疗敏感性的分子生物学机制,将携带wt p53基因的质粒分别转染两种p53基因突变的人大肠癌细胞系HT-29及SW620,分析细胞中p53及细胞周期蛋白D1（cyclin D1）蛋白的表达水平;将化疗药物5 氟尿嘧啶（5-fluorouracil,5-FU）以不同浓度、不同时间分别作用于HT-29及SW620细胞,另外将已转染wt-p53基因的大肠癌细胞用5-FU进行诱导,Western印迹分析上述干预条件下细胞中p53蛋白及细胞周期蛋白D1表达水平的变化;流式细胞术检测wt p53基因联合5-FU组及对照组中细胞凋亡的改变情况.结果表明,wt-p53基因能增加癌细胞中细胞周期蛋白D1的表达,与wt-p53基因呈剂量依赖性关系;5-FU则降低其蛋白表达,与5-FU呈时间和剂量依赖性关系,而5-FU所致的细胞周期蛋白D1表达水平的降低在细胞预先转染了wt- p53基因时会被抑制;wt-p53基因与5-FU联合使用能提高大肠癌细胞凋亡率.结果提示,wt-p53基因可提高大肠癌细胞中细胞周期蛋白D1的表达水平,并抑制5-FU所致的细胞周期蛋白D1降解,从而提高大肠癌细胞对化疗药物5-FU的敏感性.     

Spectral karyotyping of the human colon cancer cell lines SW480 and SW620

The cell lines SW480 and SW620, derived from different stages of colon carcinoma in the same patient, have been used for a number of biochemical, immunological, and genetic studies on colon cancer. A comparative analysis of their karyotypes may identify chromosomal aberrations that might represent markers for metastatic spread. In the present study spectral karyotyping (SKY) was applied to these two colon cancer cell lines. Compared to previously reported G-banded karyotypes, 9 (SW480) and 7 (SW620) markers were identical, 3 (SW480) and 3 (SW620) markers could be redefined, 5 (SW480) and 8 (SW620) markers were newly identified, and 4 (SW480) and 5 (SW620) of the previous described markers could not be confirmed. The redefined aberrations include very complex rearrangements, such as a der(16) t(3;16;1;16;8;16; 1;16;10) and a der(18)t(18;15;17)(q12; p11p13;??) in SW620 and a der(19)t(19;8;19;5) in SW480, that have not been identified by conventional banding techniques. The resulting chromosome gains (5q11-->5q15, 7pter-->q22, 11, 13q14-->qter, 20pter-->p12, X) and losses (8pter-->p2, 18q12-->qter, Y) found in both SW480 and SW620 were in good agreement with those frequently described in colorectal tumors as primary changes in the stem cell. Abnormalities found exclusively in SW620 cells only (gains of 5pter-->5q11, 12q12-->q23, 15p13-->p11, and 16q21-->q24 and losses of 2pter-->2p24, 4q28-->qter, and 6q25-->qter) can be viewed as changes that occurred in a putative metastatic founder cell.