Metastatic Colorectal Cancer Cells Research Articles

Colon cancer cell-derived 12(S)-HETE induces the retraction of cancer-associated fibroblast via MLC2, RHO/ROCK and Ca2+ signalling

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca2+-calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour–stroma interaction.

Interferon-induced transmembrane protein 1 (IFITM1) is required for the progression of colorectal cancer.

Interferon-induced transmembrane protein 1 (IFITM1) has been shown to be implicated in multiple cancers, yet little is known about biological significance of IFITM1 in colorectal cancer. Here, we show that IFITM1 is highly expressed in metastatic colorectal cancer cell lines as well as colorectal patient-derived tumor samples, and its expression is associated with a poor prognosis of the disease. Also, IFITM1 depletion resulted in a significant reduction in the mobility of cancer cell lines, whereas ectopic expression of IFITM1 promoted the migration of cancer cells. Epithelial-mesenchymal transition (EMT) signature was dysregulated by both loss and gain of function of IFITM1, which was partially reverted by Caveolin-1 (CAV1). Therefore, these results suggest that IFITM1 may be a prognostic marker and an attractive target to achieve better therapeutic outcomes in colorectal cancer.

Identifying N-Glycan Biomarkers in Colorectal Cancer by Mass Spectrometry.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide. Delineating biological markers (biomarkers) for early detection, when treatment is most effective, is key to prevention and long-term survival of patients. Development of reliable biomarkers requires an increased understanding of the CRC biology and the underlying molecular and cellular mechanisms of the disease. With recent advances in new technologies and approaches, tremendous efforts have been put in proteomics and genomics fields to deliver detailed analysis of the two major biomolecules, genes and proteins, to gain a more complete understanding of cellular systems at both genomic and proteomic levels, allowing a mechanistic understanding of the human diseases, including cancer, and opening avenues for identification of novel gene and protein based prognostic and therapeutic markers. Although the importance of glycosylation in modulating protein function has long been appreciated, glycan analysis has been complicated by the diversity of the glycan structures and the large number of potential glycosylation combinations. Driven by recent technological advances, LC-MS/MS based glycomics is gaining momentum in cancer research and holds considerable potential to deliver new glycan-based markers. In our laboratory, we investigated alterations in N-glycosylation associated with CRC malignancy in a panel of CRC cell lines and CRC patient tissues. In an initial study, LC-MS/MS-based N-glycomics were utilized to map the N-glycome landscape associated with a panel of CRC cell lines (LIM1215, LIM1899, and LIM2405). These studies were subsequently extended to paired tumor and nontumorigenic CRC tissues to validate the findings in the cell line. Our studies in both CRC cell lines and tissues identified a strong representation of high mannose and α2,6-linked sialylated complex N-glycans, which corroborate findings from previous studies in CRC and other cancers. In addition, certain unique glycan determinants such as bisecting β1,4-GlcNAcylation and α2,3-sialylation, identified in the metastatic (LIM1215) and aggressive (LIM2405) CRC cell lines, respectively, were shown to be associated with epidermal growth factor receptor (EGFR) expression status. In this Account, we will describe the mass spectrometry based N-glycomics approach utilized in our laboratory to accurately profile the cell- and tissue-specific N-glycomes associated with CRC. We will highlight altered N-glycosylation observed by our studies, consistent with findings from other cancer studies, and discuss how the observed alterations can provide insights into CRC pathogenesis, opening new avenues to identify novel disease-associated glycan markers.

Discovery of genes from feces correlated with colorectal cancer progression.

Colorectal cancer (CRC) is considered to develop slowly via a progressive accumulation of genetic mutations. Markers of CRC may serve to provide the basis for decision-making, and may assist in cancer prevention, detection and prognostic prediction. DNA and messenger (m)RNA molecules that are present in human feces faithfully represent CRC manifestations. In the present study, exogenous mouse cells verified the feasibility of total fecal RNA as a marker of CRC. Furthermore, five significant genes encoding solute carrier family 15, member 4 (SLC15A4), cluster of differentiation (CD)44, 3-oxoacid CoA-transferase 1 (OXCT1), placenta-specific 8 (PLAC8) and growth arrest-specific 2 (GAS2), which are differentially expressed in the feces of CRC patients, were verified in different CRC cell lines using quantitative polymerase chain reaction. The present study demonstrated that the mRNA level of SLC15A4 was increased in the majority of CRC cell lines evaluated (SW1116, LS123, Caco-2 and T84). An increased level of CD44 mRNA was only detected in an early-stage CRC cell line, SW1116, whereas OXCT1 was expressed at higher levels in the metastatic CRC cell line CC-M3. In addition, two genes, PLAC8 and GAS2, were highly expressed in the recurrent CRC cell line SW620. Genes identified in the feces of CRC patients differed according to their clinical characteristics, and this differential expression was also detected in the corresponding CRC cell lines. In conclusion, feces represent a good marker of CRC and can be interpreted through the appropriate CRC cell lines.

Silencing of ST6Gal I enhances colorectal cancer metastasis by down-regulating KAI1 via exosome-mediated exportation and thereby rescues integrin signaling.

Aberrant sialylation has long been correlated with human cancer. Increased ST6 Gal I (β-galactoside α 2, 6 sialyltransferase) and consequently higher levels of cell-surface α 2, 6 sialylation has been associated with human colorectal cancer (CRC) metastasis. We have extensive circumstantial data that sialylation is connected to cancer metastasis, but we do not understand in detail how sialylation can switch on/off multiple steps in cancer metastasis. To investigate the molecular mechanism underlying the ST6Gal I-mediated metastasis of CRC, we silenced the ST6Gal I gene in a metastatic SW620 CRC cell line (SW620-shST6Gal I) and examined the metastatic behavior of the cells. We found that various hallmarks of metastatic ability were considerably enhanced in ST6Gal 1-depleted SW620 clones, as assessed both in vitro and in vivo . In particular, the metastasis suppressor, KAI1, was down-regulated in ST6Gal I-deficient SW620 clones. This reflected the increased exosome-mediated exportation of KAI1, and was associated with a decrease in the KAI1-mediated inhibition of integrin. These findings indicate that gene silencing of ST6Gal I could enhance metastasis of CRC by down-regulating KAI1 activity and rescuing its negative effects on integrin signaling.

Abstract C21: IL-33 promotes growth and liver metastasis of colorectal cancer in mice by remodeling the tumor microenvironment and inducing angiogenesis

Abstract The major cause of death from colorectal cancer (CRC) is liver metastasis and understanding its mechanisms is necessary for early diagnosis and development of new therapies. Interleukin-33 (IL-33) is an IL-1 cytokine that functions as a cytokine and nuclear factor. It is released by necrotic epithelial cells and activated innate immune cells, functioning as an alarmin or early danger signal. Its role in invoking type 2 immune response is firmly established; however, its role in tumor development and metastasis in only beginning to be elucidated. We identified IL-33 as a potently upregulated cytokine in highly metastatic CRC cells and examined its role in tumor growth and metastasis to the liver. We expressed IL-33 in murine and human CRC cell lines and their growth and spontaneous metastasis to the liver was assessed in orthotopic models of CRC in wild type and IL33 knockout mice. The results suggest that tumor- rather than host- derived IL-33 promoted growth and metastasis by inducing expression of cytokines that stimulated mobilization of CD11b+ GR1+ and CD11b+F4/80+ myeloid cells to remodel the tumor microenvironment, and increase tumor angiogenesis, potentially by activating endothelial cells. Conversely, pro-tumorigenic cytokines derived from the microenvironment induced IL-33 expression in tumor cells to create a sustained pro-tumorigenic loop that promotes tumor growth and metastasis. Our findings underscore the potential of IL-33 as a novel and viable therapeutic target for CRC and liver metastasis. Citation Format: Maria Marjorette O. Pena, Yu Zhang, Celestia Davis, James C. Ryan, Diego Altomare, Sapana Shah, Daniel Titus Hughes. IL-33 promotes growth and liver metastasis of colorectal cancer in mice by remodeling the tumor microenvironment and inducing angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C21.

Quantitative Multi-targeted Imaging of Metastatic Colorectal Cancer Cells Using Aptamer Probes in Combination†

Quantitative Multi-targeted Imaging of Metastatic Colorectal Cancer Cells Using Aptamer Probes in Combination†

Glycine inhibits angiogenesis in colorectal cancer: role of endothelial cells.

Neo-angiogenesis is important for tumor growth. Glycine is a non-toxic amino acid with suspected anti-angiogenic effects. This study was designed to evaluate anti-angiogenic effects of glycine in colorectal cancer. Glycine was added to cultures of human and rat colorectal cancer cells (CRC), and endothelial cells (HUVEC). Glycine's direct impact was monitored using MTT assays. Angiogenesis in HUVEC was monitored using 3D sprouting and migration assays. VEGF and CRC-conditioned media were used to stimulate angiogenesis. The glycine receptor (GlyR) was detected using Western blotting and inhibited using strychnine. The WAG-Rij/CC-531 model of metastatic CRC was used to evaluate glycine's impact in vivo. Tumor growth and vessel density were monitored in rats fed with or without 5% glycine for 14days. VEGF and conditioned media significantly increased proliferation, migration, and capillary formation to up to 267%. Glycine completely neutralized this effect and strychnine completely blunted glycine's effect. GlyR was detected in HUVEC. Tumor volume, weight, and vessel density decreased by 35% (p=0.02), 34% (p=0.03), and 55% (p=0.04) in glycine-fed animals. Glycine inhibits angiogenic signaling of endothelial cells and tumor growth. Glycine would be a promising additive to standard and targeted cancer therapies.

MicroRNA-544a Regulates Migration and Invasion in Colorectal Cancer Cells via Regulation of Homeobox A10.

MicroRNAs (miRNAs) are a group of small RNA molecules that post-transcriptionally regulate gene expression. Aberrant expression of miRNAs has been associated with tumorigenesis in various cancers. miR-544a is an understudied miRNA that has recently been implicated in regulating invasion in lung cancer. However, its role in regulating invasion and the underlying mechanism have not been investigated in colorectal cancer (CRC) cells. Microarray analysis was performed in metastatic colorectal tumor samples and their matched normal tissues to identify differentially expressed miRNAs. Quantitative real-time PCR was used to detect miR-544a levels in tumor samples and CRC cell lines with varying metastatic properties. miR-544a mimic or inhibitor was transfected into SW480 and HCT116 cells, respectively, followed by wound healing and invasion assays. Western Blot and luciferase assay were performed to investigate the direct target of miR-544a. Xenograft mouse models was used to examine in vivo function of miR-544a. Our data showed that expression of miR-544a was significantly up-regulated in metastatic tumor samples and CRC cell lines. Inhibition of miR-544a reduced migration and invasion in HCT116 cells. Homeobox A10 (HOXA10) was the direct target of miR-544a which was required for the function of miR-544a in regulating invasiveness. miR-544a inhibitor and/or HOXA10 overexpression reduced lung metastases in HCT116 xenografts. Our study demonstrates that miR-544a regulates invasive and metastatic properties of CRC cells by modulating HOXA10 expression level both in vitro and in vivo. miR-544a may represent a new therapeutic target for the intervention of metastatic colorectal cancer.

IL-33 promotes growth and liver metastasis of colorectal cancer in mice by remodeling the tumor microenvironment and inducing angiogenesis.

Liver metastasis is the major cause of death from colorectal cancer (CRC). Understanding its mechanisms is necessary for timely diagnosis and development of effective therapies. Interleukin-33 (IL-33) is an IL-1 cytokine family member that uniquely functions as a cytokine and nuclear factor. It is released by necrotic epithelial cells and activated innate immune cells, functioning as an alarmin or an early danger signal. Its role in invoking type 2 immune response has been established; however, it has contrasting roles in tumor development and metastasis. We identified IL-33 as a potently upregulated cytokine in a highly metastatic murine CRC cell line and examined its role in tumor growth and metastasis to the liver. IL-33 was transgenically expressed in murine and human adenocarcinoma and carcinoma cell lines and their growth and spontaneous metastasis to the liver were assessed in orthotopic models of CRC in wild-type C57Bl/6 and Il33 knockout mice. The results showed that increased expression of IL-33 in CRC cells enhanced their tumor take, growth, and liver metastasis. Tumor- rather than host-derived IL-33 induced the enhanced recruitment of CD11b+ GR1+ and CD11b+ F4/80+ myeloid cells to remodel the tumor microenvironment by increased expression of mobilizing cytokines, and tumor angiogenesis by activating endothelial cells. IL-33 expression was elevated in patient tumor tissues, induced early in adenoma development, and activated by pro-inflammatory cytokines derived from the tumor microenvironment. The data suggest that tumor-derived IL-33 modulates the tumor microenvironment to potently promote colon carcinogenesis and liver metastasis, underscoring its potential as a therapeutic target. © 2016 Wiley Periodicals, Inc.

Gene therapy for colorectal cancer using adenovirus-mediated full-length antibody, cetuximab.

Cetuximab is a chimeric monoclonal antibody, approved to treat patients with metastatic colorectal cancer (mCRC), head and neck squamous cell carcinoma (HNSCC), non-small-cell lung cancer (NSCLC) for years. It functions by blocking the epidermal growth factor receptor (EGFR) from receiving signals or interacting with other proteins. Although the demand for cetuximab for the treatment of cancer patients in clinics is increasing, the complicated techniques involved and its high cost limit its wide applications. Here, a new, cheaper form of cetuximab was generated for cancer gene therapy. This was achieved by cloning the full-length cetuximab antibody into two serotypes of adenoviral vectors, termed as AdC68-CTB and Hu5-CTB. In vivo studies showed that a single dose of AdC68-CTB or Hu5-CTB induced sustained cetuximab expression and dramatically suppressed tumor growth in NCI-H508– or DiFi-inoculated nude mice. In conclusion, gene therapy using adenovirus expressing full-length cetuximab could be a novel alternative method for the effective treatment of colorectal cancer.

Sciellin mediates mesenchymal-to-epithelial transition in colorectal cancer hepatic metastasis.

Hepatic metastasis is the major cause of mortality in colorectal cancer (CRC) patients. Using proteomic analysis, we found sciellin (SCEL) to be specifically expressed in hepatic metastatic CRC cell lines. SCEL knockdown increased CRC cell migration and invasion, while overexpression had the opposite effect. SCEL knockdown also caused cancer cells to form more invasive structures within 3D cultures, increased the mesenchymal marker vimentin, and attenuated the epithelial marker E-cadherin. SCEL increased WNT signaling by activating β-catenin and its downstream target c-myc, and activated mesenchymal-to-epithelial transition (MET) through a SCEL-β-catenin-E-cadherin axis. SCEL showed higher expression in late stage primary CRC than in its hepatic metastatic counterpart. SCEL expression is dynamically modulated by TGF-β1 and hypoxia, revealing a plastic MET mechanism for tumor colonization. Intrahepatic injection in immunodeficient mice revealed that SCEL is necessary for metastatic CRC tumor growth in the liver. These results demonstrate that SCEL is a MET inducer dynamically regulated through the metastasis process. They suggest SCEL may be a useful therapeutic target for preventing or eliminating CRC hepatic metastasis.

Loss of TINCR expression promotes proliferation, metastasis through activating EpCAM cleavage in colorectal cancer.

Long non-coding RNAs (lncRNAs) are involved in kinds of human diseases, including colorectal cancer (CRC). TINCR, a 3.7 kb long non coding RNA, was associated with cell differentiation in keratinocyte and gastric cancer cells. However, little is known about the role of TINCR in regulation CRC progression. Here, we showed that lncRNA TINCR was associated with CRC proliferation and metastasis. TINCR was statistically downregulated in CRC tissues and metastatic CRC cell lines compared with their counterparts. TINCR was reversely correlated with CRC progression and promoted tumor cells growth, metastasis in vivo and in vitro. While overexpression of TINCR had opposite effect. In addition, we also found that TINCR specifically bound to EpCAM through RNA IP and RNA pull down assays. Loss of TINCR promoted hydrolysis of EpCAM and then released EpICD, subsequently, activated the Wnt/β-catenin pathway. Further studies shown that c-Myc repressed the expression of TINCR through repressing sp1 transcriptive activity, which established a positive feedback loop controlling c-Myc and TINCR expression. These findings elucidate that loss of TINCR expression promotes proliferation and metastasis in CRC and it could be considered as a potential cancer suppressor gene.

PDLIM1 Stabilizes the E-Cadherin/β-Catenin Complex to Prevent Epithelial-Mesenchymal Transition and Metastatic Potential of Colorectal Cancer Cells.

Metastasis is a major cause of death in patients with colorectal cancer, and increasing evidence supports the contribution of the epithelial-mesenchymal transition (EMT) to cancer progression. The dissociation of the E-cadherin/β-catenin adhesion complex represents a key step in EMT and promotes cancer invasion and metastasis, but the upstream signaling pathways regulating this interaction are poorly understood. Here, we show that PDLIM1, a member of the PDZ and LIM protein family, was downregulated in highly metastatic colorectal cancer cells and liver metastases from colorectal cancer patients. We found that loss of PDLIM1 promoted the expression of EMT markers and increased the invasive and migratory properties of multiple colorectal cancer cell lines. Furthermore, PDLIM1 knockdown increased colon-derived liver metastasis in an orthotopic colorectal cancer model and promoted distant metastatic colonization in an experimental lung metastasis model. Mechanistic investigations revealed that PDLIM1 interacted with and stabilized the E-cadherin/β-catenin complex, thereby inhibiting the transcriptional activity of β-catenin and preventing EMT. Accordingly, PDLIM1 overexpression attenuated EMT of colorectal cancer cells. Moreover, the downregulation of PDLIM1 in colorectal cancer samples correlated with reduced E-cadherin and membrane β-catenin levels, and was associated with shorter overall survival. In conclusion, our study demonstrates that PDLIM1 suppresses EMT and metastatic potential of colorectal cancer cells by stabilizing β-catenin at cell-cell junctions, and its loss in metastatic tissues may represent a potential prognostic marker of aggressive disease.

Inhibitor of growth 4 suppresses colorectal cancer growth and invasion by inducing G1 arrest, inhibiting tumor angiogenesis and reversing epithelial-mesenchymal transition.

Previous studies have found that inhibitor of growth 4 (ING4), a tumor suppressor, is reduced in human colorectal cancer (CRC), and is inversely correlated with clinical Dukes' stage, histological grade, lymph node metastasis and microvessel density (MVD). However, its underlying mechanism remains undetermined. In the present study, we analyzed ING4 expression in a panel of human CRC cells using low (LS174T and SW480) and high (LoVo and SW620) metastatic cell lines. We demonstrated that both the low and high metastatic CRC cells exhibited a lower level of ING4 compared to the level in normal human colorectal mucous epithelial FHC cells. Furthermore, ING4 expression in high metastatic CRC cells was less than that in low metastatic CRC cells. We then generated a lentivirus construct expressing ING4 and green fluorescent protein (GFP), established a ING4-stably transgenic LoVo CRC cell line, and investigated the effect of lentiviral-mediated ING4 expression on high metastatic LoVo CRC cells. Gain-of-function studies revealed that ING4 significantly inhibited LoVo CRC cell growth and invasion in vitro and induced cell cycle G1 phase arrest. Moreover, ING4 obviously suppressed LoVo CRC subcutaneously xenografted tumor growth and reduced tumor MVD in vivo in athymic BALB/c nude mice. Mechanistically, ING4 markedly upregulated P21 and E-cadherin but downregulated cyclin E, interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), Snail1, N-cadherin and vimentin in the LoVo CRC cells. Our data provide compelling evidence that i) ING4 suppresses CRC growth possibly via induction of G1 phase arrest through upregulation of P21 cyclin-dependent kinase (CDK) inhibitor and downregulation of cyclin E as well as inhibition of tumor angiogenesis through reduction of IL-6, IL-8 and VEGF proangiogenic factors; ii) ING4 inhibits CRC invasion and metastasis probably via a switch from mesenchymal marker N-cadherin to epithelial marker E-cadherin through downregulation of Snail1 epithelial-mesenchymal transition (EMT)-inducing transcription factor (EMT-TF).

Characterization of novel low passage primary and metastatic colorectal cancer cell lines.

In vitro models are essential to understanding the molecular characteristics of colorectal cancer (CRC) and the testing of therapies for CRC. Many efforts to establish and characterize primary CRC cell lines have been published, most describing a small number of novel cell lines. However, there remains a lack of a large panel of uniformly established and characterized cell lines. To this end we established 20 novel CRC cell lines, of which six were derived from liver metastases. Genetic, genomic and transcriptomic profiling was performed in order to characterize these new cell lines. All data are made publically available upon publication.By combining mutation profiles with CNA and gene expression profiles, we generated an overall profile of the alterations in the major CRC-related signaling pathways. The combination of mutation profiles with genome, transcriptome and methylome data means that these low passage cell lines are among the best characterized of all CRC cell lines. This will allow researchers to select model cell lines appropriate to specific experiments, facilitating the optimal use of these cell lines as in vitro models for CRC. All cell lines are available for further research.

Mesenchymal Stem/Stromal Cells Exert Trophic Effect on Colorectal Cancer Metastasis to the Liver

Colorectal Cancer (CRC) is the third most common cancer in the world. CRC tends to metastasize to the liver, which may occur in 20% to 70% of patients and represents the major cause of death. Mesenchymal Stem/stromal cells (MSCs) have shown to be able to migrate to CRC site and play an important role in tumor progression. We have previously identified a resident MSC population in the liver. Therefore, this study aims to investigate whether there is infiltration of MSCs into patient CRC Liver Metastasis (CRC-LM) and their potential effects on tumor cell growth. By culturing resected patient CRC-LM tissue, we observed the emerging of fibroblast-like cells. Further phenotype and functional characterization confirmed their bonafide MSCs features. In situ staining with a well-established MSCs marker showed a significant enrichment of candidate MSCs in patient CRC-LM, particularly the tumor-stromal area. Moreover, MSCs secreted trophic factors significantly increased colony formation and growth of a metastatic CRC cell line. In summary, we found infiltration and enrichment of MSCs in CRC-LM patient, which could in turn nourish tumor cells.

Generation of a human antibody that inhibits TSPAN8-mediated invasion of metastatic colorectal cancer cells

Tetraspanin 8 (TSPAN8) is a tumor-associated antigen implicated in tumor progression and metastasis. However, the validation of TSPAN8 as a potential therapeutic target in metastatic colorectal cancer (mCRC) has not yet been studied. In this study, through several in vitro methodologies, we identified a large extracellular loop of TSPAN8 (TSPAN8-LEL) as a key domain for regulating mCRC invasion. Using phage display technology, we developed a novel anti-TSPAN8-LEL human antibody with subnanomolar affinity that specifically recognizes amino acids 140–205 of TSPAN8-LEL in a conformation-dependent manner. Finally, we demonstrated that the antibody specifically reduces invasion in the HCT116 and LoVo mCRC cell lines more potently than in the HCT-8 and SW480 non-mCRC cell lines. Our data suggest that TSPAN8-LEL may play an important role in mCRC cell invasion, and that the antibody we have developed could be a useful tool for inhibiting the invasion of TSPAN8-expressing mCRCs.

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.

Alteration of O-GlcNAcylation affects serine phosphorylation and regulates gene expression and activity of pyruvate kinase M2 in colorectal cancer cells.

O-GlcNAcylation is a dynamic post-translational modification that has extensive crosstalk with phosphorylation either at the same or adjacent sites of various proteins. We have previously reported that O-GlcNAcylation level was increased in primary breast and colorectal cancer, but the interplay of the two modifications remains unclear. Therefore, we explored crosstalk of the modifications by RNA interference against O-GlcNAc transferase (OGT) in colorectal cancer cells. Two-dimensional immunoblotting and mass spectrometric analysis showed that the levels of O-GlcNAc and serine phosphorylation of many proteins including serine hydroxymethyltransferase, cytokeratin-8, pyruvate kinase M2 (PKM2), heterogeneous nuclear ribonucleoprotein L, and lamin-B1, were reduced in siOGT cells compared to siScramble cells. In HT29 cells, immunoprecipitated PKM2 revealed decreased O-GlcNAc and serine phosphorylation levels after siOGT knockdown, but increased levels after treatment with Thiamet-G, an inhibitor of O-GlcNAcase (OGA). In addition, when global O-GlcNAcylation was enhanced by treating cells with Thiamet-G, PKM2 expression level was upregulated, but PKM2-specific activity was decreased. On the other hand, in OGT knockdown cells, PKM2 expression level was downregulated, but PKM2-specific activity was increased. Moreover, the metastatic colorectal cancer cells, SW620, had more O-GlcNAc-PKM2 and showed lower PKM2-specific activity compared to the non-metastatic colorectal cancer SW480 cells. These results suggested roles of O-GlcNAcylation in modulating serine phosphorylation, as well as in regulating PKM2 activity and expression. Interfering levels of O-GlcNAcylation of PKM2 may be a novel target in controlling cancer metabolism and tumorigenesis of colorectal cancer.