Marine bacterial exopolysaccharide EPS11 inhibits migration and invasion of liver cancer cells by directly targeting collagen I

Abstract

Many natural polysaccharides have significant anticancer activity with low toxicity, but the complex chemical structures make in-depth studies of the involved mechanisms extremely difficult. The purpose of this study was to investigate the effect of the marine bacterial exopolysaccharide (exopolysaccharide 11 [EPS11]) on liver cancer metastasis to explore the underlying target protein and molecular mechanism. We found that EPS11 significantly suppressed cell adhesion, migration, and invasion in liver cancer cells. Proteomic analysis showed that EPS11 induced downregulation of proteins related to the extracellular matrix–receptor interaction signaling pathway. In addition, the direct pharmacological target of EPS11 was identified as collagen I using cellular thermal shift assays. Surface plasmon resonance and pull-down assays further confirmed the specific binding of EPS11 to collagen I. Moreover, EPS11 was shown to inhibit tumor metastasis by directly modulating collagen I activity via the β1-integrin–mediated signaling pathway. Collectively, our study demonstrated for the first time that collagen I could be a direct pharmacological target of polysaccharide drugs. Moreover, directly targeting collagen I may be a promising strategy for finding novel carbohydrate-based drugs. Many natural polysaccharides have significant anticancer activity with low toxicity, but the complex chemical structures make in-depth studies of the involved mechanisms extremely difficult. The purpose of this study was to investigate the effect of the marine bacterial exopolysaccharide (exopolysaccharide 11 [EPS11]) on liver cancer metastasis to explore the underlying target protein and molecular mechanism. We found that EPS11 significantly suppressed cell adhesion, migration, and invasion in liver cancer cells. Proteomic analysis showed that EPS11 induced downregulation of proteins related to the extracellular matrix–receptor interaction signaling pathway. In addition, the direct pharmacological target of EPS11 was identified as collagen I using cellular thermal shift assays. Surface plasmon resonance and pull-down assays further confirmed the specific binding of EPS11 to collagen I. Moreover, EPS11 was shown to inhibit tumor metastasis by directly modulating collagen I activity via the β1-integrin–mediated signaling pathway. Collectively, our study demonstrated for the first time that collagen I could be a direct pharmacological target of polysaccharide drugs. Moreover, directly targeting collagen I may be a promising strategy for finding novel carbohydrate-based drugs. Liver cancer is a major health problem, with an incidence of more than 850,000 new cases worldwide each year (1Torre L.A. Bray F. Siegel R.L. Ferlay J. Lortet-Tieulent J. Jemal A. Global cancer statistics, 2012.CA Cancer J. Clin. 2015; 65: 87-108Crossref PubMed Scopus (22943) Google Scholar). It is currently the second leading cause of cancer-related mortality in the world (approximately 800,000 per year), a figure that is on the rise (2Bruix J. Han K.H. Gores G. Llovet J.M. Mazzaferro V. Liver cancer: Approaching a personalized care.J. Hepatol. 2015; 62: S144-S156Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Over the past decades, there has been an improvement in the treatment of the disease. Five treatments can extend the life expectancy of patients with liver cancer: early stage tumors that are amenable to curative therapies—resection, liver transplantation, or local ablation; at more-developed stages, only chemoembolization (for intermediate tumors) and sorafenib (for advanced tumors) have shown survival benefits (3Forner A. Reig M. Bruix J. Hepatocellular carcinoma.Lancet. 2018; 391: 1301-1314Abstract Full Text Full Text PDF PubMed Scopus (2503) Google Scholar). There are still major unmet needs in the management of liver cancer that might be addressed through discovery of new therapies. The intensive communication between tumor microenvironment and tumor cells regulates growth, differentiation, and migration of tumor cells. An increasing amount of recent research has concentrated on the function of the tumor microenvironment in favoring cancer progression (4Xu S. Xu H. Wang W. Li S. Li H. Li T. Zhang W. Yu X. Liu L. The role of collagen in cancer: From bench to bedside.J. Transl. Med. 2019; 17: 309Crossref PubMed Scopus (202) Google Scholar). The extracellular matrix (ECM), a three-dimensional structure with distinct biochemical and biomechanical properties, is an important component of tumor microenvironment (5Zheng X. Liu W. Xiang J. Liu P. Ke M. Wang B. Wu R. Lv Y. Collagen I promotes hepatocellular carcinoma cell proliferation by regulating integrin β1/FAK signaling pathway in nonalcoholic fatty liver.Oncotarget. 2017; 8: 95586-95595Crossref PubMed Scopus (28) Google Scholar). ECM is increasingly recognized as an important contributor to tumor behaviors, including tumor growth, angiogenesis, and metastatic progression. The major receptors mediating ECM–cell interactions are integrins, a family of heterodimeric transmembrane proteins composed of noncovalently associated α and β subunits. β1-integrin is the key transmembrane protein that conducts extracellular biochemical and biomechanical signals into the cell, regulating cell proliferation, migration, invasion, and survival (6Casar B. Rimann I. Kato H. Shattil S.J. Quigley J.P. Deryugina E.I. In vivo cleaved CDCP1 promotes early tumor dissemination via complexing with activated β1 integrin and induction of FAK/PI3K/Akt motility signaling.Oncogene. 2014; 33: 255-268Crossref PubMed Scopus (77) Google Scholar). The β1-integrin subfamily includes a variety of receptors, such as collagen, fibronectin, and laminin for ECM proteins. The expression level of β1-integrin was higher in cancer tissues than that in adjacent nontumor tissues (5Zheng X. Liu W. Xiang J. Liu P. Ke M. Wang B. Wu R. Lv Y. Collagen I promotes hepatocellular carcinoma cell proliferation by regulating integrin β1/FAK signaling pathway in nonalcoholic fatty liver.Oncotarget. 2017; 8: 95586-95595Crossref PubMed Scopus (28) Google Scholar). And researchers have explored the possibility of developing integrin-targeted therapy to manage cancer (7Keely P.J. Mechanisms by which the extracellular matrix and integrin signaling act to regulate the switch between tumor suppression and tumor promotion.J. Mammary Gland Biol. Neoplasia. 2011; 16: 205-219Crossref PubMed Scopus (105) Google Scholar). Previous study has demonstrated that the activation of β1-integrin by collagen I promoted the epithelial–mesenchymal transition (EMT) in pancreatic cancer (8Duan W. Ma J. Ma Q. Xu Q. Lei J. Han L. Li X. Wang Z. Wu Z. Lv S. Ma Z. Liu M. Wang F. Wu E. The activation of β1-integrin by type I collagen coupling with the Hedgehog pathway promotes the epithelial-mesenchymal transition in pancreatic cancer.Curr. Cancer Drug Targets. 2014; 14: 446-457Crossref PubMed Scopus (26) Google Scholar). In breast cancer, inhibition of the β1-integrin activity by monoclonal antibody AIIB2 dramatically enhances radiotherapy efficacy and increases sensitivity to human epidermal growth factor receptor 2–targeting agents (9Wu T. Dai Y. Tumor microenvironment and therapeutic response.Cancer Lett. 2017; 387: 61-68Crossref PubMed Scopus (628) Google Scholar). Collagen, as a large family of triple helical proteins, is the major constituent of the tumor stroma environment. The collagen superfamily now includes more than 20 collagen types. Collagen I, as a major component of ECM, plays vital roles on the disruption of cellular adhesion and EMT (10Koenig A. Mueller C. Hasel C. Adler G. Menke A. Collagen type I induces disruption of E-cadherin-mediated cell-cell contacts and promotes proliferation of pancreatic carcinoma cells.Cancer Res. 2006; 66: 4662-4671Crossref PubMed Scopus (206) Google Scholar, 11Shintani Y. Maeda M. Chaika N. Johnson K.R. Wheelock M.J. Collagen I promotes transition in cancer cells via growth J. Biol. PubMed Scopus Google Scholar, I collagen and promotes a in human carcinoma J. PubMed Scopus Google which and tumor cells to the in invasion and metastasis A. T. L. J. Collagen type I regulates and to migration and proliferation of carcinoma Google which are for of liver the expression of collagen I was in liver cancer It has been that the expression of collagen I was significantly in of human liver cancer with adjacent nontumor tissues (5Zheng X. Liu W. Xiang J. Liu P. Ke M. Wang B. Wu R. Lv Y. Collagen I promotes hepatocellular carcinoma cell proliferation by regulating integrin β1/FAK signaling pathway in nonalcoholic fatty liver.Oncotarget. 2017; 8: 95586-95595Crossref PubMed Scopus (28) Google Scholar). of the role of ECM in the progression of liver cancer the of new and therapeutic in cancer of tumor ECM has shown promising in tumor metastasis in studies L. J. tumor microenvironment to epithelial-mesenchymal transition in Cancer Res. 2015; PubMed Scopus (206) Google Scholar). a was in metastasis of cancer in vivo N. H. E. E. N. Y. the of on metastasis of PubMed Scopus Google Scholar). a novel and was to inhibit EMT through the direct inhibition of collagen I expression H. N. Y. T. S. H. S. H. Y. H. S. of collagen type I and protein in Med. PubMed Scopus Google Scholar). pharmacological of collagen I can of ECM to The a of to large and have been recognized as an important of new with and therapeutic carbohydrate-based with 2018; 16: Scopus (26) Google Scholar). carbohydrate-based are of important pharmacological such as Y. Wang X. W. X. Xu N. and activity of polysaccharide 2017; Scopus Google in and by PubMed Scopus Google of and polysaccharides marine Res. 65: PubMed Scopus Google V. E. with and 2011; 17: PubMed Scopus Google and G. S. J. Y. and activity of the PubMed Scopus Google Scholar). to the marine for in the there are marine carbohydrate-based including Y. Zhang L. W. in Biol. Transl. 2019; PubMed Scopus Google and by are in marine and can be the In recent marine the efficacy as and that increasing as a promising of new for cancer novel marine and J. Cancer 2017; Google Scholar). In our a novel marine bacterial (exopolysaccharide 11 [EPS11]) marine It has been that EPS11 only significantly cancer growth in and in vivo R. W. Y. Wang J. Liu G. S. C. bacterial polysaccharide EPS11 cancer cell growth via cell adhesion and 2018; 16: Scopus Google but tumor metastasis in and in vivo J. Liu G. Ma W. Z. C. bacterial polysaccharide EPS11 cancer cell growth and metastasis via cell adhesion and structure 2019; 17: Scopus Google that EPS11 has of cancer growth and Tumor metastasis for the of cancer in and EPS11 significant activity with low and the of EPS11 on the metastasis in human cells explored in this Moreover, that EPS11 directly to collagen I and activity via β1-integrin signaling that collagen I protein is a pharmacological target for the effect of EPS11 on tumor In our found that EPS11 the growth and migration of liver cancer cells via cell adhesion and structure J. Liu G. Ma W. Z. C. bacterial polysaccharide EPS11 cancer cell growth and metastasis via cell adhesion and structure 2019; 17: Scopus Google Scholar). In to EPS11 has the inhibition on liver cancer further in this and liver cancer cells in the growth and with and the cells the structures with cellular of the of the to the ECM in the of hepatocellular carcinoma cells. structure is a key factor cell adhesion and migration in cancer cells Han S. B. J. Liu B. F. Zhang inhibition of function invasion and metastatic 2015; PubMed Scopus Google Scholar). to the of EPS11 on the migration and invasion of liver cancer cells using migration and invasion with the the of and cells and liver cancer with EPS11 dramatically in a and and We the of EPS11 on the of liver cancer cells. And the of showed that and liver cancer cells in the and was in the cells in that EPS11 could significantly the and of cells. the of EPS11 in the EPS11 with and was as a on the that was a the of each of on cells by And the showed that the of the with the of on the and the for was further with and was as a with in the the the of of EPS11 with more liver cancer cells first with and with for The of showed that induced cell to that of EPS11 that the of the activity and structure of The by and the by on the of cells that EPS11 is with was in our and the showed that the signals of only in of cell and cell but in of cellular further the between EPS11 and analysis of ECM and EPS11 in liver cancer cells. analysis showed that EPS11 the interaction signaling pathway. cells with of EPS11 and for and cell and identified using The of proteins and for analysis using with collagen I on the of the the cell cell adhesion and cell inhibition induced by EPS11 in with laminin and The as of ECM, extracellular exopolysaccharide the of EPS11 on the liver cancer a analysis to proteins EPS11 for protein are as the of proteins, with that in the there and proteins and than in and of and analysis that the significantly pathway in the was the interaction signaling pathway. proteins significantly in this pathway liver cancer cells with EPS11 and for that ECM is to be involved in the anticancer activity of of proteins by and than in a new and than Collagen I, and are the ECM proteins that can in interaction and a key role in cell with collagen I the of cells induced by EPS11 was In laminin and showed on the cell The of ECM proteins on cancer cell adhesion by with the collagen I significantly the cell adhesion in cells laminin showed further and In addition, the effect of EPS11 on the of cells was by collagen I, of cell was in the of laminin and demonstrated that collagen I, but laminin and fibronectin, to be involved in tumor and could and cell adhesion and in cells. further the of collagen I on of liver cancer induced by first with of collagen I and and cells with EPS11 treatment and showed that with the with collagen I significantly the suppression of cell adhesion induced by and collagen I the collagen I was in the The effect of collagen I on cell migration was by the shown in with the time is the of cell Collagen I significantly the of cancer cells by with the with or collagen I on to cell The showed that EPS11 suppressed cell invasion in cells collagen I cell invasion in cells with collagen I was by EPS11 of collagen I was by and the of analysis showed that the expression of collagen I cells with of collagen I for The inhibition on cell adhesion, migration, and invasion in cells by EPS11 was further with the of collagen I In collagen I to be the promising in of liver cancer induced by Moreover, and showed that the and protein expression of collagen I by EPS11 and that EPS11 effect on the and expression of collagen I. that collagen I was a key target for EPS11 the interaction of EPS11 with collagen I, first target by cellular thermal shift that EPS11 treatment collagen I protein showed that collagen I was and EPS11 with was more on or the cells The of EPS11 and collagen I and on or the cells the direct interaction between EPS11 with collagen I, plasmon resonance analysis that target of EPS11 binding to collagen I was a specific binding of EPS11 with collagen I. In addition, pull-down was further to the binding of EPS11 with collagen I in cells. first with cell and a of the of and target for shown in collagen I was in including cell through and further the interaction between EPS11 and collagen I. is a transmembrane which plays a vital role in It is that β1-integrin is with the level of collagen I in liver cancer further figure β1-integrin is involved in the of liver cancer induced by β1-integrin is and in liver cancer cell and through shown in the expression of β1-integrin in liver cancer cells dramatically for the cell adhesion by EPS11 in liver cancer cells was by of β1-integrin the of β1-integrin the of EPS11 on cell migration and invasion in liver cancer cells. antibody β1-integrin was to the signaling related with β1-integrin in our shown in antibody β1-integrin could the and of liver cancer cells and that β1-integrin is involved in cell migration and EPS11 the of cell adhesion in liver cancer cell but on cell adhesion cells with antibody β1-integrin the inhibition on cell migration and invasion of cell induced by EPS11 by of antibody further that EPS11 the of liver cancer via In the that the of liver cancer cells induced by EPS11 is directly on collagen I via β1-integrin signaling pathway. the cancer-related metastasis is the in our to cancer as a L. Y. Tumor new into the Med. PubMed Scopus Google Scholar). of therapeutic agents and for the of metastatic cancer be a promising strategy for In the has been demonstrated that natural marine carbohydrate-based EPS11 could inhibit metastasis of liver cancer by directly targeting collagen I through β1-integrin signaling a novel and key for metastatic liver Previous studies on the discovery of new agents directly targeting cancer tumor ECM proteins, has been In this our that EPS11 could target extracellular collagen I to metastasis of liver our have tumor tumor cells with the microenvironment through biochemical and L. J. tumor microenvironment to epithelial-mesenchymal transition in Cancer Res. 2015; PubMed Scopus (206) Google Scholar). And in the tumor microenvironment can local invasion and metastasis by cancer cells to a EMT to a cellular and molecular transition tumor by which cells to L. J. tumor microenvironment to epithelial-mesenchymal transition in Cancer Res. 2015; PubMed Scopus (206) Google Scholar, B. G. EMT cancer and PubMed Scopus Google Scholar). In the tumor ECM plays an important role in and tumor S. Dai Y. Y. M. L. H. X. Li J. and role of type I collagen in and 2019; PubMed Scopus Google Scholar). The role of ECM in EMT was in the by and showed that cells and in three-dimensional collagen can and as cells G. in collagen can and of Biol. PubMed Scopus Google Scholar). Zhang P.J. The collagen receptor receptor to breast cancer Biol. PubMed Scopus Google that ECM protein collagen I could tumor cell invasion by to in breast tumor cells. ECM fibronectin, was to EMT by expression in tumor cells J. Mammary cell interactions with epithelial-mesenchymal 2014; 33: PubMed Scopus Google Scholar). the therapy may be a strategy for the treatment of tumor In our analysis that EPS11 inhibition effect on the interaction signaling pathway of liver cancer cells that there was a between the of EPS11 and which component of ECM is for the metastasis cells with EPS11 on with collagen I, or showed that cells on collagen I a higher proliferation and adhesion than on laminin and study showed that collagen I was an important target of EPS11 and pull-down studies that there was a direct interaction between EPS11 and collagen I and a adhesion in cancer cancer cell with collagen (4Xu S. Xu H. Wang W. Li S. Li H. Li T. Zhang W. Yu X. Liu L. The role of collagen in cancer: From bench to bedside.J. Transl. Med. 2019; 17: 309Crossref PubMed Scopus (202) Google Scholar). of collagen to in cancer mediating a of cellular such as differentiation, proliferation, and progression B. Tumor 2019; Scopus Google Scholar). The expression level of β1-integrin was in human liver cancer with the level of collagen I. the of β1-integrin and collagen I in liver cancer cells associated with cancer proliferation and metastasis (5Zheng X. Liu W. Xiang J. Liu P. Ke M. Wang B. Wu R. Lv Y. Collagen I promotes hepatocellular carcinoma cell proliferation by regulating integrin β1/FAK signaling pathway in nonalcoholic fatty liver.Oncotarget. 2017; 8: 95586-95595Crossref PubMed Scopus (28) Google Scholar). In our β1-integrin is identified as the receptor for EPS11 via binding to collagen I therapy has promising in and studies in breast cancer, and tumors J. S. M. H. T. cells of cancer cells through collagen J. 2014; PubMed Scopus Google Scholar). It has been that tumor cell migration and invasion was by monoclonal in the pancreatic carcinoma E. A. V. M. J. of tumor cell migration by extracellular matrix proteins in human pancreatic PubMed Scopus Google Scholar). of β1-integrin significantly tumor growth and pancreatic cancer metastasis S. F. P. M. of the integrin tumor growth and pancreatic cancer J. 2011; PubMed Scopus Google Scholar). our to the inhibition of β1-integrin as a for cancer In recent studies have shown that natural polysaccharides have significant anticancer and the of the human are the activity of polysaccharides a of and and in the Y. M. Q. J. and of polysaccharide natural 2018; PubMed Scopus Google Scholar). with low and are to as a to the agents Y. A. B. of and health of the 2019; PubMed Scopus Google and have a of in the of tumor anticancer have been including W. G. Zhang P. and effect of an exopolysaccharide J. Biol. PubMed Scopus (26) Google B. R. In in vivo and of exopolysaccharide by type and Full Text Full Text PDF PubMed Scopus Google and marine exopolysaccharide marine for the and treatment of breast Cancer Google Scholar). The of of of the of the induction of cell and activation of but there are the of of are with an composed of the and and in or such as and Y. A. B. of and health of the 2019; PubMed Scopus Google the studies of in-depth mechanisms extremely difficult. study has demonstrated for the first time that can directly target collagen I to which to the research and of new polysaccharides in In that marine bacterial can directly with collagen I. And the of collagen I by in large the metastasis of liver cancer cells via β1-integrin signaling pathway. the research of polysaccharides in our a novel underlying the metastasis of polysaccharides in and a for developing more carbohydrate-based drugs. and was Collagen I, and of and by was by collagen I, and the of monoclonal was was in the of or liver cancer cells in with and liver cancer cells and liver cancer cells in with and with the of cell between and and cells in the the and with of EPS11 and for or by by and in and for at each was to the of cells on the Hepatocellular carcinoma cells and in which with or for cells by with cells with for at and with for 20 was was into each with for The at was by a human cell and in a was on the of each using with the cells to with or for or of each and with an with a the of of with Hepatocellular carcinoma cells and in with or EPS11 and into the of each further for or to cell migration through the to the of the with cells with and with the cells on the of the using and the cells on the of the and by an The of cells was in the the cells to through a with of was on the for at and in or EPS11 and into the of each The with of or for or cells on the with for by for 20 at the cells on the via with of the cells on the of the using an The was in The cells and at for to which the with for 20 at and of by an EPS11 was to the R. W. Y. Wang J. Liu G. S. C. bacterial polysaccharide EPS11 cancer cell growth via cell adhesion and 2018; 16: Scopus Google Scholar). The of was with a of to EPS11 was with to and further this for with to EPS11 was with a in the The was in for and was for at the was to cells in with for cell at for cells first with and with for with cells by the for Proteomic analysis of cells was by cells in with EPS11 and for and cells and to cellular and the in and in directly a with a to in to in and to in at for the at a of on an The in with a The was The was to for a of to to 20 for further with The higher was at of and The in the at a of first was as target was at with an of and a time of The using the human with was as to The for was as 20 in first and in and the for was as on was as on protein and on as was discovery was to and for was as The and analysis of the the of each in to this the of the protein is by the of the in is by and the of the in is The is as the and the to the of the amount of in the the of the needs to be by the of the protein is as the of the of the specific to the The is as the specific to which the protein of with 20 extracellular I, and was as F. P. S. M. E. via and in human Res. PubMed Scopus Google Scholar). laminin or collagen I with into each and for at the with and and or cells. cells in of growth in the at for and with treatment for 20 was into each and for of and was to each for to of at was by a The and human collagen I by cells in the at to with human collagen I using to the collagen I and a was as a for The of was the cells further in and the expression of collagen I in cells by was to the of was into using a with the as the was as was in with and by cells by and with and with protein on to and with and and by using the target for anticancer was as J. M. B. Li C. S. Wang L. Li X. Yu R. activity in and in vivo of a of protein J. Google Scholar). In cells with EPS11 for by for at the and and on the of treatments was the using and further by The was first by with and for In was at Collagen I protein in was on a of EPS11 to at a of with and to was by to a binding with for at by to the cell with at the proteins, and the proteins by and by of and of as Y. Liu G. Liu R. M. Zhang J. C. a novel bacterial liver cancer cell growth and PubMed Scopus Google Scholar). The of β1-integrin was by The with and into the The was into and the was confirmed by The was into liver cancer cells and using the to the The cells at and the of β1-integrin in liver cancer cell further confirmed by β1-integrin cells for with 20 antibody at and cell adhesion, migration, and invasion as are as are by using of significant of analysis are to the via the The that have of with the of this is by the of and of the of the of the of of and the of G. L. and C. S. R. L. and Y. S. G. L. G. L. G. L. R. Y. and C. S. and C. S. C. S. with with with

Topics & Concepts