Control of the Human Osteopontin Promoter by ERRα in Colorectal Cancer

Abstract

Colorectal cancer is the second leading cause of death from cancer. Osteopontin (OPN) is a component of tumor extracellular matrix identified as a key marker of cancer progression. The estrogen-related receptor α (ERRα) has been implicated in endocrine-related cancer development and progression, possibly through modulation of cellular energy metabolism. Previous reports that ERRα regulates OPN expression in bone prompted us to investigate whether ERRα controls OPN expression in human colorectal cancer. Using a tissue microarray containing 83 tumor-normal tissue pairs of colorectal cancer samples, we found that tumor epithelial cells displayed higher staining for ERRα than normal mucosa, in correlation with elevated OPN expression. In addition, knocking down endogenous ERRα led to reduced OPN expression in HT29 colon cancer cells. Promoter analysis, inhibition of ERRα activity, and expression and mutation of potential ERRα response elements in the proximal promoter of human OPN showed that ERRα and its obligate co-activator, peroxisome proliferator-activated receptor γ co-activator-1 α, positively control human OPN promoter activity. Furthermore, chromatin immunoprecipitation experiments confirmed in vivo occupancy of the OPN promoter by ERRα in HT29 cells, suggesting that OPN is a direct target of ERRα in colorectal cancer. These findings suggest an additional mechanism by which ERRα participates in the development and progression of colorectal cancer, further supporting the relevance of targeting ERRα with antagonists as anticancer agents. Colorectal cancer is the second leading cause of death from cancer. Osteopontin (OPN) is a component of tumor extracellular matrix identified as a key marker of cancer progression. The estrogen-related receptor α (ERRα) has been implicated in endocrine-related cancer development and progression, possibly through modulation of cellular energy metabolism. Previous reports that ERRα regulates OPN expression in bone prompted us to investigate whether ERRα controls OPN expression in human colorectal cancer. Using a tissue microarray containing 83 tumor-normal tissue pairs of colorectal cancer samples, we found that tumor epithelial cells displayed higher staining for ERRα than normal mucosa, in correlation with elevated OPN expression. In addition, knocking down endogenous ERRα led to reduced OPN expression in HT29 colon cancer cells. Promoter analysis, inhibition of ERRα activity, and expression and mutation of potential ERRα response elements in the proximal promoter of human OPN showed that ERRα and its obligate co-activator, peroxisome proliferator-activated receptor γ co-activator-1 α, positively control human OPN promoter activity. Furthermore, chromatin immunoprecipitation experiments confirmed in vivo occupancy of the OPN promoter by ERRα in HT29 cells, suggesting that OPN is a direct target of ERRα in colorectal cancer. These findings suggest an additional mechanism by which ERRα participates in the development and progression of colorectal cancer, further supporting the relevance of targeting ERRα with antagonists as anticancer agents. CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Despite advanced early diagnosis and clinical treatment, colorectal cancer (CRC) remains a worldwide health concern. More than 608,700 deaths were estimated to occur in 2008 from CRC,1Jemal A. Bray F. Center M.M. Ferlay J. Ward E. Forman D. Global cancer statistics.CA Cancer J Clin. 2011; 61: 69-90Crossref PubMed Scopus (30539) Google Scholar representing the fourth leading cause of death after lung, stomach, and liver cancers. CRC development and progression are complex events that involve multistep accumulations of numerous genetic mutations and epigenetic events that result in unregulated transcription of genes involved in cancer cell growth and in alterations in growth factor-regulated signaling pathways. Osteopontin (OPN) is a secreted matricellular glycophosphoprotein produced by osteoblasts and osteoclasts which plays an important role in bone mineral deposit.2McKee M.D. Pedraza C.E. Kaartinen M.T. Osteopontin and wound healing in bone.Cells Tissues Organs. 2011; 194: 313-319Crossref PubMed Scopus (74) Google Scholar OPN has also been found to be expressed in various cell types, therefore contributing to numerous physiological processes and to the pathogenesis of a variety of disease states, including chronic inflammation and cancer.3Lund S.A. Giachelli C.M. Scatena M. The role of osteopontin in inflammatory processes.J Cell Commun Signal. 2009; 3: 311-322Crossref PubMed Scopus (357) Google Scholar OPN is involved in almost all steps of tumor progression by regulating cell–matrix interactions and cell signaling through binding with integrins and CD44 receptors.4Kim J.Y. Bae B.N. Kim K.S. Shin E. Park K. Osteopontin. CD44, and NFkappaB expression in gastric adenocarcinoma.Cancer Res Treat. 2009; 41: 29-35Crossref PubMed Google Scholar In particular, OPN has been reported to promote metastasis by increasing tumor growth, cell migration, degradation of the extracellular matrix, cell survival in blood stream, and angiogenesis (reviewed in Wai and Kuo5Wai P.Y. Kuo P.C. Osteopontin: regulation in tumor metastasis.Cancer Metastasis Rev. 2008; 27: 103-118Crossref PubMed Scopus (276) Google Scholar). OPN is overexpressed in a number of cancers, including CRC in which it has been identified as one of the leading markers in cancer progression.6Coppola D. Szabo M. Boulware D. Muraca P. Alsarraj M. Chambers A.F. Yeatman T.J. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies.Clin Cancer Res. 2004; 10: 184-190Crossref PubMed Scopus (310) Google Scholar, 7Agrawal D. Chen T. Irby R. Quackenbush J. Chambers A.F. Szabo M. Cantor A. Coppola D. Yeatman T.J. Osteopontin identified as lead marker of colon cancer progression, using pooled sample expression profiling.J Natl Cancer Inst. 2002; 94: 513-521Crossref PubMed Scopus (369) Google Scholar, 8Rohde F. Rimkus C. Friederichs J. Rosenberg R. Marthen C. Doll D. Holzmann B. Siewert J.R. Janssen K.P. Expression of osteopontin, a target gene of de-regulated Wnt signaling, predicts survival in colon cancer.Int J Cancer. 2007; 121: 1717-1723Crossref PubMed Scopus (69) Google Scholar, 9Likui W. Hong W. Shuwen Z. Clinical significance of the upregulated osteopontin mRNA expression in human colorectal cancer.J Gastrointest Surg. 2010; 14: 74-81Crossref PubMed Scopus (46) Google Scholar Of note, OPN can be expressed by both cancer cells and cellular components of the tumor’s microenvironment.10Anborgh P.H. Mutrie J.C. Tuck A.B. Chambers A.F. Role of the metastasis-promoting protein osteopontin in the tumour microenvironment.J Cell Mol Med. 2010; 14: PubMed Scopus Google Scholar in from the in OPN gene expression has been to be of epithelial of the from normal to and in the PubMed Scopus Google Scholar suggesting that OPN is a target of implicated in epithelial Of it has been reported that A.F. of expression of osteopontin secreted in Res. Google Scholar and J. for growth PubMed Scopus Google Scholar, J. Osteopontin is overexpressed in colorectal and is with by Med. 3: Google Scholar can OPN expression. Furthermore, in of the OPN proximal promoter as as suggest that and human OPN gene are by numerous including and receptor α, and the C. Z. Kuo P.C. regulates osteopontin expression in human colon 2007; PubMed Scopus Google Scholar, C. M. T. M. Osteopontin early and be in Mol 2010; PubMed Scopus Google Scholar, P. P. P. B. cancer.J PubMed Scopus Google Scholar, A. C. M. is osteopontin expression in J Cancer. 2011; PubMed Scopus Google Scholar, Chen regulation of human osteopontin promoter by and in cancer 2004; PubMed Scopus Google Scholar, R. W. A. B. B. C. D. osteopontin and for colorectal 2009; PubMed Scopus Google Scholar, M. A. gene with and in regulation of osteopontin 2004; PubMed Scopus Google Scholar OPN regulation also a number of transcription relevant to bone including estrogen-related receptor α for which the to bone has been in the E. B. Expression of the estrogen-related receptor 1 receptor PubMed Scopus Google Scholar, C. of the osteopontin promoter by the receptor receptor Google Scholar, receptor regulates osteopontin expression through a response in a cell Mol 2008; PubMed Scopus Google Scholar Despite its with ERRα is an of the receptor it is by P. of a of PubMed Scopus Google Scholar The activity of ERRα is by the with of energy as peroxisome proliferator-activated receptor γ 1 which are as protein of D. K. T. A. The regulates the expression and activity of the receptor estrogen-related receptor PubMed Scopus Google Scholar, J. F. response in the human estrogen-related receptor promoter peroxisome proliferator-activated receptor control of 2004; PubMed Scopus Google Scholar no ERRα has been reported to ERRα including have been to ERRα in R. P. R. of a for the receptor estrogen-related receptor 2004; PubMed Scopus Google Scholar, the estrogen-related receptor as a target in Cancer Res. PubMed Scopus Google Scholar ERRα is a of that controls involved in energy control of energy by the estrogen-related Rev. 2008; PubMed Scopus Google Scholar In addition, the role of ERRα as a ERRα is overexpressed and with and clinical in various cancer cell types, including receptor and estrogen-related receptor with and in human Res. 2002; Google Scholar, T. T. T. receptor in human as a Res. 2004; PubMed Scopus Google Scholar, control of the by and Res. 2010; PubMed Scopus Google Scholar, A. D. B. A. F. P. E. of in cancer and bone metastasis of and Res. 2011; PubMed Scopus Google Scholar M. Chen Expression and of in human cells and PubMed Scopus Google Scholar, T. T. J. T. K. T. M. expression of estrogen-related receptor is a in human cancer.Int J Cancer. 2007; PubMed Scopus Google Scholar J. E. T. Clinical of estrogen-related receptor expression in Mol 2007; PubMed Scopus Google Scholar M. P. J. D. Expression of receptor and clinical significance in J Cancer. PubMed Scopus Google Scholar and A. M. R. D. A. F. receptor and and gene expression in human colorectal tumour J Cancer. 41: PubMed Scopus Google Scholar in higher ERRα expression in than in normal A. M. R. D. A. F. receptor and and gene expression in human colorectal tumour J Cancer. 41: PubMed Scopus Google Scholar In of its ERRα has also been reported to in cancer cell and as as in growth tumor A. D. B. A. F. P. E. of in cancer and bone metastasis of and Res. 2011; PubMed Scopus Google Scholar, C. activity cell 2009; PubMed Scopus Google Scholar, J. T. M. receptor is for the growth of Res. 2008; PubMed Scopus Google Scholar, D. expression is by estrogen-related receptor and and in an to cancer cell Res. 2010; PubMed Scopus Google Scholar, A. J. of estrogen-related in response to and growth of Natl A. 2008; PubMed Scopus Google Scholar the of ERRα as a of energy is a of ERRα has also been to be relevant to tumor J. T. M. receptor is for the growth of Res. 2008; PubMed Scopus Google Scholar, J. M. Park M. M. of direct target genes estrogen-related receptor as a of cancer Res. 2009; PubMed Scopus Google Scholar This that ERRα key of cancer and a for the development of cancer agents. In the of we whether ERRα protein expression is in colon cancer epithelial cells and whether ERRα is a of human OPN expression. of 83 CRC and normal from the were from Tissues were after to a by the of the were with in in and in were and to and Furthermore, of tumor cells from of tissue with the of a for and Tissues and by of mutations in tumor by direct in mutation of to containing mutation and and mutations of and mutations of and of the are in and of in This of CRC stage in a microarray as tissue a J PubMed Scopus Google Scholar were for and staining to tumor and normal tissue to be tissue with a of were from tissue using a a with and down for 1 to a the were and were in in were with Tissues were with and from were with and and with the and for 1 were with and with with and with a were with a The for protein expression in tumor and normal staining staining staining staining staining and of expression in epithelial cells to to to to of from 1 to The expression for as the the expression for tumor and normal no expression in cancer and expression in cancer The human cells and the human colon cell HT29 were from the American The HT29 cell of its ERRα and OPN expression. were in with and and in a were also with the ERRα for 48 to ERRα activity and to its R. P. R. of a for the receptor estrogen-related receptor 2004; PubMed Scopus Google Scholar The expression targeting and control were from were produced by the expression and the in cells. HT29 cells were with for and in with with the to the with and were with the OPN and ERRα and human and were in with the of in a mRNA expression were with the for in Res. PubMed Scopus Google Scholar to expression and expressed as to that of control cells were in with were to and were with by with with the were and were and The human OPN promoter for ERRα response elements with the of the from OPN human promoter in the and of and potential ERRα response elements were with the of the to the of were and and and The of confirmed by cells were with to the were in and in with the of of of of and of In of of were with and to the with cell were to for by with were an additional 48 for using a experiments immunoprecipitation HT29 cells as J.C. C. E. receptor is a of and controls in the 2004; PubMed Scopus Google Scholar after chromatin of to in were with of no for by with of for 1 were and as J.C. C. E. receptor is a of and controls in the 2004; PubMed Scopus Google Scholar were with for and with for were with and were and of OPN with the and for this a control and of the ERRα promoter as as J.C. C. E. receptor is a of and controls in the 2004; PubMed Scopus Google Scholar with and were with of with for to tumor and normal for The for correlation to the and the expression for ERRα and were expressed as the of were as the expression of ERRα and OPN the protein in tissue we a that one of normal and one of CRC for 83 in which the mutation in key genes involved in colorectal were ERRα found to be expressed in the of CRC samples, the expression higher in than in and In a for ERRα expression were to be higher in than in in in and in only ERRα expression also in the from with epithelial ERRα expression as as epithelial cells, cells, and the tumor displayed ERRα a second protein of only CRC displayed no OPN in epithelial cells and expression were higher in than in normal colon and In the 83 staining for epithelial OPN were higher in than in in in and in only OPN in and cells were from normal and in than in epithelial cells Expression for ERRα and OPN with including tumor and stage as as and mutations in and Of for ERRα and OPN were in of in this and both were in of to normal of and OPN staining in with tissue from and Cancer with with 3: Cancer with with The number of is the were for ERRα and OPN and OPN in Colorectal Cancer and number of in which ERRα and OPN are with number of in which ERRα and OPN are with number of in which ERRα and OPN are with for ERRα and OPN expression in cancer to tissue no 1 in in ERRα and OPN were higher in and of with normal The of both were in of ERRα and OPN both in of The number of in which ERRα and OPN are with in a Correlation for ERRα and OPN expression in cancer to tissue no 1 in in ERRα and OPN were higher in and of with normal The of both were in of ERRα and OPN both in of investigate whether OPN expression is by ERRα in colon cancer cells, its endogenous expression in HT29 cells, using a that down ERRα mRNA and protein with HT29 cells with ERRα in a in OPN gene and protein expression and which to after of HT29 cells These findings a of ERRα to the expression of OPN in colon cancer cells. In of reports that ERRα controls the OPN promoter in cells through a of an ERRα response C. of the osteopontin promoter by the receptor receptor Google Scholar a response receptor regulates osteopontin expression through a response in a cell Mol 2008; PubMed Scopus Google Scholar we the human OPN promoter in to potential of the its potential ERRα response elements were found the proximal promoter of to to and to from the transcription a in the significance of the human proximal OPN in of a gene The binding of ERRα to this by in cells, using as ERRα ERRα a and its activity In led to a of activity possibly of endogenous ERRα in cells. Furthermore, of and ERRα promoter activity activity with Of note, of increasing of and ERRα in of the promoter whether ERRα is for promoter cells were with and with the ERRα in ERRα activity and expression in in promoter activity and ERRα response elements be found in the proximal promoter we to in further the for ERRα control of expression. therefore mutation of the in cells. in and of led to of the promoter activity by ERRα to the mutation in displayed reduced and ERRα activity and These findings suggest that by is to control through the the Furthermore, the be than the its activity, mutation in the further the in activity in In the activity, suggesting that this potential binding is a ERRα also promoter activity through a of the and ERRα activity. suggest that by positively regulates the activity of the human OPN proximal that the and are for ERRα in and that ERRα OPN expression in HT29 colon cancer cells, we a to whether ERRα to the promoter in the chromatin of HT29 colon cancer cells. in with a human in a in the that the and in the promoter a we found endogenous ERRα to be to the promoter of the human ERRα gene with the of no suggest that ERRα to the of the endogenous OPN promoter in HT29 and this In the we that the expression of the receptor ERRα is elevated in epithelial cells of a of CRC samples, in correlation with higher OPN expression in normal Furthermore, we showed with the of that ERRα to OPN expression in HT29 colon cancer cells. from the and mutation of the OPN promoter and also suggest that OPN is a direct target gene of ERRα in colon cancer cells. ERRα with and reports of prompted the of ERRα expression in cancers. ERRα is overexpressed in cancer types, and elevated expression has been with clinical in and receptor and estrogen-related receptor with and in human Res. 2002; Google Scholar, T. T. T. receptor in human as a Res. 2004; PubMed Scopus Google Scholar, J. E. T. Clinical of estrogen-related receptor expression in Mol 2007; PubMed Scopus Google Scholar Furthermore, ERRα gene expression has been by in and found to be higher in than in normal and to with higher and stage of A. M. R. D. A. F. receptor and and gene expression in human colorectal tumour J Cancer. 41: PubMed Scopus Google Scholar In the we a tissue microarray with 83 tumor-normal tissue pairs from with CRC and the by ERRα This the of of samples, and its to is In with reported ERRα gene expression analysis, we that for ERRα expression were higher in than in normal epithelial cells, the of the tumor ERRα in cells, and This is with of ERRα in in and and J. J. F. receptor and are of 2007; PubMed Scopus Google Scholar, M. D. J.C. is a of and Natl A. 2011; PubMed Scopus Google Scholar ERRα is in the staining also in tumor cells with ERRα expression. this involve a in which ERRα and its in cancer M. D. C. A. F. A. M. controls estrogen-related receptor cellular and its 2011; PubMed Scopus Google Scholar we correlation ERRα expression and including tumor and in a of CRC be to ERRα and pathways. OPN has been an important in bone and tumor progression. In with reported in colorectal tumor D. Chen T. Irby R. Quackenbush J. Chambers A.F. Szabo M. Cantor A. Coppola D. Yeatman T.J. Osteopontin identified as lead marker of colon cancer progression, using pooled sample expression profiling.J Natl Cancer Inst. 2002; 94: 513-521Crossref PubMed Scopus (369) Google Scholar, 8Rohde F. Rimkus C. Friederichs J. Rosenberg R. Marthen C. Doll D. Holzmann B. Siewert J.R. Janssen K.P. Expression of osteopontin, a target gene of de-regulated Wnt signaling, predicts survival in colon cancer.Int J Cancer. 2007; 121: 1717-1723Crossref PubMed Scopus (69) Google Scholar, 9Likui W. Hong W. Shuwen Z. Clinical significance of the upregulated osteopontin mRNA expression in human colorectal cancer.J Gastrointest Surg. 2010; 14: 74-81Crossref PubMed Scopus (46) Google Scholar, J. Osteopontin is overexpressed in colorectal and is with by Med. 3: Google Scholar a in epithelial OPN expression in with normal correlation OPN and Of note, OPN expression with ERRα in of suggest that ERRα and OPN in CRC and that the expression of the is positively by OPN is a target of ERRα in also a role for ERRα as a possibly by contributing to the of and to promote cancer cell In addition, and physiological have found that the complex can positively the expression of a of genes which are key to tumor ERRα in colon cancer cells gene including gene with reduced cell cell growth, and in T. J. The and its colon J Scholar the role of ERRα in bone and the of ERRα and OPN in CRC samples, we that ERRα to OPN expression in HT29 colon cancer cells, in which both are In of ERRα expression reduced OPN gene and protein expression in this cell ERRα target genes have been to to the of cancer cell including growth A. D. B. A. F. P. E. of in cancer and bone metastasis of and Res. 2011; PubMed Scopus Google Scholar, receptor the expression of growth in cancer Mol 2009; PubMed Scopus Google Scholar and the matrix 1 and A. D. B. A. F. P. E. of in cancer and bone metastasis of and Res. 2011; PubMed Scopus Google Scholar and D. expression is by estrogen-related receptor and and in an to cancer cell Res. 2010; PubMed Scopus Google Scholar Of the regulation of OPN expression by ERRα has been in the of bone In particular, E. F. A. P. of the receptor in and Mol 2010; PubMed Scopus Google Scholar reported that of ERRα the expression of a of ERRα in OPN in the cell that in Furthermore, the of the regulation of OPN expression by ERRα has been in cell of In the showed that the complex positively the human OPN In of ERRα human OPN promoter activity, its activity is to the of its including D. K. T. A. The regulates the expression and activity of the receptor estrogen-related receptor PubMed Scopus Google Scholar in to ERRα transcription of its including can also ERRα expression through a its promoter ERRα response J. F. response in the human estrogen-related receptor promoter peroxisome proliferator-activated receptor control of 2004; PubMed Scopus Google Scholar including peroxisome proliferator-activated receptor liver receptor α, and Z. A. and cell in cancer, disease and 2011; PubMed Scopus Google Scholar The that the of OPN by the complex is by of ERRα a role for ERRα in OPN The role of ERRα in colorectal tumor growth therefore be in by expression of is as to whether ERRα regulates OPN expression in a and through response receptor regulates osteopontin expression through a response in a cell Mol 2008; PubMed Scopus Google Scholar The the to by a in which can be control of energy by the estrogen-related Rev. 2008; PubMed Scopus Google Scholar The of the proximal human OPN promoter found and in which only the the its This receptor to be for mutation showed that of this activity. In of in of activity, mutation of the reduced activity, possibly to the of endogenous ERRα and in cells. These response elements are pairs suggesting a in the of the OPN promoter by findings suggest that ERRα to the and the proximal promoter of human OPN and that the complex positively regulates its activity. with and the in vivo and chromatin In a reported that the activity of the OPN promoter can be by a in P. regulation of human osteopontin promoter by in cancer Cancer. 2010; PubMed Scopus Google Scholar therefore whether ERRα the human OPN promoter in HT29 cells, using a with from we found that ERRα to the human OPN promoter in a containing the and These are to ERRα binding of the the ERRα with transcription to CRC to OPN expression. In particular, the promoter has been reported to be by A. C. M. is osteopontin expression in J Cancer. 2011; PubMed Scopus Google Scholar involved in early of Furthermore, ERRα and has been to to the expression of and to cancer cell D. expression is by estrogen-related receptor and and in an to cancer cell Res. 2010; PubMed Scopus Google Scholar are to the ERRα and as as the of of the in the regulation of gene expression involved in CRC development and progression. In with the of of that ERRα and OPN are and higher in tumor epithelial cells as to normal Furthermore, provide that OPN is a direct target of the of which to CRC These findings also the in ERRα antagonists as in cancer for in and for the