Comparative Proteomics of Colon Cancer Stem Cells and Differentiated Tumor Cells Identifies BIRC6 as a Potential Therapeutic Target

Abstract

Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring.We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software.Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that “cell death ” regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin.This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells. Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that “cell death ” regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells. Treatment of colorectal cancer patients with chemotherapy is characterized by highly divergent tumor responses, but tumor recurrence is almost always observed. Therefore, chemotherapy is not considered to be a curative modality in the treatment of colorectal cancer (1Chau I. Cunningham D. Treatment in advanced colorectal cancer: what, when and how?.Br. J. Cancer. 2009; 100: 1704-1719Crossref PubMed Scopus (91) Google Scholar). Tumor recurrence may be because of the presence of therapy-resistant, genetically distinct tumor subclones. These subclones may either be pre-existent or may be generated as a direct result of the chemotherapy itself. More recently, it has been suggested that therapy resistance and subsequent tumor recurrence could be mediated by the “cancer stem cell ” fraction of colorectal tumors. Cancer stem cells make up only a few percent of the total tumor cell mass, but are uniquely endowed with tumor-initiating capacity (2Ricci-Vitiani L. Lombardi D.G. Pilozzi E. Biffoni M. Todaro M. Peschle C. De Maria R. Identification and expansion of human colon-cancer-initiating cells.Nature. 2007; 445: 111-115Crossref PubMed Scopus (3400) Google Scholar, 3O'Brien C.A. Pollett A. Gallinger S. Dick J.E. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice.Nature. 2007; 445: 106-110Crossref PubMed Scopus (3449) Google Scholar, 4Todaro M. Francipane M.G. Medema J.P. Stassi G. Colon cancer stem cells: promise of targeted therapy.Gastroenterology. 2010; 138: 2151-2162Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 5Vermeulen L. Todaro M. de Sousa M.F. Sprick M.R. Kemper K. Perez A.M. Richel D.J. Stassi G. Medema J.P. Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13427-13432Crossref PubMed Scopus (602) Google Scholar). Interestingly, normal intestinal stem cells have been identified as the cell-of-origin of intestinal tumors (6Barker N. Ridgway R.A. van Es J.H. van de W.M. Begthel H. van den B.M. Danenberg E. Clarke A.R. Sansom O.J. Clevers H. Crypt stem cells as the cells-of-origin of intestinal cancer.Nature. 2009; 457: 608-611Crossref PubMed Scopus (1634) Google Scholar, 7Zhu L. Gibson P. Currle D.S. Tong Y. Richardson R.J. Bayazitov I.T. Poppleton H. Zakharenko S. Ellison D.W. Gilbertson R.J. Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation.Nature. 2009; 457: 603-607Crossref PubMed Scopus (550) Google Scholar). Cancer stem cells may therefore be transformed descendants of normal tissue stem cells. Although normal stem cells give rise to differentiated cells lacking tissue-regenerating capacity, cancer stem cells give rise to differentiated tumor cells lacking tumor-regenerating capacity (8Clarke M.F. Dick J.E. Dirks P.B. Eaves C.J. Jamieson C.H. Jones D.L. Visvader J. Weissman I.L. Wahl G.M. Cancer stem cells–perspectives on current status and future directions: AACR Workshop on cancer stem cells.Cancer Res. 2006; 66: 9339-9344Crossref PubMed Scopus (2503) Google Scholar, 9Hamburger A.W. Salmon S.E. Primary bioassay of human tumor stem cells.Science. 1977; 197: 461-463Crossref PubMed Scopus (1713) Google Scholar). Normal colon stem cells are exposed to toxins and drugs for an entire lifetime. To cope with this continuous challenge, stem cells must possess intrinsic resistance mechanisms that protect their DNA from being mutated and that allow prolonged survival. Inheritance of these resistance mechanisms by cancer stem cells may protect them from the cytotoxic action of chemotherapeutic drugs. If cancer stem cells are indeed the major driving force behind tumor recurrence, novel strategies are required to target this subset of cancer cells. Indeed, several studies have shown that residual tumor tissue after chemotherapy is enriched for cancer stem cell-like cells (10Todaro M. Alea M.P. Di Stefano A.B. Cammareri P. Vermeulen L. Iovino F. Tripodo C. Russo A. Gulotta G. Medema J.P. Stassi G. Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4.Cell Stem Cell. 2007; 1: 389-402Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar, 11Dylla S.J. Beviglia L. Park I.K. Chartier C. Raval J. Ngan L. Pickell K. Aguilar J. Lazetic S. Smith-Berdan S. Clarke M.F. Hoey T. Lewicki J. Gurney A.L. Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy.PLoS. ONE. 2008; 3: e2428Crossref PubMed Scopus (504) Google Scholar). However, the relationship between chemo-resistance and tumor-initiating potential and the mechanisms underlying cancer stem cell selective drug resistance are currently poorly understood (12Dean M. Fojo T. Bates S. Tumour stem cells and drug resistance.Nat. Rev. Cancer. 2005; 5: 275-284Crossref PubMed Scopus (3065) Google Scholar, 13Dean M. ABC transporters, drug resistance, and cancer stem cells.J. Mammary. Gland. Biol. Neoplasia. 2009; 14: 3-9Crossref PubMed Scopus (353) Google Scholar, 14Liu G. Yuan X. Zeng Z. Tunici P. Ng H. Abdulkadir I.R. Lu L. Irvin D. Black K.L. Yu J.S. Analysis of gene expressio! and chemoresistance of CD133+ cancer stem cells in glioblastoma.Mol. Cancer. 2006; 5: 67Crossref PubMed Scopus (1479) Google Scholar, 15Hermann P.C. Huber S.L. Herrler T. Aicher A. Ellwart J.W. Guba M. Bruns C.J. Heeschen C. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.Cell Stem Cell. 2007; 1: 313-323Abstract Full Text Full Text PDF PubMed Scopus (2274) Google Scholar). Here we set out to address the relationship between colorectal cancer stem cells and drug resistance. To this end, we have generated cancer stem cell enriched human colonosphere cultures from colorectal liver metastases. In addition, we have generated colonosphere-derived stably differentiated progeny. These isogenic cell pairs were then used to identify proteome differences using mass spectrometry. Analysis of the data revealed that proteins governing cell survival are overrepresented in the cancer stem cell cultures. The most prominently overexpressed survival protein, BIRC6/BRUCE/Apollon, was identified as a key mediator of cancer stem cell resistance to cisplatin and oxaliplatin. Collection of tumor specimens, isolation and expansion of colorectal cancer stem cell and differentiated cell cultures was performed as described in Emmink et al. (16Emmink B.L. van Houdt W.J. Vries R.G. Hoogwater F.J.H. Nijkamp M.W. Govaert. K.M. Verheem A. Steller E.J.A. Jimenez C.R. Clevers H. Borel Rinkes I.H.M. Kranenburg O. Differentiated colorectal cancer cells protect tumor-initiating cells from irinotecan.Gastroenterology. 2011; 141: 269-278Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Human colorectal tumor specimens were obtained from patients undergoing a liver resection for metastatic adenocarcinoma, in accordance with the ethical committee on human experimentation. Informed consent was obtained from all patients. All tumors were diagnosed as colorectal adenocarcinomas. Liver metastases were excised from segment VII (L145), segment IV (L146), and segment II-IV (L167). Differentiation status was not determined. The obtained tissue fragments were washed extensively with phosphate-buffered saline and were mechanically dissociated using scalpels and vigorous trituration to yield small fragments (<1 mm3) and single cells. Enzymatic digestion was performed using thermolysin 0.05% (Sigma, Type X) in Dulbecco's modified Eagle's medium/F12 containing 5 mm Hepes (Invitrogen, Carlsbad, CA) for 2 h at 37 °C. The suspension was then filtered through a 40-μm-pore size nylon cell strainer (BD Falcon) to separate the tissue fragments from the single cells. The single cell suspension was cultured in advanced Dulbecco's modified Eagle's medium/F12 (Invitrogen) supplemented with 0.6% glucose (BDH Lab. Supplies), 2 mm l-glutamine (Biowhittaker, Rockland, ME), 9.6 μg/ml putrescin (Sigma), 6.3 ng/ml progesterone (Sigma), 5.2 ng/ml sodium selenite (Sigma), 25 μg/ml insulin (Sigma), 100 μg/ml apotransferrin (Sigma), 5 mm hepes (Invitrogen), 0,005 μg/ml trace element A (Cellgro), 0.01 μg/ml trace element B (Cellgro), 0.01 μg/ml trace element C (Cellgro), 100 μm β-mercaptoethanol (Merck), 10 ml antibiotic-antimycotic (Invitrogen), 4 μg/ml (Invitrogen), (Sigma), 5 μg/ml and 4 μg/ml factors ng/ml (Invitrogen) and 10 ng/ml were to the cell All cell was out in (BD Falcon) at 37 in a In differentiation was by colon for on in Dulbecco's modified Eagle's medium/F12 supplemented with of the used cultures and the differentiated tumor cell cultures were all and differentiated tumor cell cultures were in 10 tissue and cultured for h in stem cell were washed with phosphate-buffered and washed with to of the mm mm 5 mm containing was used to cells. of were on were with washed and was into using a to the The gel and was performed in a MS proteins were as described A. M. O. M. of proteins PubMed Scopus Google Scholar). to the different were into The were washed and three in mm were with 10 mm for 1 h at and with mm for at in the two subsequent and the were 10 in a and with at 25 °C. were in and two in in The was to in a to liquid chromatography tandem MS analysis A. M. O. M. of proteins PubMed Scopus Google Scholar). were by an The with a μm with μm were at on a 5 mm μm at B 0.05% in 0.05% in and at in a B in were at in a using a mass and were on a mass were at in the cell using a target of 1 In after an the 5 and were to in the isolation of and a of was with a of 1 and an of by has as a to with and analysis is in which proteins are in when small differences in are However, with a is and for at a were against the human using which is of the data analysis were with a of 10 for the mass and 1 for and were as variable two were and the of was the and were into was used to the data and to using the A. E. R. to the of by and PubMed Scopus Google Scholar). with a were the A. E. R. A for proteins by tandem mass PubMed Scopus Google was and with a of with two or in at least one of the were that and could not be differentiated based on analysis were the of in gel was to the were on the of the for that The of proteins, was in the Ingenuity and and analysis was performed with only direct the of proteins was in the of and The direct and between The of containing used of of of were obtained from the The target set used for BIRC6 and these of cells with and the the we used the containing a of containing of of of and their differentiated were in mm mm 5 mm of were out on (Invitrogen) and were analyzed by using against BIRC6 and was cells were using and cell and were using cells were analyzed to the by using and The cell were with and BIRC6 and differentiated tumor cells were cultured in the presence of oxaliplatin or cisplatin at the for 5 activity was using All are as of To the proteome of colon cancer stem cells to their differentiated we analyzed three isogenic pairs of and differentiated tumor cells derived from liver metastases. All colonosphere cultures were enriched for cancer stem cells based on their and potential (16Emmink B.L. van Houdt W.J. Vries R.G. Hoogwater F.J.H. Nijkamp M.W. Govaert. K.M. Verheem A. Steller E.J.A. Jimenez C.R. Clevers H. Borel Rinkes I.H.M. Kranenburg O. Differentiated colorectal cancer cells protect tumor-initiating cells from irinotecan.Gastroenterology. 2011; 141: 269-278Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The of these cultures were on an gel by Analysis of the was performed by by In 3048 proteins were identified in all of cells with an of proteins isogenic The of proteins that was at least twofold up-regulated in the of isogenic from to these up-regulated proteins, 32 proteins were up-regulated in the of all isogenic pairs The of that the between isogenic pairs of colonosphere cells and differentiated tumor cells is the the different cultures In all and proteins that all colonosphere cultures and differentiated cultures This that it may be to identify a cancer stem cell in colorectal differences between and isogenic differentiated cells. The to all identified and proteins all up-regulated proteins and all up-regulated The of proteins in all three that were identified in and in pairs are shown in the two in a of from differentiated tumor cells by of analysis of all identified of analysis of all and proteins to the The of proteins from differentiated tumor we the up-regulated proteins in three twofold up-regulated in three out of three isogenic up-regulated in three out of three isogenic and up-regulated in two out of three isogenic A total of proteins was to be enriched in in in and in Interestingly, the up-regulated proteins is which was identified by and as a colon stem cell S.J. Beviglia L. Park I.K. Chartier C. Raval J. Ngan L. Pickell K. Aguilar J. Lazetic S. Smith-Berdan S. Clarke M.F. Hoey T. Lewicki J. Gurney A.L. Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy.PLoS. ONE. 2008; 3: e2428Crossref PubMed Scopus (504) Google Scholar, B.L. van Houdt W.J. Vries R.G. Hoogwater F.J.H. Nijkamp M.W. Govaert. K.M. Verheem A. Steller E.J.A. Jimenez C.R. Clevers H. Borel Rinkes I.H.M. Kranenburg O. Differentiated colorectal cancer cells protect tumor-initiating cells from irinotecan.Gastroenterology. 2011; 141: 269-278Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, T. C. G. H. B.M. 1 is a for normal and human stem cells and colon Res. 2009; PubMed Scopus Google Scholar). The of the of to identify up-regulated factors in cancer stem proteins up-regulated in in to differentiated tumor cells. The all proteins that are up-regulated in when compared to differentiated tumor cells to the following up-regulated in three out of three isogenic pairs W.J. a 2010; PubMed Scopus Google and up-regulated in three out of three isogenic pairs Z. M. S. T. T. T. A human in human cancer Res. PubMed Scopus Google Scholar). colonosphere enriched for cancer stem cells, isogenic differentiated of colonosphere up-regulated proteins in 2 4 with containing 1 1 C up-regulated proteins in B 1 1 and in a we used the Ingenuity Pathway to identify and that from differentiated cells. this analysis all proteins were to proteins ” T. C. G. H. B.M. 1 is a for normal and human stem cells and colon Res. 2009; PubMed Scopus Google and proteins ” (12Dean M. Fojo T. Bates S. Tumour stem cells and drug resistance.Nat. Rev. Cancer. 2005; 5: 275-284Crossref PubMed Scopus (3065) Google were most frequently of and were identified as the that are overrepresented in colonosphere cultures analysis of All up-regulated proteins were analyzed by The and by this are T. C. G. H. B.M. 1 is a for normal and human stem cells and colon Res. 2009; PubMed Scopus Google and (12Dean M. Fojo T. Bates S. Tumour stem cells and drug resistance.Nat. Rev. Cancer. 2005; 5: 275-284Crossref PubMed Scopus (3065) Google Scholar). The by this are and BIRC6, of identified of in of in a the was used to identify and the up-regulated analysis of all up-regulated proteins showed major in DNA and regulation of in drug resistance we on proteins cell death and survival revealed that the of overrepresented ” have activity as or an Inhibitor of Apoptosis is a key of the intrinsic and has been implicated in drug resistance L. J. L. C. X. against tumor cell growth and of 2008; PubMed Scopus Google Scholar, Z. M. S. T. T. T. A human in human cancer Res. PubMed Scopus Google of The cell death that were identified by Ingenuity were to their based on 2 containing 1 B of of in a we BIRC6 a in chemotherapy resistance in colorectal cancer stem cells. we analyzed BIRC6 in the three of isogenic cell pairs by In with the BIRC6 was highly up-regulated in when compared with differentiated cells analysis of other up-regulated proteins and the data not and (16Emmink B.L. van Houdt W.J. Vries R.G. Hoogwater F.J.H. Nijkamp M.W. Govaert. K.M. Verheem A. Steller E.J.A. Jimenez C.R. Clevers H. Borel Rinkes I.H.M. Kranenburg O. Differentiated colorectal cancer cells protect tumor-initiating cells from irinotecan.Gastroenterology. 2011; 141: 269-278Abstract Full Text Full Text PDF PubMed Scopus (78) Google have shown that activity by the the and cancer stem cell (16Emmink B.L. van Houdt W.J. Vries R.G. Hoogwater F.J.H. Nijkamp M.W. Govaert. K.M. Verheem A. Steller E.J.A. Jimenez C.R. Clevers H. Borel Rinkes I.H.M. Kranenburg O. Differentiated colorectal cancer cells protect tumor-initiating cells from irinotecan.Gastroenterology. 2011; 141: 269-278Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Therefore, we used to separate from cells and analyzed BIRC6 in tumor cell that BIRC6 is highly in cells, but not in nontumorigenic cells. we the of BIRC6 in colonosphere resistance to oxaliplatin and two frequently used chemotherapeutic drugs. To this end, of BIRC6 was in by using a set of and were to BIRC6 and BIRC6 and differentiated tumor cells were with of oxaliplatin or cisplatin and cell was by Knockdown of BIRC6 in a of colonosphere cells to oxaliplatin and cisplatin and The of BIRC6 cells to drugs was to that of the differentiated tumor cells. These identify BIRC6 as an important mediator of resistance to oxaliplatin and cisplatin and that BIRC6 may protect the cancer stem cell fraction against these drugs. In the study we have used a to identify potential of the cancer stem cell in colorectal tumors. We identified and novel factors enriched in cancer stem cell cultures when compared with stably differentiated tumor cells. Interestingly, analysis revealed that distinct in DNA and are enriched in when compared with differentiated tumor cells. the of these in the of colorectal cancer stem cells. cancer stem cells were characterized by of a set of survival proteins, the most of which was BIRC6 is with to chemotherapy in in and in studies L. J. L. C. X. against tumor cell growth and of 2008; PubMed Scopus Google Scholar, Z. M. S. T. T. T. A human in human cancer Res. PubMed Scopus Google Scholar). BIRC6 and A. M. P. P. M.G. M. N. gene in cancer cells through and J. Cancer. 2009; 100: PubMed Scopus Google Scholar). data show that colorectal cancer stem cell cultures resistance to oxaliplatin and to cisplatin and that BIRC6 is an important mediator of resistance. it was shown by gene that and BIRC6 are up-regulated in colorectal tumors when compared with normal intestinal M. E. C. C. De L. P. J. study of gene by in human colorectal cancer and normal J. 2006; Google Scholar). that it is the cancer stem in colorectal tumors that this survival BIRC6, as or belongs to the of are major of at least in to their to the to Rev. Biol. 3: PubMed Scopus Google Scholar, of PubMed Scopus Google Scholar). Human as 1 as as as and proteins one to three that are required for their activity the to Rev. Biol. 3: PubMed Scopus Google Scholar). are in with studies that cancer stem cells of proteins and resist A. L. A. R. F. G. Pilozzi E. Peschle C. De M.R. resistance of stem 2006; PubMed Scopus Google Scholar, M. L. De M.R. Targeting in cancer stem cells.Cancer 2011; PubMed Google Scholar). it was that of colorectal cancer stem cells against (10Todaro M. Alea M.P. Di Stefano A.B. Cammareri P. Vermeulen L. Iovino F. Tripodo C. Russo A. Gulotta G. Medema J.P. Stassi G. Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4.Cell Stem Cell. 2007; 1: 389-402Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar, M. L. De M.R. Targeting in cancer stem cells.Cancer 2011; PubMed Google Scholar, Stefano A.B. Iovino F. Y. T. F. Stassi G. Todaro M. is by in colon cancer stem cells.J. Cell. 2010; PubMed Scopus Google Scholar). The described not identify as a cancer stem different tumors resist by the of distinct an important in tumor and therapy resistance for targeted are highly in several cancer L. Z. H. of of and of proteins in human tumor cells: for cancer Res. Google Scholar). small have been and a of D.J. S. as cancer 2009; PubMed Scopus (91) Google Scholar, W.J. a 2010; PubMed Scopus Google Scholar). studies in tumors have shown in the treatment of cell and However, most of these studies have on and on BIRC6 C. H. A. T. A.R. H. of of the for the treatment of PubMed Scopus Google Scholar, S. M. K.M. for PubMed Scopus Google Scholar). are being for their and in either in with or with chemotherapy M. P. from to of and Rev. Cancer. 2010; PubMed Scopus Google Scholar, S. Targeting of proteins for cancer 2008; PubMed Scopus Google Scholar). is not established these target of to and the in to BIRC6 D.J. S. as cancer 2009; PubMed Scopus Google it is not that on the data we that BIRC6 the cancer stem cell fraction of colorectal tumors against oxaliplatin and cisplatin. Targeting BIRC6 by or by novel BIRC6 may therefore be in with drugs. This may help eradicating the cancer stem cell fraction in colorectal tumors. with