CD44-Positive Cancer Stem Cells Expressing Cellular Prion Protein Contribute to Metastatic Capacity in Colorectal Cancer

Published OnlineFirst February 15, 2013; DOI: 10.1158/0008-5472.CAN-12-3759

Cancer Tumor and Stem Cell Biology Research

Lei Du1, Guanhua Rao3, Hongyi Wang2, Baowei Li1, Weili Tian3, Jiantao Cui2, Leya He4, Brian Lafﬁn5, Xiuyun Tian2, Chunyi Hao2, Hongmin Liu1, Xin Sun1, Yushan Zhu3, Dean G. Tang5, Maryam Mehrpour1,6, Youyong Lu2, and Quan Chen1,3

Abstract Cancer stem cells are implicated in tumor progression, metastasis, and recurrence, although the exact mechanisms remain poorly understood. Here, we show that the expression of cellular prion protein (PrPc, PRNP) is positively correlated with an increased risk of metastasis in colorectal cancer. PrPc defines a þ þ subpopulation of CD44-positive cancer stem cells that contributes to metastatic capacity. PrPc CD44 þ colorectal cancer stem cells displayed high liver metastatic capability, unlike PrPc CD44 stem cells, that was inhibited by RNAi-mediated attenuation of PrPc. Notably, administration of PrPc monoclonal antibodies significantly inhibited tumorigenicity and metastasis of colorectal cancer stem cells in mouse models of orthotopic metastasis. PrPc promoted epithelial to mesenchymal transition (EMT) via the ERK2 (MAPK1) pathway, thereby conferring high metastatic capacity. Our findings reveal the function of PrPc in regulating EMT in cancer stem cells, and they identify PrPc as candidate therapeutic target in metastatic colorectal cancer. Cancer Res; 73(8); 2682–94. 2013 AACR.

Introduction and have now been reported in most human solid tumors on – Cancer is a major cause of mortality, and most human the basis of their functional properties (5 7). Colorectal cancer-related deaths are related to metastasis (1, 2). The CSCs (CCSC) have been distinguished using CD44 or CD133 – difficulty in diagnosing and therapeutically eliminating either alone (8 10), or combined with other markers, such as metastasis is related to our limited understanding of the EpCAM, CD166, CD29, CD24, LGR5 and aldehyde dehydro- fi genase 1 (ALDH1; refs. 11–13). Previous studies by ourselves molecules speci cally expressed on the surface of metastatic þ cancer cells. Accumulating evidence suggests that a subset and other authors have shown that as few as 100 CD44 of cancer cells, operationally termed cancer stem cells (CSC) cancer cells isolated from patients were able to develop into a heterogeneous tumor, and that spheroids derived from a or tumor-initiating cells, possesses enhanced ability to þ regenerate tumors and survive anticancer therapies (3). single CD44 cancer cell could recapitulate the heteroge- CSCs were first identified in the hematopoietic system (4), neous hierarchy of tumor cells (8, 14). These studies indicate that CCSCs may trigger the formation of colorectal cancer (CRC), and that CD44 is a functionally important surface Authors' Affiliations: 1State Key Laboratory of Biomembrane and Mem- biomarker of CCSCs. Importantly, an antibody specificto brane Biotechnology, Institute of Zoology, Chinese Academy of Sciences; in vivo 2Laboratory of Molecular Oncology, Key Laboratory of Carcinogenesis and CD44 selectively eradicated leukemic stem cells by Translational Research (Ministry of Education), Peking University Cancer altering the fate of leukemic stem cells (15). Monoclonal 3 Hospital & Institute, Beijing; Tianjin Key Laboratory of Protein Sciences, antibodies targeting surface antigens specifically expressed College of Life Sciences, Nankai University, Tianjin; 4Cancer Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of in CSCs offer a potentially powerful strategy to eliminate Science and Technology, Wuhan, China, 5Department of Molecular Car- CSCs with minimal toxicity. Besides CD44, monoclonal cinogenesis, University of Texas M.D. Anderson Cancer Center, Science Park, Smithville, Texas; and 6INSERM U984, Faculte de Pharmacie, Uni- antibodies targeting CD123 and CD47 have been reported versite Paris-Sud, Chatenay-Malabry, France to eradicate leukemia stem cells (16). Recent evidence suggests that CSCs may be endowed Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). with enhanced disseminating capacity enabling them to invade neighboring tissues or metastasize to distant organs L. Du, G. Rao, H. Wang, and B. Li contributed equally to this work. (17–19). Although CD44 and some other adhesion mole- Corresponding Author: Quan Chen, Institute of Zoology, CAS, 1 Beichen cules, such as E-cadherin, could act as migration suppres- West Road, Chaoyang District, Beijing 100101 China. Phone: 86-10-6480- 7321; Fax: 86-10-6480-7321; E-mail: [email protected] and Youyong Lu, sors by restricting cancer cells to their primary sites (20), Peking, University Cancer Hospital & Institute. Phone: 86-10-8819-6765; these same molecules with certain modifications (e.g., E-mail: [email protected] splice variants of CD44) or in combination with other doi: 10.1158/0008-5472.CAN-12-3759 adhesion molecules, such as CD26, may result in metastatic 2013 American Association for Cancer Research. phenotypes (21–24).

2682 Cancer Res; 73(8) April 15, 2013

PrPcþ Metastatic Cancer Stem Cells

Cellular prion protein (PrPc) is a highly conserved glyco- a whole-body fluorescence imaging system (In-Vivo FX PRO, protein present in all vertebrates and has the same protein Carestream). sequence as the scrapie prion protein (PrPsc), a pathogenic factor in scrapie in sheep, bovine spongiform encephalop- Results athy in cattle, and kuru in human beings (25, 26). In addition The PrPc expression level is correlated with clinical and to the central involvement of PrPc in prion diseases, several pathologic features and outcomes in CRC patients lines of evidence have intriguingly implicated PrPc in human On the basis of our previous observations that CD44 is a tumors including glioblastoma, breast, prostate, gastric and robust marker for CCSCs (14), we hypothesized that additional colorectal cancers (CRC; 27). PrPc interacts homophilically factors may exist that are coexpressed with CD44 in the with itself or heterotypically with components of the extra- promotion of migration and metastasis of CSCs. We therefore cellular matrix (ECM) such as laminin and vitronectin, to compared gene expression in colorectal cancer, liver metas- mediate cell adhesion (28). PrPc expression is required for tases, and normal tissues by cDNA array conducted in our lab. þ the self-renewal of CD34 hematopoietic stem cells (29, 30). In addition, we conducted a CD44 coexpression analysis from However, despite tantalizing evidence linking PrPc to Oncomine datasets (Ki_Colon, Koinuma_Colon, and Smith_ human cancers, its physiologic function and exact role in Colorectal_2 datasets; refs., 33–35). Interestingly, we found cancer development remain elusive. Here, we report that that several genes were in fact coexpressed with CD44 and þ high levels of PrPc expression (in PrPc cells) identify a upregulated in metastases; one of these genes was PRNP (data þ functionally distinct subpopulation of CD44 CCSCs, which not shown), which encodes PrPc. contributes to CRC initiation and metastasis. Our findings To find out whether PrPc and CD44 are associated with the thus reveal hitherto-unknown roles of PrPc in CCSCs and clinical and pathologic features, we carried out immunohis- epithelial–mesenchymal transition (EMT), and suggest that tochemical (IHC) staining in a TMA containing 309 colorectal PrPc could provide a potential therapeutic target in meta- tumor specimens. CD44 and PrPc were found to be coex- static CRCs. pressed in most of the tumors, especially in those that were poorly differentiated (correlation coefficient ¼ 0.73; Fig. 1A). Materials and Methods The PrPc expression level was significantly higher in poorly Cells, animals, and samples differentiated than in moderately and well-differentiated sam- Fresh colon samples from patients with CRC and paired ples (P < 0.001; Fig. 1A and Supplementary Fig. S1A), whereas normal subjects were collected from the tumor bank of the level of CD44 was not associated with clinical stage (P ¼ Beijing Cancer Hospital (Beijing, China), as approved by the 0.143; Fig. 1A). This observation was supported by examining Research Ethics Board at the Beijing Institute for Cancer multiple Oncomine cDNA microarray datasets, which revealed Research. A tissue microarray (TMA) was constructed as that the PRNP mRNA expression levels were significantly described previously (31, 32). The NCI-60 human tumor cell elevated in high-grade and advanced-stage CRCs (Supplemen- line panel was kindly provided by Dr. Jacque Grassi (National tary Fig. S1B). Importantly, the patients with CRC with the Cancer Institute, Bethesda, MD). SW480 and MDA-MB-231 highest PrPc expression were also found to have the poorest cell lines were purchased from American Type Culture prognosis, with a median survival of only 12 months (Fig. 1B), Collection, authenticated using polyphasic (genotypic and which was significantly (P < 0.0001) shorter than that of phenotypic) testing every 6 months, and cultured according patients whose primary tumor was PrPc-negative (median to the manufacturer's instructions. Human primary CRC survival >5 years). cells were isolated from patients and cultured in with CRC-related death is usually attributable to recurrence serum-free medium. All CCSCs were subcultured for fewer and distant metastasis. Out of 309 specimens, there were 228 than 20 passages. Four-week-old female nude mice (BALB/c- primary CRCs without metastasis versus 81 cases with nu/nu) and 5-week-old female Nonobese diabetic/severe concurrent distant metastasis. Strikingly, we found that PrPc combined immunodeficient (NOD/SCID) mice were pur- was highly expressed in the primary tumors of the patients chased from Chinese Academy of Medical Sciences (Beijing, withliverorlungmetastasis(P ¼ 0.0007; Fig. 1C). Oncomine China), and all experiments were conducted under standard cDNA microarray data analysis also showed a close corre- conditions in accordance with the National and Institutional lation of the PrPc level with disease recurrence and early regulations. patient death (Supplementary Fig. S1C). We also observed that PrPc expression was most prominent at the interface Tumorigenesis and metastasis in orthotopic xenograft and invasion fronts of tumor nodules (identified by cytoker- models atin 20 staining; Fig. 1D), which may constitute a reservoir of NOD/SCID mice were anesthetized with pentobarbital and mobile CSCs (36). In the lymph nodes that had been invaded þ the cecums were exteriorized by laparotomy. Various numbers by CRC cells, PrPc cells were also observed in vascular þ þ þ þ of CD44 PrPc , CD44 PrPc , CD44 PrPc , and CD44 PrPc invasions (Fig. 1E and Supplementary Fig. S1D). In addition, subpopulations freshly sorted from patients, or 50,000 DsRed- high levels of PrPc were present in CRC cells and could be labeled PrPc overexpressing or knockdown CCSCs were seen invading the tumor stroma (Fig. 1F). Collectively, these injected into the cecal wall. All the animals were monitored data indicate that high PrPc expression is associated with for up to 80 to 100 days or until tumor-associated death advanced clinical stages, increased metastatic risk, and occurred. Primary tumors and metastasis were evaluated by reduced patient survival.

www.aacrjournals.org Cancer Res; 73(8) April 15, 2013 2683

Du et al.

A 01 23

CD44

PrPc

Grade 3 Grade 2 Grade 1

B C 150

100 ** M n = 23 23/80 27/77 Non-M n = 41 100 PrPc null n = 34 PrPc low 9/49 50 PrPc moderate 22/22 PrPc high 50

n = 11 Number of patients Percentage survival % survival Percentage 0 0 0 123 0 20 40 60 Month

PrPc expression level D

Figure 1. PrPc is associated with high grade tumors and poor prognosis in patients. A, heatmap of CD44 and PrPc protein expression in CRCs (n ¼ 309 patients). Data are grouped by the following tumor grades: grade 1, well-differentiated; grade 2, moderately differentiated; grade 3, poorly differentiated. CD44 and PrPc expression are semiquantitatively described as 0, negative; 1, low; 2, moderate; and 3, high. B, Kaplan–Meier plot showing the survival percentage of 109 patients with CRC (>5 years after surgery) grouped by PrPc expression level. The number of patients (n) in each group is indicated in parentheses. C, the PrPc expression level is positively correlated with metastasis. Patients with a concurrent metastasis (M, red) and those withouta metastasis (Non-M, blue) are grouped by PrPc expression levels. y-axis, number of patients. , P < 0.001. D, double immunofluorescence staining of cytokeratin 20 (green) and PrPc (red) in CRC showing preferential localization of PrPc-positive cells in the invasive fronts of tumor islets (indicated by dotted white lines). Scale bar, 200 mm. E, representative immunofluorescence images of PrPc expression in invasive vasculatures in the draining lymph node þ þ þ (white dotted outline). PrPc /cytokeratin 20 tumor cells (yellow) are magnified and indicated (arrows). Scale bar, 200 mm. F, IHC staining shows PrPc CRC cell invasion into stromal cells (red arrows). Scale bar, 100 mm.

þ þ A subpopulation of PrPc CRC cells possesses CSC properties of PrPc-expressing cells in CRC. We detected CD44 þ properties PrPc cells in the cryosections of CRCs by double immuno- We next examined PrPc and CD44 coexpression patterns at ﬂuorescence (IF) staining of CD44 and PrPc (Fig. 2A). Flow þ the cellular level, and determined the biologic and tumorigenic cytometry analysis revealed only a small percentage of PrPc

2684 Cancer Res; 73(8) April 15, 2013 Cancer Research

PrPcþ Metastatic Cancer Stem Cells

þ þ Figure 2. The CD44 PrPc subpopulation of CRC cells possesses CSC properties. A, double immunofluorescence staining of CD44 (green) and PrPc (red) in a representative CRC cryosection. The section was counterstained with 40,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 100 mm. B, a representative 4-color flow cytometric analysis of a CRC. Cells were gated by CD45 negativity and EpCAM positivity to remove leukocytes and stromal cells (left and middle) before the indicated subpopulations were analyzed and sorted out on the basis of PrPc and CD44 expression (right). C, Venn diagram illustrating the degree of overlap between the CD44 and PrPc populations. The percentage of each subpopulation is shown with the median values (M) and ranges indicated. D, clonogenic efficiency of indicated subpopulations freshly isolated from CRC (day 42). Single colonies are magnified to show the detailed morphology. Colonies with diameter 0.5 mm were counted for statistical analysis. , P < 0.001; , P < 0.0001. E, representative H&E images of regenerated tumors from CD44þPrPcþ and CD44þPrPc subpopulations in NOD/SCID mice (day 90). Note that the cancer cells from CD44þPrPcþ subpopulation have infiltrated into the underlying muscle tissue. Scale bar, 100 mm. F, growth curve of xenograft tumor initiated from 1 106 CD44 RNAi (black), PrPc RNAi (blue), CD44 and PrPc RNAi (bright red) CRC cells in nude mice (n ¼ 6). Parental (brown) and scramble (pink) shRNA cells were used as controls. , P < 0.01; , P < 0.0001.

www.aacrjournals.org Cancer Res; 73(8) April 15, 2013 2685

Du et al.

Table 1. Tumor-initiating incidence and liver metastasis in the orthotopic model

þ þ þ þ Cecal tumor CD44 PrPc CD44 PrPc CD44 PrPc CD44 PrPc Case 500 2,000 10,000 500 2,000 10,000 500 2,000 10,000 500 2,000 10,000 24 1/3 1/3 3/3 0/3 1/3 2/3 0/3 1/3 2/3 0/3 0/3 0/3 25 1/3 2/3 3/3 0/3 1/3 2/3 0/3 1/3 2/3 0/3 0/3 0/3 26 1/3 2/3 3/3 1/3 1/3 2/3 0/3 0/3 2/3 0/3 0/3 0/3 27 1/3 1/3 2/3 0/3 1/3 1/3 0/3 0/3 2/3 0/3 0/3 0/3 28 0/3 1/3 2/3 0/3 0/3 2/3 0/3 0/3 1/3 0/3 0/3 1/3 29 1/3 1/3 3/3 0/3 1/3 3/3 0/3 1/3 3/3 0/3 0/3 1/3 Total 5/18 8/18 16/18 1/18 5/18 12/18 0/18 3/18 12/18 0/18 0/18 2/18

Metastasis CD44þPrPcþ CD44þPrPc CD44PrPcþ CD44PrPc Case 500 2,000 10,000 500 2,000 10,000 500 2,000 10,000 500 2,000 10,000 24 0/3 0/3 2/3 0/3 0/3 0/3 03 1/3 2/3 0/3 0/3 0/3 25 1/3 1/3 3/3 0/3 0/3 0/3 0/3 1/3 2/3 0/3 0/3 0/3 26 0/3 0/3 3/3 0/3 0/3 0/3 0/3 0/3 2/3 0/3 0/3 0/3 27 0/3 0/3 1/3 0/3 0/3 0/3 0/3 0/3 1/3 0/3 0/3 0/3 28 0/3 0/3 1/3 0/3 0/3 0/3 0/3 0/3 1/3 0/3 0/3 0/3 29 0/3 1/3 2/3 0/3 1/3 2/3 0/3 1/3 2/3 0/3 0/3 1/3 Total 1/18 2/18 12/18 0/18 1/18 2/18 0/18 3/18 10/18 0/18 0/18 1/18

NOTE: Indicated numbers (500, 2,000 and 10,000) of CD44þPrPcþ, CD44þPrPc, CD44PrPcþ, and CD44PrPc subpopulations freshly isolated from CD45EpCAMþ CRC cells in 7 patients (i.e., case 24–30, Supplementary Table S1) were transplanted into cecal wall of NOD/SCID mice. In 6 of 7 cases, xenograft tumors were regenerated (day 100). The incidence of tumor regeneration at the cecum and the incidence of metastasis to the liver are shown.

þ þ þ þ þ cells in the CD45 EpCAM CD44 population in patient tumors were generated from the CD44 PrPc , CD44 PrPc , and þ þ þ (Fig. 2B), and the abundance of CD44 PrPc cells ranged from CD44 PrPc cells, but not from the CD44 PrPc cells in 6 of 0.02% to 10.83% (median 0.28%; Fig. 2C and Supplementary 9 cases (Supplementary Table S2). In limited-dilution assays, þ þ þ þ Table S1). Interestingly, the subpopulation of CD44 PrPc cells the CD44 PrPc cells displayed greater tumor-initiating in the 6 patients with concomitant liver metastasis was signif- capacity than the other subpopulations (P < 0.001) (Supple- icantly higher than that in the 25 primary CRC patients without mentary Table S2). We frequently observed that tumor cells metastasis (3.19% vs. 0.79%, mean, P ¼ 0.03; Supplementary had invaded the underlying muscle tissue in tumors regener- þ þ Table S1). Furthermore, PrPc and CD44 were closely associated ated from CD44 PrPc (but not in those regenerated from þ in the NCI-60 human tumor cell line panel (correlation coeffi- CD44 PrPc ) cells (Fig. 2E). To further test their self-renewal þ þ cient ¼ 0.68; Supplementary Fig. S2A–S2C). capabilities in vivo, 100 CD44 PrPc cancer cells purified from We next asked whether PrPc is associated with CD44 in a first-generation xenograft tumor were transplanted into contributing to CSC properties. The abilities to form anchor- NOD/SCID mice. Our findings indicated that 8 of 9 transplan- age-independent spheres/spheroids in vitro and to regenerate tations grew secondary xenograft tumors that recapitulated serially transplantable xenograft tumors in immunodeficient the phenotype of the original tumor, and even higher tumor- mice are 2 of the hallmarks of CSCs (37). We therefore used igenic capacities were detected in the third transplantation fluorescence-activated cell sorting (FACS) to purify 4 sub- (Supplementary Fig. S2D and data not shown). þ þ þ þ populations: CD44 PrPc ,CD44PrPc ,CD44PrPc ,and Similar tumor-initiating capacities were also observed in CD44 PrPc from patient tumors (Fig. 2B). The purity of each nude mice. We isolated the 4 subpopulations prepurified from subpopulation was more than 97%, as assessed by post-sort CD45 tumor cells, and injected them subcutaneously into FACS and fluorescence microscopic analyses (data not shown). nude mice (cases 1–11, Supplementary Table S1). Tumor þ þ As expected, CD44 PrPc CRC cells displayed significantly development was observed in 8 cases (Supplementary Fig. þ þ þ greater ability to form colonies compared with CD44 PrPc or S2E). Again, the CD44 PrPc CRC cells exhibited the greatest þ CD44 PrPc cells (P ¼ 0.02 and P ¼ 0.007, respectively), whereas tumor-initiating capacity (P < 0.05; Supplementary Table S2). CD44 PrPc cancer cells failed to generate colonies (Fig. 2D). As shown in Supplementary Figure S2E, the reconstituted þ þ To evaluate their tumorigenic potential, the CD44 PrPc , tumors were histologically heterogeneous. PrPc and CD44 þ þ CD44 PrPc , CD44 PrPc , and CD44 PrPc subpopulations coexpression was detected in only a minor subset of cells isolated from 9 patients (i.e., cases 22–30 and Supplementary in regenerated tumors (Supplementary Fig. S2F–S2G), also þ þ Table S1) were subcutaneously injected intoNOD/SCID mice. suggesting that the CD44 PrPc cells were capable of self- As predicted by their clonogenicity data, xenograft tumors renewal.

2686 Cancer Res; 73(8) April 15, 2013 Cancer Research

PrPcþ Metastatic Cancer Stem Cells

þ þ Figure 3. PrPc confers greater metastatic capacity. A, 10,000 freshly sorted CD44 PrPc cells from CRCs were transplanted into the cecal wall of NOD/SCID mice. The regenerated cecal tumor (black arrow) and the liver metastatic nodules (white arrow) are shown. B, metastatic nodules in the liver formed after injecting 2,000 of the cells into the cecal wall (day 100). Metastatic cancer cells are distinguished from normal mouse liver tissue by having a higher nucleus/cytoplasm ratio. Scale bar, 300 mm. C, metastatic efficiency. The number of cecal tumors with liver metastasis (white bar) vs. cecal tumors without metastasis (black bar) determined 100 days after CRC cell injections. D, metastatic areas were determined by Image-Pro Plus software based on H&E staining. The metastatic area was normalized to CD44þPrPcþ cells. , P < 0.001. E, PrPcþ cells exhibit a higher metastatic index than PrPc cells. Metastatic lesions were determined under a microscope on the basis of H&E staining, and the metastatic index was defined as the number of metastatic nodules per primary tumor volume. , P < 0.0001. F, cell migration assays with the indicated subpopulations freshly isolated from CRCs. Each sample was analyzed in triplicate, and the experiment was carried out using 7 patient tumors. , P < 0.0001. G, PrPc and CD44 expression in migrated vs. nonmigrated CRC cells determined by flow cytometry analysis. This experiment was conducted in triplicate using 6 patient tumors.

www.aacrjournals.org Cancer Res; 73(8) April 15, 2013 2687

Du et al.

2688 Cancer Res; 73(8) April 15, 2013 Cancer Research

PrPcþ Metastatic Cancer Stem Cells

Figure 5. PrPc modulates EMT in CCSCs. A, phase-contrast images of CCSCs in which PrPc was either overexpressed or knocked down. Note the morphologic changes after the PrPc expression level was modulated. Scale bar, 50 mm. B, Western blot analysis of N-cadherin and E-cadherin in CCSCs in which PrPc was either overexpressed or knocked down. Monoglycosylated (30 kDa) and unglycosylated (28 kDa) PrPc expressed on CCSCs were detected by anti-PrPc (Clone5) antibody. C, double immunoﬂuorescence staining of N- cadherin (green) and E-cadherin (red) in CCSCs in which PrPc was either overexpressed or knocked down. Scale bar, 50 mm. D, qRT-PCR analysis of EMT related genes in Twist or PrPc manipulated CCSCs. The mRNA expression levels were normalized to that of parental CCSCs. Purple bars, knock-in of twist in PrPc RNAi CCSCs. Yellow bars, knock-in of PrPc in Twist RNAi cells. , P < 0.001; , P < 0.0001. E, relative mRNA expression levels of N-cadherin, E-cadherin, and Twist in the indicated subpopulations freshly sorted from patients. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a common control. The mRNA expression levels of each gene were normalized to those of the cell population with the lowest mRNA expression. This experiment was conducted using cells from 6 patient tumors. , P < 0.001; , P < 0.0001. F, representative ﬂow cytometry analysis showing that the N- cadherin-positive CRC cells were also PrPc positive, whereas most E-cadherin-positive cells were PrPc negative. This experiment was carried out in 8 freshly sorted CRCs þ by gating for CD44 EpCAM .

Figure 4. PrPc confers greater metastatic capacity. A, representative whole-body fluorescence imaging of orthotopically growing (dotted circles) and metastasizing CCSCs (n ¼ 9, day 80). B, quantitative imaging data of tumor metastases (day 60). Relative intensity of fluorescence (excluding the cecum region) was calculated. , P < 0.001; , P < 0.0001. C, liver metastasis with manipulated levels of PrPc. Macroimaging of metastatic nodules in liver (top) with boxed regions observed under a stereofluorescence microscope (middle; scale bar, 1 mm). Metastases were verified by H&E staining and indicated by dotted circles (bottom). Scale bar, 100 mm. D, metastatic index of CCSCs with PrPc overexpression or knockdown. Metastatic index was defined as number of metastatic nodules in the liver vs. primary tumor volume. , P < 0.001; , P < 0.0001. E, migratory abilities of CCSCs with PrPc overexpression or knockdown. , P < 0.001. F, monoclonal anti-PrPc antibodies inhibit the migration of CCSCs in Transwell migration assays. Mouse IgG was used as control. , P < 0.001; , P < 0.0001. G, monoclonal anti-PrPc antibodies inhibit tumor initiation and liver metastasis in vivo (n ¼ 15). Scale bars, 5 mm. www.aacrjournals.org Cancer Res; 73(8) April 15, 2013 2689

Du et al.

2690 Cancer Res; 73(8) April 15, 2013 Cancer Research

PrPcþ Metastatic Cancer Stem Cells

As in leukemic stem cells (15), knocking down CD44 in CRC cells (Fig. 3G). Collectively, these observations imply that PrPc cells significantly inhibited tumor regeneration (14). To inves- is crucial for CCSC migration in vitro and for metastasis in vivo. tigate the role of PrPc in tumor initiation, we isolated primary CRC cells from patients and cultured them in ultra-low attach- PrPc is functionally important for cell migration and ment dishes without serum. Remarkably, PrPc knockdown in cancer metastasis the CRC cells also retarded tumor growth in nude mice, Because CSC is generally rare and it is difficult to label and þ although the inhibitory effect was slightly less apparent than trace its growth in animals, we generated a cell line of CD44 with CD44 knockdown (Fig. 2F). However, combined knock- CCSCs isolated from a CRC patient. These cells have all the down of both CD44 and PrPc completely abolished tumor characteristics of CSCs including constitutive expression of development (Fig. 2F). Taken together, these data suggest that CD44, and the capacity to form spheres and generate xenograft þ þ the CD44 PrPc subpopulation of CRC cells does possess CSC tumors (14). The CCSCs were grown in ultra-low attachment properties. dishes without serum, and were subcultured once a week for fewer than 20 passages. To investigate the unique role of PrPc þ PrPc CSC is more metastatic in metastasis, we used this cell line prelabeled with DsRed and Because PrPc is highly expressed in metastatic tissues, we in which PrPc had either been knocked down by shRNAs or was attempted to find out whether it contributes to metastasis overexpressed via the pBABE-PURO retrovirus system. As in CRC. To recapitulate human cancers with greater fidelity, shown in Fig. 4A–C, PrPc knockdown strongly inhibited tumor we transplanted 500, 2,000 and 10,000 cells from each of the growth and liver metastasis as assessed by whole-body fluo- þ þ þ þ CD44 PrPc ,CD44 PrPc ,CD44 PrPc and CD44 PrPc rescence imaging and hematoxylin and eosin (H&E) staining, subpopulations isolated from 7 patients (i.e., case 24–30, suggesting that PrPc play a central role in CSC metastasis. In Supplementary Table S1), into the cecal wall of NOD/SCID contrast, both tumor growth and liver metastasis were pro- mice (38). As shown in Table 1, 6 of the 7 patient-derived moted by PrPc overexpression. Moreover, the level of PrPc CRC cells produced cecal tumors, which was consistent expression was correlated with the metastatic index (Fig. 4D). with the results obtained from subcutaneously-implanted Similar results were observed using the spleen metastasis þ þ tumor cells (Supplementary Table S2). The CD44 PrPc model (Supplementary Fig. S4A–S4C). In addition, Transwell cells clearly formed tumors with the greatest efficiency migration assays revealed that the levels of PrPc correlated (P < 0.01; Table 1 and Supplementary Table S2). Remarkably, with the migratory capacity of CCSCs (Fig. 4E). It is noteworthy macroscopic and microscopic analysis revealed that liver that the PrPc levels did not alter the cell-cycle profiles (Sup- metastases were almost exclusively observed in the mice plementary Fig. S4D). Using CRC cells with CSC properties, we þ þ þ implanted with PrPc cells (i.e., CD44 PrPc and CD44 were able to show that PrPc is functionally required for the þ PrPc ;Fig.3A–C; Table 1). Furthermore, semiquantitative migration of CCSCs and contributes to CRC metastasis. measurement of the metastatic area and the metastatic index þ confirmed that the tumors derived from PrPc CRC cells were highly metastatic (Fig. 3D–E). PrPc antibodies inhibit migration and metastasis The spleen transplantation model is another commonly Surface markers unique to CSCs would provide ideal ther- used approach to test the proliferative and metastatic potential apeutic targets on these cells, and indeed monoclonal anti- þ þ þ þ of CSCs (39, 40). CD44 PrPc , CD44 PrPc , CD44 PrPc , and bodies have shown therapeutic efficacy in both preclinical CD44 PrPc subpopulations were sorted from the tumors of tumor models and patients with cancer (41). We were therefore 10 patients (i.e., cases 12–21, Supplementary Table S1), and 500 interested in addressing the question of whether PrPc could be cells from each subpopulation were then transplanted into the used as a therapeutic target, in a manner similar to CD44 (15). spleen of nude mice. Similar to the results obtained using the We generated several monoclonal antibodies against PrPc, out orthotopic model, liver metastasis occurred within 70 days in of which we selected the IgG1 antibody. The specificity of the þ þ þ 28 of 30 cases of CD44 PrPc , 3 of 30 cases of CD44 PrPc ,15 antibodies was verified by ELISA, Western blotting (WB), and þ of 30 cases of CD44 PrPc , and 2 of 30 cases of CD44 PrPc IHC staining (Supplementary Fig. S4E–S4G). The migration of þ cells (Supplementary Fig. S3A–S3B). These results suggest that CD44 CCSCs in vitro was strongly inhibited after they had þ PrPc is intimately associated with the CRC liver metastasis. been treated with 1 mg/mL anti-PrPc antibodies. The inhibitory þ þ Transwell analysis showed that CD44 PrPc cells displayed effect obtained was greater than that associated with the anti- þ about 16-fold greater migratory ability than CD44 PrPc cells CD44 antibody (1.7 fold). A combination of 1 mg/mL anti- (Fig. 3F). Most of the migrating cells (84.25 3.14%) were found CD44 and 1 mg/mL anti-PrPc antibodies was able to inhibit to be PrPc-positive, versus only 1.25 0.22% of the nonmigrant migration by up to approximately 80% (Fig. 4F); whereas PrPc

Figure 6. PrPc-regulated EMT is ERK2 dependent. A, Western blot analysis of the proteins indicated in CCSCs in which PrPc was either overexpressed or knocked down. B, parental CCSCs and those overexpressing PrPc were treated with the ERK2 inhibitor U0126 for 24 hours, and a whole-cell lysate was used for Western Blot analysis of the molecules indicated. C, CCSCs were treated with 5 mg/mL anti-PrPc antibody (Ab), 2 mg/ml laminin (LN), and a combination of Ab and LN for 24 hours, after which cells were harvested for Western Blot analysis. D, phase-contrast images of CCSCs maintained under the conditions indicated. Note that the anti-PrPc antibodies rescued the morphologic changes in the presence of LN. Scale bar, 100 mm. E, Double immunoﬂuorescence staining of N-cadherin (green) and E-cadherin (red) in CCSCs in the presence of 10 mmol/L U0126, 2 mg/mL LN, and indicated dose of anti-PrPc antibodies (mg/mL). Scale bar, 20 mm. F, migration of CCSCs in the presence of U0126, 2 mg/mL LN, and indicated dose of anti-PrPc antibodies (mg/mL).

www.aacrjournals.org Cancer Res; 73(8) April 15, 2013 2691

Du et al.

antibodies did not show any effect on cell migration in PrPc Twist was indeed able to induce EMT in PrPc-knocked down knockdown cells (data not shown). CCSCs, leading to increased levels of Snail and N-cadherin It was essential to find out whether PrPc monoclonal anti- (Fig. 5D). However, overexpression of PrPc in the Twist RNAi bodies can prevent CRC metastases in vivo. NOD/SCID mice cells was unable to restore EMT, probably because Snail and bearing orthotopic implantations of CCSCs were administered N-cadherin expression was lower than in the parental cells 200 mg antibodies intraperitoneally on a weekly basis. ELISA (Fig. 5D). Overall, we conclude that PrPc regulates the EMT analysis revealed that the concentration of anti-PrPc antibody phenotype by modulating Twist. in the serum of tumor-bearing animals was maintained at 0.87 Next, we used qRT-PCR to measure the mRNA levels of þ þ þ to 1 mg/mL (data not shown). The orthotopic and metastatic Twist, N-cadherin, and E-cadherin in CD44 PrPc , CD44 þ tumors were monitored by whole-body fluorescence imaging PrPc , CD44 PrPc , and CD44 PrPc subpopulations of CRC every week and after 8 to 10 weeks the mice were sacrificed and cells freshly purified from 6 patients. The results showed that underwent pathology examinations. The presence of anti-PrPc both Twist and N-cadherin were expressed at significantly þ þ antibodies dramatically decreased tumorigenicity and liver higher levels in the PrPc-positive (i.e. CD44 PrPc and CD44 þ metastases (Fig. 4G), whereas their bodyweight did not PrPc ) CRC cells than in the PrPc subpopulations (Fig. 5E). þ decrease (not shown). CCSCs precoated with anti-PrPc anti- Furthermore, approximately 82% of N-cadherin cells were þ þ bodies before orthotopic implantation produced a similar also PrPc , whereas more than 93% of E-cadherin cells were þ inhibitory effect (Supplementary Fig. S4H). PrPc in CD45 EpCAM CRC cells as analyzed by flow cyto- To make sure that the observed metastasis-inhibiting effects metry (Fig. 5F). These findings suggest that PrPc is positively of anti-PrPc antibodies were not due to inhibition of primary correlated with the mesenchymal properties of cells from CRC tumor growth, we preinoculated the CCSCs orthotopically into patients. the cecal wall for 3 weeks. Mice with equivalent tumors were then grouped and injected with 100 or 200 mg antibodies Evidence that PrPc-induced EMT is ERK2 dependent weekly. The results clearly showed that the anti-PrPc antibody EMT is regulated by the TGF-b and ERK signaling pathways inhibited tumor growth and metastasis at the lower dosage (44). To understand the molecular mechanisms of PrPc- (100 mg/week) and eliminated tumor growth at higher dosage induced EMT, we first assessed the activation of these 2 (200 mg/week; Supplementary Fig. S4I). pathways (45). Knocking down PrPc seemed to result in a reduction of both Phospho-smad2 and ERK1/2; however, over- PrPc promotes EMT in CCSCs expression of PrPc activated ERK1/2 but not smad2, suggesting We consistently observed that the PrPc-overexpressing that ERK signaling may act downstream of PrPc (Fig. 6A). In CCSCs exhibited an elongated mesenchymal-like morphology, support of this, we found that U0126, an inhibitor of ERK whereas the PrPc knockdown cells were flatter(Fig.5A;Sup- signaling, suppressed the expression of several EMT-associat- þ plementaryFig.S4G). Also, PrPc CRCcells generated branching ed molecules (Fig. 6B) as well as EMT itself, especially when colonies with loose cell–cell interaction (see Fig. 2D), charac- PrPc was overexpressed. teristic of the EMT phenotype. This prompted us to investigate Laminin is a PrPc ligand (46), which, we reasoned, could whether PrPc promotes EMT, a known mechanism of CSC activate events occurring downstream of PrPc, such as EMT. migration and invasion. Cells undergoing EMT retain stem Laminin did enhance both ERK2 phosphorylation and the EMT cell functionality and form invasive front colonies (42). We first phenotype (Fig. 6C and D). Interestingly, PrPc antibody used quantitative reverse transcription-PCR (qRT-PCR; primers blocked the laminin-induced ERK activation, EMT protein shown in Supplementary Table S3) to measure several EMT- expression and EMT morphology in a dose-dependent manner, related genes by manipulating PrPc levels. In PrPc overexpres- (Fig. 6C and D), probably via interference with the interaction sing cells, mesenchymal genes such as N-cadherin and Twist between laminin and its receptor PrPc. Immunofluorescence were significantlyupregulated, whereasE-cadherin,anepithelial staining confirmed that the anti-PrPc antibody attenuated the marker, was downregulated (Supplementary Fig. S5A). In addi- laminin-induced N-cadherin expression (Fig. 6E). In addition, tion, there were increased levels of matrix metalloproteinase 2 Transwell migration assays revealed that U0126 blocked the (MMP2) and MMP11 (Supplementary Fig. S5A), which may have PrPc overexpression-induced migration, and that the anti-PrPc contributed to the metastasis observed in PrPc overexpressing antibody also attenuated laminin-enhanced migration (Fig. cells. In contrast, knockdown of PrPc resulted in a reversed 6F). Our findings thus suggest that ERK2 signaling pathway is expression pattern (Supplementary Fig. S5A). Western blot involved in PrPc-induced EMT. analysis and double immunofluorescence staining further confirmed the correlation of PrPc with the expression of EMT- Discussion related molecules (Fig. 5B and C; Supplementary Fig. S5B). It has been shown that CSCs contribute to both tumor Twist is a key transcription factor regulating mesenchymal initiation and cancer metastasis in many tumor systems. cell fate, differentiation, and morphogenesis (43). Because PrPc However, in most cases, the underlying mechanisms are not expression had been found to influence the expression of Twist well understood. In this study, we have identified a distinct þ þ (Supplementary Fig. S5A), we reasoned that PrPc might reg- PrPc subpopulation in CD44 CCSCs that displays enhanced ulate EMT via Twist. To test this, Twist was ectopically capacity to regenerate tumors. These results emphasize the expressed in CCSCs in which PrPc was stably knocked down functionalities of PrPc as an "enrichment" factor for CCSC. Our by shRNA. We observed that constitutively overexpressed data show that PrPc is a novel CCSC biomarker, which, by itself,

2692 Cancer Res; 73(8) April 15, 2013 Cancer Research

PrPcþ Metastatic Cancer Stem Cells

functionally contributes to both CRC tumorigenicity and targeting surface molecules expressed in CSCs show powerful þ metastasis. Interestingly, the PrPc CRC cells tend to localize therapeutic efficacy by eliminating CSCs and leading to at the invasion fronts in patient tumors, are more mesenchy- tumor resolution (16). Our findings therefore have significant þ mal, and behave like other cancer cells undergoing EMT, clinical implications, as PrPc CSCs may provide important properties that have probably given these cells a greater new targets for therapy in metastatic CRC. Indeed, PrPc capacity to disseminate and metastasize. These observations monoclonal antibodies display strong inhibitory effects on indicate PrPc's role in regulating CCSC likely occurring CCSC tumorigenesis and metastasis in orthotopic xenograft through its ability to promote EMT and the exact mechanism models, suggesting possible therapeutic applications in the warrants future investigations. In terms of the mechanism clinic. Overall, our findings throw new light on the mechan- involved, our work implies that PrPc may regulate EMT isms underlying CRC cell metastasis and are of clinical through ERK2- and Twist-dependent signaling pathways. importance for the design of therapeutic strategies for treat- Importantly, the interaction of PrPc with its ligand, laminin, ing CRC. directly activates ERK2 signaling and promotes EMT. There- fore, conceptually, interference with PrPc expression/func- Disclosure of Potential Conflicts of Interest tions, the PrPc-laminin interaction or its downstream signaling No potential conflicts of interest were disclosed. events, may be a promising avenue in developing new therapies þ targeting PrPc CRC cell-mediated metastasis. Authors' Contributions Previous work has shown that PrP1, a PrPc ortholog in the Conception and design: L. Du, B. Laffin, M. Mehrpour, Q. Chen fi Development of methodology: L. Du, G. Rao, B. Li, Y. Zhu, D.G. Tang zebra sh, regulates the stability of E-cadherin mediated cell Acquisition of data (provided animals, acquired and managed patients, adhesion (47), suggesting that PrPc expression may endow the provided facilities, etc.): G. Rao, H. Wang, B. Li, W. Tian, J.T. Cui, L. He, X. Tian, cell with properties of EMT, which is a developmentally C. Hao, H. Liu, Y. Zhu, D.G. Tang, Y. Lu c Analysis and interpretation of data (e.g., statistical analysis, biostatistics, conserved regulatory mechanism. In addition, PrP -null computational analysis): L. Du, G. Rao, H. Wang, B. Li, B. Laffin, D.G. Tang, þ þ (PRNP0/0) cells display less migratory activity than PRNP / M. Mehrpour PRNPþ/þ Writing, review, and/or revision of the manuscript: L. Du, G. Rao, B. Li, cells (48). The more aggressive phenotype of cells is M. Mehrpour, Y. Lu, Q. Chen consistent with our observations that PrPc contributes to Administrative, technical, or material support (i.e., reporting or orga- þ CCSC migration. Recently, CD26 CRC cells have also been nizing data, constructing databases): G. Rao, B. Li, X. Sun, D.G. Tang Study supervision: Y. Lu, Q. Chen found to possess CSC properties, with greatly increased metastatic capacity, and, intriguingly, like PrPc, CD26 also func- Acknowledgments tions as an adhesion molecule that binds to ECM (24). These The authors would like to thank Drs. G. Ge and K.-J. Wu for providing Twist findings, together with our present study, strongly suggest that plasmids, Dr. S. L. Holbeck for providing CMA slides, Dr. J. Grassi for providing the enhanced tumorigenic and metastatic capacities of CCSCs antibody against PrPc (SAF69), Mrs. X. Zhang, T. Zhao, J. Wang, and L. Dou for FACS assistance, Mrs. Y. Zhou and X. Shi for whole-body fluorescence imaging are probably determined by the CSC-intrinsic expression of assays, Dr. W. Zhang and Dr. C. Han for biostatistical analysis, Dr. A. Lu, Dr. W. functional molecules such as PrPc and CD26, which in turn Xue, and Dr. L. Zhou for pathologic determination, and Dr. A. Zhou for his mediate the interactions of CSCs with their microenvironment. suggestions and revision of our manuscript. Future work will attempt to elucidate the potential relation- þ þ ship between PrPc and CD26 CCSCs. Grant Support This work was supported by initiative of stem cell and regenerative medicine Previous studies have shown that the expression of PrPc is from Chinese Academy of Sciences (XDA01040409), the 973 project from the positively correlated with invasiveness and drug resistance in Ministry of Science and Technology (MOST) of China (2009CB512800 and both gastric and breast cancers (27, 49). In addition, Liao and 2010CB912204), and a grant from the National Natural Science Foundation of China (NSFC; 81130045 to Q. Chen and 31000614 to L. Du), from MOST colleagues have reported that endoglin and PrPc are enriched 2010CB529403 (X. Tian), MOST 2010CB912204 (Y. Zhu), and from Institut in mammosphere-initiating cells (50). Although PrPc has been National de la Sante et de la Recherche Medicale, Partenariat Hubert Curien - Programme Francais¸ de Cooperation avec la Chine, Fondation Franco-Chinoise reported to be highly expressed in some CRCs (51), as far as we pour la Science et ses Applications (M. Mehrpour). are aware this is the first report linking PrPc to CCSCs, and our The costs of publication of this article were defrayed in part by the payment of data help to explain how PrPc is associated with cancer page charges. This article must therefore be hereby marked advertisement in metastasis. The level of PrPc was inversely correlated with accordance with 18 U.S.C. Section 1734 solely to indicate this fact. patient survival, suggesting a potential prognostic value in Received September 26, 2012; revised January 31, 2013; accepted February 2, CRC. It has recently emerged that monoclonal antibodies 2013; published OnlineFirst February 15, 2013.

References 1. Sporn MB. The war on cancer. Lancet 1996;347:1377–81. 5. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. 2. Jemal A, Thun MJ, Ward EE, Henley SJ, Cokkinides VE, Murray TE. Prospective identification of tumorigenic breast cancer cells. Proc Natl Mortality from leading causes by education and race in the United Acad Sci U S A 2003;100:3983–8. States, 2001. Am J Prev Med 2008;34:1–8. 6. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al. 3. Pardal R, Clarke MF, Morrison SJ. Applying the principles of stem-cell Identification of a cancer stem cell in human brain tumors. Cancer Res biology to cancer. Nat Rev Cancer 2003;3:895–902. 2003;63:5821–8. 4. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a 7. Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, hierarchy that originates from a primitive hematopoietic cell. Nat Med et al. Identification of bronchioalveolar stem cells in normal lung and 1997;3:730–7. lung cancer. Cell 2005;121:823–35.

www.aacrjournals.org Cancer Res; 73(8) April 15, 2013 2693

Du et al.

8. Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, et al. Phenotypic 31. Tang Z, Zhao M, Ji J, Yang G, Hu F, He J, et al. Overexpression of characterization of human colorectal cancer stem cells. Proc Natl Acad gastrin and c-met protein involved in human gastric carcinomas and Sci U S A 2007;104:10158–63. intestinal metaplasia. Oncol Rep 2004;11:333–9. 9. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C. 32. Wang HY, Zhang JY, Cui JT, Tan XH, Li WM, Gu J, et al. Expression Identification and expansion of human colon-cancer-initiating cells. status of S100A14 and S100A4 correlates with metastatic potential Nature 2007;445:111–5. and clinical outcome in colorectal cancer after surgery. Oncol Rep 10. O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell 2010;23:45–52. capable of initiating tumour growth in immunodeficient mice. Nature 33. Ki DH, Jeung HC, Park CH, Kang SH, Lee GY, Lee WS, et al. Whole 2007;445:106–10. genome analysis for liver metastasis gene signatures in colorectal 11. Huang EH, Hynes MJ, Zhang T, Ginestier C, Dontu G, Appelman H, cancer. Int J Cancer 2007;121:2005–12 et al. Aldehyde dehydrogenase 1 is a marker for normal and 34. Koinuma K, Yamashita Y, Liu W, Hatanaka H, Kurashina K, Wada T, malignant human colonic stem cells (SC) and tracks SC over- et al. Epigenetic silencing of AXIN2 in colorectal carcinoma with population during colon tumorigenesis. Cancer Res 2009;69: microsatellite instability. Oncogene 2006;25:139–46. 3382–9. 35. Smith JJ, Deane NG, Wu F, Merchant NB, Zhang B, Jiang A, et al. 12. Takeda K, Kinoshita I, Shimizu Y, Matsuno Y, Shichinohe T, Dosaka- Experimentally derived metastasis gene expression profile predicts Akita H. Expression of LGR5, an Intestinal Stem Cell Marker, During recurrence and death in patients with colon cancer. Gastroenterology Each Stage of Colorectal Tumorigenesis. Anticancer Res 2011;31: 2010;138:958–68 263–70. 36. Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A, 13. Vermeulen L, Todaro M, de Sousa MF, Sprick MR, Kemper K, Perez et al. The EMT-activator ZEB1 promotes tumorigenicity by repres- AM, et al. 14. Single-cell cloning of colon cancer stem cells reveals a sing stemness-inhibiting microRNAs. Nat Cell Biol 2009;11: multi-lineage differentiation capacity. Proc Natl Acad Sci U S A 1487–95. 2008;105:13427–32. 37. Malanchi I, Santamaria-Martínez A, Susanto E, Peng H, Lehr HA, 14. Du L, Wang H, He L, Zhang J, Ni B, Wang X, et al. CD44 is of functional Delaloye JF, et al. Interactions between cancer stem cells and their importance for colorectal cancer stem cells. Clin Cancer Res niche govern metastatic colonization. Nature 2012;481:85–9. 2008;14:6751–60. 38. Fu X, Besterman JM, Monosove A, Hoffman RM. Models of human 15. Jin L, Hope KJ, Zhai Q, Smadja-Joffe F, Dick JE. Targeting of CD44 metastatic colon cancer in nude mice orthotopically constructed by eradicates human acute myeloid leukemic stem cells. Nat Med using histologically intact patient specimens. Proc Natl Acad Sci U S A 2006;12:1167–74. 1991;88:9345–9. 16. Majeti R. Monoclonal antibody therapy directed against human acute 39. Brand MI, Casillas S, Dietz DW, Milsom JW, Vladisavljevic A. Devel- myeloid leukemia stem cells. Oncogene 2011;30:1009–19. opment of a reliable colorectal cancer liver metastasis model. J Surg 17. Dalerba P, Clarke MF. Cancer stem cells and tumor metastasis: first Res 1996;63:425–32. steps into uncharted territory. Cell Stem Cell 2007;1:241–2. 40. Giavazzi R, Jessup JM, Campbell DE, Walker SM, Fidler IJ. Experi- 18. Wicha MS. Cancer stem cells and metastasis: lethal seeds. Clin Cancer mental nude mouse model of human colorectal cancer liver metasta- Res 2006;12:5606–7. ses. J Natl Cancer Inst 1986;77:1303–8. 19. Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, et al. 41. Chao MP, Alizadeh AA, Tang C, Myklebust JH, Varghese B, Gill S, Distinct populations of cancer stem cells determine tumor growth and et al. Anti-CD47 antibody synergizes with rituximab to promote metastatic activity in human pancreatic cancer. Cell Stem Cell phagocytosis and eradicate non-Hodgkin lymphoma. Cell 2007;1:313–23. 2010;142:699–713. 20. Gao AC, Lou W, Dong JT, Isaacs JT. CD44 is a metastasis suppressor 42. Brabletz T, Jung A, Spaderna S, Hlubek F, Kirchner T. Opinion: gene for prostatic cancer located on human chromosome 11p13. migrating cancer stem cells - an integrated concept of malignant Cancer Res 1997;57:846–9. tumour progression. Nat Rev Cancer 2005;5:744–9. 21. Bankfalvi A, Krassort M, Buchwalow IB, Vegh A, Felszeghy E, Piffko J. 43. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, Gains and losses of adhesion molecules (CD44, E-cadherin, and beta- et al. Twist, a master regulator of morphogenesis, plays an essential catenin) during oral carcinogenesis and tumour progression. J Pathol role in tumor metastasis. Cell 2004;117:927–39. 2002;198:343–51. 44. Zavadil J, Bottinger€ EP. TGF-b and epithelial-to-mesenchymal transi- 22. Jothy S. CD44 and its partners in metastasis. Clin Exp Metastasis tions. Oncogene 2005;24:5764–74. 2003;20:195–201. 45. Shin S, Dimitri CA, Yoon SO, Dowdle W, Blenis J. ERK2 but not ERK1 23. Martin TA, Harrison G, Mansel RE, Jiang WG. The role of the CD44/ induces epithelial-to-mesenchymal transformation via DEF motif- ezrin complex in cancer metastasis. Crit Rev Oncol Hematol dependent signaling events. Mol Cell 2010;38:114–27. 2003;46:165–86. 46. Rieger R, Edenhofer F, Lasmezas CI, Weiss S. The human 37-kDa 24. Pang R, Law WL, Chu AC, Poon JT, Lam CS, Chow AK, et al. laminin receptor precursor interacts with the prion protein in eukaryotic A subpopulation of CD26þ cancer stem cells with metastatic cells. Nat Med 1997;3:1383–8. capacity in human colorectal cancer. Cell Stem Cell 2010;6: 47. Malaga-Trillo E, Sempou E. PrPs: Proteins with a purpose: Lessons 603–15. from the zebrafish. Prion 2009;3:129–33. 25. Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, 48. Muras AG, Hajj GN, Ribeiro KB, Nomizo R, Nonogaki S, Chammas R, et al. Mice devoid of PrP are resistant to scrapie. Cell 1993;73: et al. Prion protein ablation increases cellular aggregation and embo- 1339–47. lization contributing to mechanisms of metastasis. Int J Cancer 26. Klein MA, Aguzzi A. The neuroimmune interface in prion diseases. 2009;125:1523–31. News Physiol Sci 2000;15:250–5. 49. Pan Y, Zhao L, Liang J, Liu J, Shi Y, Liu N, et al. Cellular prion protein 27. Mehrpour M, Codogno P. Prion protein: from physiology to cancer promotes invasion and metastasis of gastric cancer. FASEB J biology. Cancer Lett 2010;290:1–23. 2006;20:1886–8. 28. Aguzzi A, Polymenidou M. Mammalian prion biology: one century of 50. Liao MJ, Zhang CC, Zhou B, Zimonjic DB, Mani SA, Kaba M, et al. evolving concepts. Cell 2004;116:313–27. Enrichment of a population of mammary gland cells that form mammo- 29. Dodelet VC, Cashman NR. Prion protein expression in human leuko- spheres and have in vivo repopulating activity. Cancer Res cyte differentiation. Blood 1998;91:1556–61. 2007;67:8131–8. 30. Zhang CC, Steele AD, Lindquist S, Lodish HF. Prion protein is 51. Antonacopoulou AG, Palli M, Marousi S, Dimitrakopoulos FI, Kyria- expressed on long-term repopulating hematopoietic stem cells and kopoulou U, Tsamandas AC, et al. Prion protein expression and the is important for their self-renewal. Proc Natl Acad Sci U S A M129V polymorphism of the PRNP gene in patients with colorectal 2006;103:2184–9. cancer. Mol Carcinog 2010;49:693–9.

2694 Cancer Res; 73(8) April 15, 2013 Cancer Research

CD44-Positive Cancer Stem Cells Expressing Cellular Prion Protein Contribute to Metastatic Capacity in Colorectal Cancer

Lei Du, Guanhua Rao, Hongyi Wang, et al.

Cancer Res 2013;73:2682-2694. Published OnlineFirst February 15, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-3759

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2013/02/14/0008-5472.CAN-12-3759.DC1

Cited articles This article cites 51 articles, 13 of which you can access for free at: http://cancerres.aacrjournals.org/content/73/8/2682.full#ref-list-1

Citing articles This article has been cited by 4 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/73/8/2682.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/73/8/2682. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.