Pancreatic ductal adenocarcinoma (PDAC) is a

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1 GASTROENTEROLOGY 2013;145: A Gene Expression Signature of Epithelial Tubulogenesis and a Role for ASPM in Pancreatic Tumor Progression WEI YU WANG, 1,2, * CHUNG CHI HSU, 1, * TING YUN WANG, 1, * CHI RONG LI, 5, * YA CHIN HOU, 3 JUI MEI CHU, 1 CHUNG TA LEE, 2 MING SHENG LIU, 1 JIMMY J. M. SU, 1 KUAN YING JIAN, 1 SHENQ SHYANG HUANG, 1 SHIH SHENG JIANG, 1,6 YAN SHEN SHAN, 3 PIN WEN LIN, 3 YIN YING SHEN, 7 MICHAEL T. L. LEE, 8 TZE SIAN CHAN, 9 CHUN CHAO CHANG, 9 CHUNG HSING CHEN, 1,6 I SHOU CHANG, 1,6 YEN LING LEE, 1 LI TZONG CHEN, 1 and KELVIN K. TSAI 1,4,9 1 Laboratory for Tumor Epigenetics and Stemness, National Institute of Cancer Research and Translational Center for Glandular Malignancies, National Health Research Institutes, Tainan, Taiwan; Departments of 2 Pathology, 3 Surgery, and 4 Medicine, National Cheng-Kung University Hospital and College of Medicine, National Cheng Kung University, Tainan, Taiwan; 5 Department of Medical Education and School of Nursing, Chung Shan Medical University and Hospital, Taichung, Taiwan; 6 Taiwan Bioinformatics Core, National Health Research Institutes, Zhunan, Taiwan; 7 Pathology Core Laboratory, National Health Research Institutes, Tainan, Taiwan; 8 Department of Information Engineering, Kun Shan University, Tainan, Taiwan; and 9 Graduate Institute of Clinical Medicine, School of Medicine, and Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taipei Medical University and Hospital, Taipei, Taiwan BACKGROUND & AIMS: Many patients with pancreatic ductal adenocarcinoma (PDAC) develop recurrent or metastatic diseases after surgery, so it is important to identify those most likely to benefit from aggressive therapy. Disruption of tissue microarchitecture is an early step in pancreatic tumorigenesis and a parameter used in pathology grading of glandular tumors. We investigated whether changes in gene expression during pancreatic epithelial morphogenesis were associated with outcomes of patients with PDAC after surgery. METHODS: We generated architectures of human pancreatic duct epithelial cells in a 3-dimensional basement membrane matrix. We identified gene expression profiles of the cells during different stages of tubular morphogenesis (tubulogenesis) and of PANC-1 cells during spheroid formation. Differential expression of genes was confirmed by immunoblot analysis. We compared the gene expression profile associated with pancreatic epithelial tubulogenesis with that of PDAC samples from 27 patients, as well as with their outcomes after surgery. RESULTS: We identified a gene expression profile associated with tubulogenesis that resembled the profile of human pancreatic tissue with differentiated morphology and exocrine function. Patients with PDACs with this profile fared well after surgery. Based on this profile, we established a 6 28 gene tubulogenesis-specific signature that accurately determined the prognosis of independent cohorts of patients with PDAC (total n ¼ 128; accuracy ¼ 81.2% 95.0%). One gene, ASPM, was down-regulated during tubulogenesis but up-regulated in human PDAC cell lines and tumor samples; up-regulation correlated with patient outcomes (Cox regression P ¼.0028). Bioinformatic, genetic, biochemical, functional, and clinical correlative studies showed that ASPM promotes aggressiveness of PDAC by maintaining Wnt-b-catenin signaling and stem cell features of PDAC cells. CONCLUSIONS: We identified a gene expression profile associated with pancreatic epithelial tubulogenesis and a tissue architecturelspecific signature of PDAC cells that is associated with patient outcomes after surgery. Keywords: Pancreatic Cancer; Tumor Progression; Prognostic Factor; Biomarker. Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy. Because of the paucity of symptoms in early diseases and the aggressive behaviors of the tumors, <20% of patients with PDAC present with localized and resectable diseases at the time of diagnosis. Even with curative-intent surgery, the majority of patients with initially localized tumors developed recurrent or metastatic diseases, and only a small subset (18% 26%) of the patients could attain long-term survival. 1,2 Additional improvements in the prognosis of patients with localized PDAC might rely on elucidating the pathogenesis underlying tumor recurrence and clinically reliable prognostic prediction that can guide patient-tailored treatment plans. Loss of tissue architectures is one of the hallmark features of malignant tumors. 3 The extent to which a tumor forms tissue microarchitecture reflects the degree of malignant transformation, which dictates the clinical behavior of the tumor. Glandular differentiation has been widely used in the histopathological assessment for glandderived malignancies, including prostate cancer, breast cancer, and PDAC. 4 6 Nevertheless, assessing tissue architectures by morphological criteria is partly subjective and can fail to provide in-depth mechanistic insights. Recently, comparative genomic analysis on clinical tumor materials has led to the identification of gene profiles or tumor molecular subtypes of PDAC that carried prognostic *Authors share co-first authorship. Abbreviations used in this paper: ASPM, abnormal spindle-like microcephaly associated; C-index, concordance index; CSC, cancer stem cell; 3D, 3-dimensional; Dvl, dishevelled; HPDE, human pancreatic ductal epithelial; PDAC, pancreatic ductal adenocarcinoma; rbm, reconstituted basement membrane; shrna, short-hairpin RNA by the AGA Institute /$

2 November 2013 TUBULOGENESIS SIGNATURE IN PANCREATIC CANCER 1111 significance. 7,8 As opposed to molecular patterns identified from a developed tumor, which might reflect the accumulative effect of the malignant transformation process, knowledge-based and biology-informed approaches offer an opportunity to identify biomarkers or classifiers that additionally provide pathogenetic information. For example, gene expression patterns associated with glandular morphogenesis have been linked to clinical prognosis of patients with breast or prostate cancer. 9,10 In this study, we investigated whether the molecular changes associated with the formation of pancreatic epithelial architectures can provide prognostic information in PDAC. We identified a gene signature/subtype of PDAC that is associated with pancreatic epithelial tubulogenesis and the oncogenic role of ASPM (abnormal spindle-like microcephaly associated), which provides a mechanistic link between tissue architecture, Wnt signaling pathway, cancer stemness, and tumor aggressiveness. Materials and Methods Detailed Materials and Methods are described in the Supplementary Material. Cell Culture and Staining The sources and culture of human pancreatic ductal epithelial (HPDE) cells and other cells are described in the Supplementary Material. For organotypic cultures, the cells were grown on top of a thick layer of 3-dimensional (3D) reconstituted basement membrane (rbm; Matrigel, BD Biosciences, San Jose, CA), as described previously. 11 Gene Expression Profiling Gene expression analysis was performed on an Affymetrix Human Genome U133A 2.0 Plus GeneChip platform according to the manufacturer s protocol (Affymetrix, Santa Clara, CA). The gene expression data have been deposited in NCBIs Gene Expression Omnibus (GEO, and are accessible through GEO Series accession number GSE Quantitative Real-Time Polymerase Chain Reaction, Immunoblot, and Co-immunoprecipitation Analyses Quantitative reverse transcription polymerase chain reaction analysis was performed using the LightCycler System (Roche Diagnostics GmbH, Mannheim, Germany). The procedure and antibodies used for immunoblot and co-immunoprecipitation analysis are described in the Supplementary Material. Gene Expression Data Sets of PDAC Sources of different PDAC tumor transcriptome datasets are described in the Supplementary Material. Construction of Prognostic Predictors To identify from the differentially expressed genes, a set of gene markers that optimally predicted survival, we used a previously described supervised approach, 12 as described in the Supplementary Material. Immunohistochemistry Formalin-fixed, paraffin-embedded tissues of human PDAC from 53 patients who received tumor resection at National Cheng-Kung University Hospital (Tainan, Taiwan; the National Cheng-Kung University Hospital cohort; Supplementary Table 1) were acquired and used in conformity with Institutional Review Board approved protocols. All immunohistochemical staining was evaluated by expert pathologists (C. Lee and Y. Shen) and the staining patterns were quantified using the histological score. 13 Gene Expression Manipulations Sustained ASPM knockdown in PDAC cells was achieved by lentivirus-mediated RNA interference using validated shorthairpin RNA (shrna) oligonucleotides (MISSION shrna lentiviruses; Sigma-Aldrich, St Louis, MO). The S33Y mutant of b-catenin was subcloned from pcdna3 (Addgene, Cambridge, MA) into the pqcxih retroviral vector (Clontech, Mountain View, CA). 14 Cell Migration Assay The migration capacity of cells was measured using the modified Boyden chamber assay. Luciferase Reporter Assay Cells were transduced with Cignal Lenti TCF/LEF Reporter (Qiagen, Taipei, Taiwan) according to the manufacturer s protocol. After stimulation of the cells with recombinant human Wnt-3a (R&D Systems, Minneapolis, MN) or vehicle, the reporter activity was measured by using the ONE-Glo Luciferase Assay System (Promega, Madison, WI). Orthotopic Pancreatic Tumorigenesis Model and Bioluminescence Imaging AsPC-1 cells were retrovirally transduced with a vector encoding green fluorescence protein and firefly luciferase. Cells ( cells) were inoculated into the pancreatic body of 8-weekold nonobese diabetic/severe combined immunodeficient mice, and tumor mass and distribution were assessed by bioluminescence (IVIS Imaging System, Caliper Life Sciences, Waltham, MA). Flow Cytometry Cells were dissociated, antibody-labeled and resuspended in Hank s balanced salt solution/2% fetal bovine serum as described previously. 15 The procedure of flow cytometry and antibodies used are described in the Supplementary Material. Statistical Analysis We used the statistical programming language R (cran. r-project.org) and SPSS 10.0 software (SPSS, Inc., Chicago, IL) to conduct the statistical analysis of the data. The concordance index (C-index) was used to evaluate the predictive accuracy in the survival analysis. 16 Statistical significance was considered P <.05. Results Molecular Profiling of Pancreatic Epithelial Tubulogenesis We recapitulated the process of pancreatic epithelial morphogenesis by using a physiologically relevant 3D

3 1112 WANG ET AL GASTROENTEROLOGY Vol. 145, No. 5 organotypic culture model. 11 When pancreatic ductal epithelial HPDE cells were seeded on top of 3D rbm for a short length of time (36 48 h), they grew into unorganized cellular clusters or cords (Figure 1A and Supplementary Figure 1A). Intriguingly, HPDE cells subsequently underwent structural reorganization and formed branching and tortuous tubules after 6 8 days in culture. Confocal imaging analysis revealed that these tubules consisted of a single layer of polarized cells, indicated by the polarized expressions of the basal surface marker a6-integrin and the adherens junction protein b-catenin, and a cell-free lumen (Figure 1B and Supplementary Figure 1B). The HPDE tubulogenesis is a differentiationspecific process, as malignant PDAC cells, such as PANC- 1 cells, only formed disorganized spheroids that lacked discernible structural organization within the same context (Supplementary Figure 1C and data not shown). To molecularly dissect the pancreatic epithelial morphogenetic process, we performed comparative transcriptomic analysis and thereby identified a list of 620 unique genes, the transcript levels of which varied significantly during the tubular morphogenesis, or tubulogenesis, of HPDE cells (Figure 1C). In contrast, we found surprisingly few (n ¼ 18) genes that displayed differential expression during the formation of malignant PANC-1 spheroids. Functional clustering analysis on these 620 genes revealed a significant enrichment of the Gene Ontology terms related to cell cycle as well as epidermis developments, wound healing, and inflammatory response (Supplementary Figure 2). Importantly, several genes that specify the exocrine functions of pancreas, including CEL (bile salt-stimulated lipase), CA9 (carbonic anhydrase 9), MUC1 (mucin 1), AGR2 (anterior gradient homolog 2), and MUC20 (mucin 20), were profoundly up-regulated during epithelial tubulogenesis, and their expressions remained unaltered during the formation of malignant tumor spheroids (Figure 1D). Immunoblotting analysis confirmed the tubulogenesis-specific expressional changes in these functional markers (Figure 1E). These data support our tissue organization model as a valid way to capture the molecular signals related to the structural and functional differentiation of exocrine pancreatic epithelium. Figure 1. Pancreatic epithelial tubulogenesis and the related molecular alterations. (A, B) Representative confocal images of HPDE cell clusters (A) or tubules (B) formed in 3D rbm. The structures were immunostained with a6-integrin (red) and b-catenin (green). Nuclei were counterstained with 4 0,6-diamidino-2-phenylindole (DAPI) (blue). Asterisks: cell-free lumen. Scale bars ¼ 100 mm. (C) Heat map showing expression patterns of 620 differentially expressed genes (DEGs) during HPDE tubulogenesis or PANC-1 spheroid formation. The heat map depicts high (red) and low (green) relative levels of medium-centered gene expression in log space. (D) Fold changes in the transcript levels of CEL, CA9, MUC1, AGR2, and MUC20 in HPDE or PANC-1 organoids as measured by quantitative reverse transcription polymerase chain reaction analysis. (E) Western blot analysis of bile salt stimulated lipase, carbonic anhydrase 9, or mucin-1 in HPDE or PANC-1 3D organoids. b-tubulin was included as a loading control.

4 November 2013 TUBULOGENESIS SIGNATURE IN PANCREATIC CANCER 1113 Tubulogenesis-Specific Gene Signature of PDAC As disruption of tissue microarchitectures is one of the hallmark features of glandular cancers, including PDAC, 3 6 we investigated whether the gene expression profile associated with pancreatic epithelial tubulogenesis can carry prognostic information in PDAC. To this end, we interrogated the transcriptomes of microdissected, cancer cell-enriched tumor samples from a cohort of 27 patients with localized PDAC (the University of California San Francisco dataset). 8 We determined the degree of resemblance between these tumors and HPDE tubules by calculating Pearson s correlation coefficients (r tubules ) based on the expression of the 620 tubulogenesis-associated genes (Figure 2A). We divided the patients into 2 subgroups according to r tubules and designated the tumors with higher r tubules the tubule-like PDAC. Intriguingly, we found that the patients carrying the tubule-like tumors had much longer postoperative survival than those with lower r tubules (Figure 2B, left panel). We repeated this analysis in 2 independent series of patients with PDAC and consistently found a favorable clinical prognosis in patients with tubule-like tumors (the Johns Hopkins and the Northwestern/NorthShore cohorts; Figure 2B, right panels). 7 Next, to identify a smaller set of genes that could optimally predict the clinical outcomes of patients with PDAC, we constructed a risk score based on a Cox s model to predict patients survival after surgery. We selected a set of 28 genes whose performance in survival prediction reached the maximum as assessed by C-index (Figure 2C and Supplementary Table 2). We found that patients in the high risk-score group had poor postoperative prognosis, Figure 2. Identification of a tubulogenesis-specific gene signature in PDAC. (A) An illustration depicting the derivation of r tubules.(b) Kaplan-Meier survival curve comparing postoperative survival of PDAC patients with high or low r tubules of their tumors. (C) Selection of a 28-gene gene set with the highest accuracy (C-index) for the prediction of postoperative survival in PDAC. (D) Kaplan-Meier survival curves comparing postoperative survival in PDAC. The patients were stratified into 2 groups based on predicted risk of relapse (risk score; RS) calculated by the 28-gene signature. (E) Forest plots showing hazard ratios (with 95% confidence limits) of death according to the RS and clinicopathological criteria in a Cox proportionalhazards analysis. *P <.05; **P <.01. JHMI, Johns Hopkins Medical Institutions; NW/NSU, Northwestern Memorial Hospital/NorthShore University Health System; UCSF, University of California San Francisco.

5 1114 WANG ET AL GASTROENTEROLOGY Vol. 145, No. 5 and patients in the low risk-score group fared well (Figure 2D). A multivariate Cox proportional-hazards analysis confirmed that this tubulogenesis-specific signature was the strongest prognostic predictor and significantly outperformed clinicopathological criteria across independent PDAC datasets (C-index ¼ ; Figure 2E and Supplementary Tables 3 and 5). To further enhance clinical utility, we sought to refine the prognostic model and found that the 6 top-ranked genes from the 28-gene signature (Cox regression P <.005; Supplementary Table 2 and Supplementary Figure 3), including ATP9A, ASPM, ACOX3, CDC45L, SLC40A1, andagr2, could form a more condensed 6-gene signature that performed as excellently as the 28-gene signature in the prognostic prediction (C-index ¼ ; Supplementary Figure 4 and Supplementary Tables 4 and 5). ASPM as a Tissue-ArchitectureLSpecific Prognostic Marker in PDAC Among the constituent genes in the tubulogenesisspecific signature, ASPM exhibited the most prominent transcriptional change during pancreatic tubulogenesis (28.6-fold down-regulation; Figure 3A, arrow). This, together with a strong correlation of ASPM with the prognosis of PDAC patients (Cox regression P ¼.0028), incited us to investigate its prognostic and biological roles in PDAC. We first asked if ASPM expression was regulated in a tissue architecture dependent manner. We found that the transcript level of ASPM markedly decreased when HPDE cells were transferred from cell monolayers to 3D rbm, and the level decreased further upon tubulogenesis (Figure 3B). Interestingly, when we enzymatically digested HPDE tubules and cultured the recovered cells in 3D rbm, the transcript level of ASPM in the resultant secondary cell clusters was restored to a level similar to that previously measured in the original cellular clusters, indicating that the expression of ASPM is regulated in a contextdependent and reversible manner. The human ASPM gene encodes a large (409.8 kda) and multifunctional protein that plays a critical role in neurogenesis, neuronal migration, and the expansion of glioma stem cells. 17,18 Recently, ASPM expression has been Figure 3. ASPM as a tissue architecture specific prognostic marker in PDAC. (A) The transcript levels of selected top-ranked genes in the tubulogenesis-specific signature as measured by quantitative reverse transcription polymerase chain reaction (qrt-pcr) analysis. (B) Schematic representation of the experimental protocol for growing and manipulating different HPDE tissue assemblies. Right, the transcript levels of ASPM as measured by qrt-pcr analysis. Data are represented as mean SEM (n ¼ 3). *P <.05; **P <.01; ***P <.001 in (A) and (B). (C) Overall survival of patients stratified based on the expression levels of ASPM in the University of California San Francisco (UCSF) (C[i]), Johns Hopkins Medical Institutions (JHMI) (C[ii]), or Northwestern/NorthShore (NW/NSU) (C[iii]) cohort of patients with PDAC. (D) Representative immunostaining of ASPM in PDAC tissues (400 magnification). Shown are tumors with moderate (PDAC #1) or undetectable (PDAC #2) ASPM staining. (E) Distribution of the staining intensities of ASPM in tumors in the National Cheng-Kung University Hospital (NCKUH) cohort. (F) Kaplan-Meier survival curves comparing postoperative survival of patients in the NCKUH cohort stratified according to the staining intensities of ASPM.

6 November 2013 TUBULOGENESIS SIGNATURE IN PANCREATIC CANCER 1115 linked with poor clinical prognosis in ovarian cancer and hepatocellular carcinoma. 17,19,20 In accordance with the poor prognostic role of ASPM in human malignancies, we found that PDAC patients with their tumors expressing high transcript levels of ASPM fared poorly across 3 independent cohorts (Figure 3C). To corroborate these findings, we carried out immunohistochemical staining of the tumor tissues from another cohort of 53 patients with PDAC (National Cheng-Kung University Hospital cohort; Supplementary Table 1). We found that more than two thirds of tumors exhibited weak (1þ) or moderate (2þ) staining intensities for ASPM in cancer cells (Figure 3D and E). In line with the tumor transcriptome data, patients with their tumors exhibiting moderate ASPM staining had a significantly higher risk of death after surgery than those with weak or no ASPM staining (Figure 3F). ASPM Promotes PDAC Aggressiveness To assess if ASPM plays a role in pancreatic tumorigenesis, we surveyed a panel of PDAC cell lines and found that ASPM expression was significantly upregulated in most cancer lines relative to HPDE cells (Figure 4A and B). Consistent with the cell line data, ONCOMINE and immunohistochemistry analyses revealed significantly increased transcript and protein levels of ASPM in human PDAC tissues compared with adjacent normal pancreatic ducts (Figure 4C and Supplementary Figure 5B). 21 To investigate the functional role of ASPM in PDAC cells, we stably down-regulated its expression using lentivirus-mediated RNA interference (Figure 4D). We found that knockdown of endogenous ASPM expression in primary tumor-derived PANC-1 cells by 2 different shrna vectors could significantly attenuate cellular proliferation (Figure 4E and data not shown) and migration (Figure 4F). Silencing of ASPM also compromised the proliferative and migratory capacities of metastatic AsPC-1 cells (Figure 4E and F, right panels). To further address the oncogenic role of ASPM in vivo, we stably expressed a luciferase reporter in AsPC-1 cells and orthotopically implanted them into the pancreatic tail of nonobese Figure 4. The roles of ASPM in the malignant behaviors of PDAC cells. (A) The transcript levels of ASPM in HPDE cells and various PDAC cell lines as measured by quantitative reverse transcription polymerase chain reaction analysis. (B) Western blot analysis of ASPM in HPDE and PDAC cells. b-tubulin was included as a loading control. (C) ASPM immunostaining in a representative PDAC tissue and the adjacent normal exocrine pancreatic ducts (400 magnification). Right, the intensities of ASPM were quantified using histological score. (D) Immunoblots (left) or confocal images (right) showing effect of ASPM knockdown on PDAC cells. Cells were immunostained with ASPM (red) with nuclei counterstained with 4 0,6-diamidino- 2-phenylindole (blue). Scale bars ¼ 40 mm. (E) Line graphs showing the rate of growth of control- or ASPMshRNA transduced PDAC cells. (F) Silencing of ASPM attenuated the migratory capacity of PDAC cells. Data are represented as mean SEM (n ¼ 3 6). *P <.05; **P <.01; ***P <.001 vs HPDE (A), PDAC (C) or control (E and F).

7 1116 WANG ET AL GASTROENTEROLOGY Vol. 145, No. 5 diabetic/severe combined immunodeficient mice. As shown in Figure 5, the tumors expressing the ASPM shrna grew significantly slower than the control tumors and were associated with attenuated formation of malignant ascites. The mice carrying ASPM-deficient tumors exhibited significantly prolonged survival so that they survived, on average, 37% (16.5 days) longer than the control animals (log-rank test P <.001; Figure 5D). Together, these functional studies indicate an important role of ASPM in PDAC aggressiveness. ASPM Maintains WntLb-catenin Signaling in PDAC We performed gene set enrichment analysis comparing the transcriptomes of control- and ASPMshRNA transduced PDAC cells and found the KEGG Wnt signaling pathway significantly enriched (P <.001) in the differential gene expression profile (Supplementary Figures 6A and Supplementary Figure 7). To assess if ASPM regulates Wnt pathway activity, we expressed a Wnt reporter construct in PANC-1 and AsPC-1 cells and found that silencing of ASPM dramatically blunted Wnt-mediated luciferase reporter activation (Figure 6A). b-catenin is an essential mediator of canonical Wnt signaling and its active form frequently accumulates in PDAC tissues and contributes to PDAC maintenance and metastasis We found that silencing of ASPM led to a substantial reduction in b-catenin protein level (Figure 6B), raising the possibility that ASPM can maintain Wnt pathway activity by regulating b-catenin. To address this possibility, we conducted a series of co-immunoprecipitation experiments, thereby identifying several upstream positive regulators of b-catenin, including dishevelled (Dvl)-2, axin, and protease-activated receptor-1, which interacted with endogenous ASPM (Supplementary Figure 8 and Figure 6C). Interestingly, silencing of ASPM resulted in a dramatic decrease in the protein level of Dvl-2, but not that of axin or protease-activated receptor-1, without affecting its transcript level (Figure 6B and Supplementary Figure 9B and 9C). Given that Dvl stimulates canonical Wnt signaling principally by attenuating b-catenin degradation, 25 it follows that ASPM can promotewntpathwayactivitybyincreasingtheprotein stability of Dvl-2. Consistently, confocal imaging and reciprocal co-immunoprecipitation analyses confirmed that endogenous ASPM interacted with and vastly co-localized with endogenous Dvl-2 in the cytosolic compartment near the plasma membrane in PDAC cells (Figure 6C; Supplementary Figure 9D and E). Importantly, we found that silencing of ASPM markedly increased the protein polyubiquitination of Dvl-2 (Supplementary Figure 9F), indicating that ASPM might inhibit the proteasome-dependent degradation of Figure 5. ASPM promotes pancreatic cancer aggressiveness. (A) Representative bioluminescence images (BLI) of nonobese diabetic severe combined immunodeficient mice implanted in the pancreatic tails with firefly luciferase labeled, control-, or ASPMshRNA transduced AsPC-1 cells at the indicated time points after cell inoculation. (B) Tumor bulk quantified as BLI normalized photon counts as a function of time. (C) Representative images showing presence of bloody ascites in mice implanted with controlshrna transduced AsPC-1 cells, but not in animals implanted with ASPM-shRNA transduced cells. Right, the amounts of ascites measured at 6 weeks after cell implantation. Data are represented as mean SEM (n ¼ 6 9). *P <.05; **P <.01 vs control in (B) and (C). (D) Percent survival as a function of time in mice described in (A).

8 November 2013 TUBULOGENESIS SIGNATURE IN PANCREATIC CANCER 1117 Figure 6. ASPM maintains Wnt pathway activity in PDAC. (A) Fold Wnt-mediated luciferase expression in control- or ASPM-shRNA (shrna) transduced PANC-1 or AsPC-1 cells. Data are represented as mean SEM (n ¼ 6). ***P <.001 vs control. (B) Western blot analysis of b-catenin and Dvl-2 in control- or ASPM-shRNA transduced HEK293 or PANC-1 cells. (C) Left, endogenous Dvl-2 co-immunoprecipitated with endogenous ASPM in HEK293 or PANC-1 cells. Right, representative confocal images showing co-localization of ASPM (green) and Dvl-2 (red) in PANC-1 cells. Cell nuclei were counterstained with 4 0,6-diamidino-2-phenylindole (blue). Scale bars ¼ 40 mm. (D) Control- or ASPM-shRNA transduced PANC-1 cells were treated with MG132 (10 mm for 12 h) or co-transfected with Dvl-2, after which b-catenin and Dvl-2 were detected by immunoblotting. b-tubulin was included as a loading control in (B), (C), and (D). (E) Fold changes in the Wnt reporter activity or cellular migration of control- or ASPM-shRNA transduced PANC-1 cells with or without co-expression of Dvl-2 or b-catenin (S33Y). Data are represented as mean SEM (n ¼ 6). *P <.01 vs control shrna; P <.01 vs ASPM shrna plus empty vector. (F) Representative immunostaining of human PDAC tissues with moderate ASPM/high cytoplasmic or nuclear b-catenin staining (PDAC #1) or undetectable ASPM/low cytoplasmic or nuclear b-catenin staining (PDAC #2; 400 magnification). Bottom, scatterplot of the staining intensities of ASPM vs cytoplasmic/nuclear b-catenin of 27 PDAC tissues with the linear regression line shown. Dvl-2. Indeed, treatment with a specific proteasome inhibitor or ectopic expression of Dvl-2 in ASPMdeficient cells could restore Dvl-2 and b-catenin protein levels (Figure 6D). To further address if down-regulation of Dvl-2 and/or b-catenin mediates the cellular effects induced by ASPM deficiency, we ectopically expressed Dvl-2 or a constitutively active b-catenin (S33Y) mutant in ASPM-silenced PANC-1 cells. 14 Indeed, forced expression of Dvl-2 or functional activation of b-catenin could significantly increase Wnt reporter activity and the migratory potential of ASPM-deficient cells (Figure 6E). The clinical relevance of ASPM-dependent regulation of Wnt/b-catenin pathway activity was credentialed by a strong positive correlation between the staining intensities of ASPM and cytoplasmic/nuclear b-catenin, which indicate active Wnt signaling (Supplementary Figure 10), 23 in human PDAC tissues (n ¼ 27; r ¼ 0.539, P ¼.004; Figure 6F). ASPM Maintains Pancreatic Cancer Stemness Studies have indicated a role of ASPM in regulating neural stem cells. 18,26 Consistently, a core stem cells-like gene module that is activated in human cancers was significantly enriched by gene set enrichment analysis in the gene profile associated with silencing of ASPM in PDAC cells (Supplementary Figure 6B). 27 This finding, together with the roles of Wnt signaling in stem-like cells in PDAC and gastrointestinal malignancies, 23,24,28 prompted us to investigate whether ASPM regulates pancreatic cancer stemness. To this end, we measured the proportion of

9 1118 WANG ET AL GASTROENTEROLOGY Vol. 145, No. 5 Figure 7. ASPM maintains pancreatic cancer stemness. (A) Representative plots showing patterns of CD133, CD44, CD24, and epithelialspecific antigen (ESA) staining of control- or ASPM-shRNA transduced PANC-1 cells, with the frequency of the boxed CD44 hi CD133 hi (left) or CD44 hi CD24 hi ESA hi (right) cell population as a percentage of cancer cells shown. (B) Percentages of CD44 hi CD133 hi or CD44 hi CD24 hi ESA hi cell subpopulation in control- or ASPM-shRNA transduced PANC-1 or AsPC-1 cells. (C) Transcript levels of ASPM and other stem cell markers in CD44 hi CD133 hi and CD44 lo CD133 lo PANC-1 cells as measured by quantitative reverse transcription polymerase chain reaction analysis. (D) Representative phase-contrast images of tumorspheres formed by control- or ASPM-shRNA transduced CD44 hi CD133 hi PANC-1 cells. Bars ¼ 100 mm. (E) Bar graphs showing diameters of tumorspheres in (D). (F) The percentages of CD44 hi CD133 hi cell subpopulation in control- or ASPM-shRNA transduced PANC-1 cells and those co-transduced with Dvl-2 or b-catenin (S33Y). Data are represented as mean SEM (n ¼ 3). **P <.01; ***P <.001 vs control in (B), (C), and (E). *P <.01 vs control shrna; P <.01 vs ASPM shrna plus empty vector in (F). PDAC cells that co-expressed the surface markers CD44 and CD133 or CD44, CD24, and epithelial-specificantigen, which are known to contain the enriched cancer stem cells (CSCs) in PDAC (Figure 7A). 15,29 We confirmed that these pancreatic CSCs were capable of forming death-resistant tumorspheres in vitro, exhibited tumor-initiating potentials in vivo, and profoundly responded to Wnt pathway activation or inhibition (Supplementary Figure 11). Importantly, silencing of ASPM led to a substantial reduction of the CSC subpopulation in different PDAC cells (Figure 7B). Complementing these results, we detected higher ASPM expression in pancreatic CSCs than their non-csc counterparts (Figure 7C) and found that depletion of ASPM markedly blunted CSC tumorsphere growth in a nonadherent condition (Figure 7D and E). Importantly, forced expression of Dvl-2 or b-catenin (S33Y) in ASPM-deficient PDAC cells could restore their CSC subpopulations (Figure 7F), pointing to a pivotal role of Wnt Dvl-2 b-catenin signaling in pancreatic cancer stemness regulated by ASPM. Discussion By using a physiologically relevant tissue organization model, we identified a transcript profile specific to pancreatic epithelial tubulogenesis, and we presented evidence for the favorable outcomes of PDAC patients with tumors carrying this molecular pattern. This

10 November 2013 TUBULOGENESIS SIGNATURE IN PANCREATIC CANCER 1119 biology-informed approach led to the identification of a biologically tractable and clinically instructive molecular signature for PDAC and ASPM-mediated Wnt pathway activity as a novel mechanistic link between tissue architecture, cancer stemness, and PDAC aggressiveness. Our results offer insight toward the pathogenetic significance of tissue-architectural formation in PDAC. One of the major challenges in the care of patients with localized PDAC is the ability to identify those patients who are at risk for early relapse after surgery, such that they can benefit from more aggressive therapy. However, traditional pathological grading of PDAC fails to provide an accurate prediction of tumor relapse. Recent efforts in improving classification and outcomes prediction of PDAC have been the molecular characterizations of the excised tumors. For instance, Stratford and colleagues identified a 6-gene metastasis-associated signature that was predictive of survival in patients with early-stage PDAC. 7 By comparing the transcriptomes of 27 microdissected PDAC tissues, Collisson and colleagues identified a 62-gene signature that could be used to define molecular subtypes in PDAC. 8 In this classification scheme, tumors classified as the classical subtype, which expressed epithelium-associated markers, were associated with favorable clinical outcomes. In this study, we discovered a novel tubule-like subtype of PDAC based on criteria informed by tissue architecture. The tubule-like PDAC might be partially similar to the classical subtype of PDAC, as they both exhibit the epithelial and secretory properties of exocrine pancreas. Importantly, the biologyinformed approach used in our study could provide additional mechanistic insights into PDAC differentiation or de-differentiation, permitting identification of molecular pathways related to its pathogenesis. ASPM was initially identified as a centrosomal protein that regulates neurogenesis and brain size, and it was later known to be widely expressed in a variety of normal or malignant tissues. 19,30 For example, ASPM expression positively correlated with the pathological grade of glioma and was up-regulated in recurrent tumors. 17 ASPM expression also correlated with the pathological grade and poor survival in patients with ovarian cancer or hepatocellular carcinoma. 19,20 Interestingly, ASPM was both cytoplasmic and nuclear localized in interphase and its cytoplasmic expression levels were highly variable among tumors, 19 suggesting that it might have diverse biological functions in malignant tissues. Consistently, we provided the first evidence demonstrating that ASPM is a robust poor prognostic factor in PDAC and plays a critical role in the malignant behaviors of PDAC cells as well as pancreatic cancer aggressiveness. We dissected the mechanisms underlying the oncogenic potential of ASPM in PDAC, which was attributed to its ability to promote Wnt pathway activity and cancer stemness by positively regulating Dvl-2 and b-catenin. As Wnt signaling is commonly activated in human PDAC and plays a crucial role in cancer cell proliferation, survival, and metastasis, it is plausible that ASPM-mediated Wnt Dvl-2 b-catenin signaling plays a principal role in its oncogenic functions in PDAC. Despite these findings, additional studies are required to explore how expression of ASPM is regulated by tissue architecture at the cellular and molecular levels in normal or malignant pancreatic epithelial tissues. In conclusion, this is the first study to link tissue architecture associated molecular pattern with the clinical behavior of PDAC. The study illustrates that biology-informed molecular markers can greatly enhance outcomes prediction in malignant tumors and provide valuable diagnostic and therapeutic information. Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at and at dx.doi.org/ /j.gastro References 1. Ahmad NA, Lewis JD, Ginsberg GG, et al. Long term survival after pancreatic resection for pancreatic adenocarcinoma. Am J Gastroenterol 2001;96: Oettle H, Post S, Neuhaus P, et al. Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer: a randomized controlled trial. JAMA 2007;297: Potter JD. Morphogens, morphostats, microarchitecture and malignancy. Nat Rev Cancer 2007;7: Gleason DF. 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11 1120 WANG ET AL GASTROENTEROLOGY Vol. 145, No Li C, Heidt DG, Dalerba P, et al. Identification of pancreatic cancer stem cells. Cancer Res 2007;67: Pencina MJ, D Agostino RB. Overall C as a measure of discrimination in survival analysis: model specific population value and confidence interval estimation. Stat Med 2004;23: Bikeye SN, Colin C, Marie Y, et al. ASPM-associated stem cell proliferation is involved in malignant progression of gliomas and constitutes an attractive therapeutic target. Cancer Cell Int 2010;10: Buchman JJ, Durak O, Tsai LH. ASPM regulates Wnt signaling pathway activity in the developing brain. Genes Dev 2011;25: Bruning-Richardson A, Bond J, Alsiary R, et al. ASPM and microcephalin expression in epithelial ovarian cancer correlates with tumour grade and survival. Br J Cancer 2011;104: Lin SY, Pan HW, Liu SH, et al. ASPM is a novel marker for vascular invasion, early recurrence, and poor prognosis of hepatocellular carcinoma. Clin Cancer Res 2008;14: Grutzmann R, Pilarsky C, Ammerpohl O, et al. Gene expression profiling of microdissected pancreatic ductal carcinomas using highdensity DNA microarrays. Neoplasia 2004;6: Wang L, Heidt DG, Lee CJ, et al. Oncogenic function of ATDC in pancreatic cancer through Wnt pathway activation and beta-catenin stabilization. Cancer Cell 2009;15: Pasca di Magliano M, Biankin AV, Heiser PW, et al. Common activation of canonical Wnt signaling in pancreatic adenocarcinoma. PLoS One 2007;2:e Yu M, Ting DT, Stott SL, et al. RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis. Nature 2012;487: Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell 2012;149: Horvath S, Zhang B, Carlson M, et al. Analysis of oncogenic signaling networks in glioblastoma identifies ASPM as a molecular target. Proc Natl Acad Sci U S A 2006;103: Wong DJ, Liu H, Ridky TW, et al. Module map of stem cell genes guides creation of epithelial cancer stem cells. Cell Stem Cell 2008; 2: Vermeulen L, De Sousa EMF, van der Heijden M, et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat Cell Biol 2010;12: Hermann PC, Huber SL, Herrler T, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 2007;1: Kouprina N, Pavlicek A, Collins NK, et al. The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein. Hum Mol Genet 2005;14: Author names in bold designate shared co-first authorship. Received December 5, Accepted July 24, Reprint requests Address requests for reprints to: Kelvin K. Tsai, MD, PhD, National Institute of Cancer Research, National Health Research Institutes, 367 Shengli Road, Tainan 70456, Taiwan. tsaik@nhri.org.tw; fax: þ Acknowledgments Transcript Profiling: The gene expression data have been deposited in NCBIs Gene Expression Omnibus (GEO, geo/) and are accessible through GEO Series accession number GSE Conflicts of interest The authors disclose no conflicts. Funding Supported in part by grants CA-100-PP-19 and CA-101-PP-19 from National Health Research Institutes (K. K. Tsai); National Core Facility Program for Biotechnology Grants from National Science Council (NSC B ; I.-S. Chang); NSC B MY2 (K. K. Tsai); and Development of Cancer Research System Excellence Program from Department of Health, Taiwan (DOH 100-TD-C , DOH 101- TD-C , and DOH 102-TD-C ).

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