Volume 145, Issue 5, Pages 1110-1120 (November 2013) A Gene Expression Signature of Epithelial Tubulogenesis and a Role for ASPM in Pancreatic Tumor Progression  Wei–Yu Wang, Chung–Chi Hsu, Ting–Yun Wang, Chi–Rong Li, Ya–Chin Hou, Jui–Mei Chu, Chung–Ta Lee, Ming–Sheng Liu, Jimmy J.– M. Su, Kuan–Ying Jian, Shenq–Shyang Huang, Shih–Sheng Jiang, Yan–Shen Shan, Pin–Wen Lin, Yin–Ying Shen, Michael T.–L. Lee, Tze–Sian Chan, Chun–Chao Chang, Chung–Hsing Chen, I–Shou Chang, Yen–Ling Lee, Li–Tzong Chen, Kelvin K. Tsai  Gastroenterology  Volume 145, Issue 5, Pages 1110-1120 (November 2013) DOI: 10.1053/j.gastro.2013.07.040 Copyright © 2013 AGA Institute Terms and Conditions

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 α6-integrin (red) and β-catenin (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Asterisks: cell-free lumen. Scale bars = 100 μm. (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. β-tubulin was included as a loading control. Gastroenterology 2013 145, 1110-1120DOI: (10.1053/j.gastro.2013.07.040) Copyright © 2013 AGA Institute Terms and Conditions

Figure 2 Identification of a tubulogenesis-specific gene signature in PDAC. (A) An illustration depicting the derivation of rtubules. (B) Kaplan-Meier survival curve comparing postoperative survival of PDAC patients with high or low rtubules 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 proportional-hazards 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. Gastroenterology 2013 145, 1110-1120DOI: (10.1053/j.gastro.2013.07.040) Copyright © 2013 AGA Institute Terms and Conditions

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 = 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. Gastroenterology 2013 145, 1110-1120DOI: (10.1053/j.gastro.2013.07.040) Copyright © 2013 AGA Institute Terms and Conditions

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. β-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′,6-diamidino-2-phenylindole (blue). Scale bars = 40 μm. (E) Line graphs showing the rate of growth of control- or ASPM-shRNA−transduced PDAC cells. (F) Silencing of ASPM attenuated the migratory capacity of PDAC cells. Data are represented as mean ± SEM (n = 3−6). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 vs HPDE (A), PDAC (C) or control (E and F). Gastroenterology 2013 145, 1110-1120DOI: (10.1053/j.gastro.2013.07.040) Copyright © 2013 AGA Institute Terms and Conditions

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 ASPM-shRNA−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 control-shRNA−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 = 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). Gastroenterology 2013 145, 1110-1120DOI: (10.1053/j.gastro.2013.07.040) Copyright © 2013 AGA Institute Terms and Conditions

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 = 6). ∗∗∗P < .001 vs control. (B) Western blot analysis of β-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′,6-diamidino-2-phenylindole (blue). Scale bars = 40 μm. (D) Control- or ASPM-shRNA−transduced PANC-1 cells were treated with MG132 (10 μM for 12 h) or co-transfected with Dvl-2, after which β-catenin and Dvl-2 were detected by immunoblotting. β-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 β-catenin (S33Y). Data are represented as mean ± SEM (n = 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 β-catenin staining (PDAC #1) or undetectable ASPM/low cytoplasmic or nuclear β-catenin staining (PDAC #2; 400× magnification). Bottom, scatterplot of the staining intensities of ASPM vs cytoplasmic/nuclear β-catenin of 27 PDAC tissues with the linear regression line shown. Gastroenterology 2013 145, 1110-1120DOI: (10.1053/j.gastro.2013.07.040) Copyright © 2013 AGA Institute Terms and Conditions

Figure 7 ASPM maintains pancreatic cancer stemness. (A) Representative plots showing patterns of CD133, CD44, CD24, and epithelial-specific antigen (ESA) staining of control- or ASPM-shRNA−transduced PANC-1 cells, with the frequency of the boxed CD44hiCD133hi (left) or CD44hiCD24hiESAhi (right) cell population as a percentage of cancer cells shown. (B) Percentages of CD44hiCD133hi or CD44hiCD24hiESAhi 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 CD44hiCD133hi and CD44loCD133lo 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 CD44hiCD133hi PANC-1 cells. Bars = 100 μm. (E) Bar graphs showing diameters of tumorspheres in (D). (F) The percentages of CD44hiCD133hi cell subpopulation in control- or ASPM-shRNA−transduced PANC-1 cells and those co-transduced with Dvl-2 or β-catenin (S33Y). Data are represented as mean ± SEM (n = 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). Gastroenterology 2013 145, 1110-1120DOI: (10.1053/j.gastro.2013.07.040) Copyright © 2013 AGA Institute Terms and Conditions