1. Cancer Biology
  2. Stem Cells and Regenerative Medicine
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HNF1A is a novel oncogene that regulates human pancreatic cancer stem cell properties

  1. Ethan V Abel
  2. Masashi Goto
  3. Brian Magnuson
  4. Saji Abraham
  5. Nikita Ramanathan
  6. Emily Hotaling
  7. Anthony A Alaniz
  8. Chandan Kumar-Sinha
  9. Michele L Dziubinski
  10. Sumithra Urs
  11. Lidong Wang
  12. Jiaqi Shi
  13. Meghna Waghray
  14. Mats Ljungman
  15. Howard C Crawford
  16. Diane M Simeone  Is a corresponding author
  1. University of Michigan Health System, United States
  2. New York University Langone Health, United States
  3. New York University Langone Health, United states
Research Article
Cite this article as: eLife 2018;7:e33947 doi: 10.7554/eLife.33947
10 figures, 1 table and 3 additional files

Figures

Figure 1 with 2 supplements
HNF1A-signature dominates pancreatic CSCs.

(A) Flow cytometry analysis of CD44 and EPCAM surface expression of three primary PDA samples. (B) CD44High/EPCAMLow (P1), CD44High/EPCAMHigh (P2) and CD44Low/EPCAMHigh (P3) NY8 cells were isolated by FACS and grown in culture for 17 days, followed by flow cytometry for analysis for CD44 and EPCAM expression. (C, D) Isolated subpopulations were grown in tumorsphere media on non-adherent plates (500 cells/well) for 6 days. Representative images of resultant tumorspheres (100X magnification) are shown in (C), while quantitation of spheres (n = 3) is shown in (D). Statistical difference was determined by one-way ANOVA with Tukey’s multiple comparisons test. (E) Heat map representing relative fold differences in qRT-PCR expression of 50 cancer stem-cell-enriched genes in NY8 and NY15 cells. Per-gene values are relative to P1 or P3, whichever is higher. Gene names in red text indicate predicted HNF1A targets and asterisks (*) indicate known HNF1A targets. P1: CD44High/EPCAMLow, P2: CD44High/EPCAMHigh, P3: CD44Low/EPCAMHigh. For all genes, expression levels were normalized to an ACTB mRNA control, n = 3. Only genes with a significant (p<0.05) increase in P2 over both P1 and P3 subpopulations are shown, with statistical difference determined by one-way ANOVA with Tukey’s multiple comparisons test. (F) qRT-PCR analysis of HNF1A mRNA expression, normalized to an ACTB mRNA control, from different primary PDA subpopulations (n = 3). Statistical difference was determined by one-way ANOVA with Tukey’s multiple comparisons test. Related data can be found in Figure 1—figure supplements 1 and 2.

https://doi.org/10.7554/eLife.33947.003
Figure 1—source data 1

Quantitation of tumorspheres, P2 subpopulation-enriched transcripts, and HNF1A mRNA.

https://doi.org/10.7554/eLife.33947.006
Figure 1—source data 2

Quantitation of GFP expression in adherent cells and tumorspheres.

https://doi.org/10.7554/eLife.33947.007
Figure 1—figure supplement 1
Cancer stem cell properties of PDA cell subpopulations.

(A) Co-localization of CD24High surface expression with EPCAM and CD44 expression is highlighted in red. (B) Western blot analysis of HNF1A and target genes CDH17 and DPP4, as well as CD44 and EPCAM as subpopulation controls in sorted PDA cells. Quantitation of proteins is indicated below the respective blots.

https://doi.org/10.7554/eLife.33947.004
Figure 1—figure supplement 2
HNF1A is elevated in tumorspheres.

(A) NY8 and NY15 cells were grown either in non-adherent conditions (labeled S) or under adherent conditions (labeled A) for 7 days. Lysates were collected and western blotted for HNF1A, CDH17, and Actin. Quantitation of proteins is indicated below the respective blots. (B) Schematic representation of HNF1A-responsive reporter construct showing eight tandem copies of the HNF1A-binding site upstream of a minimal promoter (pTA) driving GFP expression. (C) HNF1A-reporter-labeled NY5, NY8, and NY15 cells were grown under adherent (A) or tumorsphere (S) conditions for 7 days, lysed and immunoblotted for HNF1A, CDH17, GFP, and Actin. Quantitation of proteins is indicated below the respective blots. (D) Reporter cells were transfected for 7 days with control (Ctl) or HNF1A-targeting siRNA, lysed and immunoblotted for HNF1A, CDH17, GFP, and Actin. Quantitation of proteins is indicated below the respective blots. (E, F) NY15 reporter cells grown under adherent or tumorsphere conditions were collected by trypsinization and analyzed by flow cytometry for GFP fluorescence and EPCAM, CD44, and CD24 surface staining. Representative flow cytometry plots and quantitation of GFP expression are shown in the upper and lower panel of (E), respectively. Statistical difference was determined by unpaired t test with Welch's correction (n = 3). (F) Flow cytometry plots showing co-localization of GFP with EPCAM, CD44, CD24 are shown. The percentage of CD44High/EPCAMHigh/GFP+ or CD44High/CD24High/GFP+ cells is indicated as a mean ± SEM (n = 3). Statistical difference between adherent and sphere cells was determined by unpaired t test with Welch's correction, and respective p values are indicated.

https://doi.org/10.7554/eLife.33947.005
Figure 2 with 1 supplement
Knockdown of HNF1A in primary PDA cells inhibits growth in vitro.

(A) Western blot analysis of HNF1A expression in a panel of primary PDA lines compared to immortalized pancreatic ductal cell line HPNE and HPDE. Quantitation of HNF1A protein is indicated below the respective blots. (B) Western blot of NY5, NY8, and NY15 cells transfected with non-targeting (Ctl) or HNF1A-targeting siRNA for 3 days, showing effective depletion of HNF1A protein by RNAi. Quantitation of HNF1A protein is indicated below the respective blots. (C) 1.5 × 105 PDA cells were transfected with control (Ctl) or HNF1A-targeting siRNA (Day 1). Cells were collected and manually counted 3 and 6 days after transfection (n = 3). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. Red and green p values indicate Ctl vs. HNF1A#1 or #2, respectively. (D) Annexin V staining was performed on NY5, NY8, and NY15 cells transfected with control (Ctl) or HNF1A-targeting siRNA (H1, H2) for 3 days. The amount of apoptotic (annexin V+) cells are quantitated (n = 4). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test, with p values relative to the control siRNA group indicated. (E) Western blot analysis of cleaved caspases in NY8 and NY15 cells following HNF1A-knockdown (3 days). Actin serves as a loading control. Quantitation of proteins is indicated below the respective blots. Related data can be found in Figure 2—figure supplement 1.

https://doi.org/10.7554/eLife.33947.008
Figure 2—source data 1

Quantitation of PDA cell growth and apoptosis following HNF1A knockdown.

https://doi.org/10.7554/eLife.33947.010
Figure 2—source data 2

Quantitative PCR analysis of HNF1A mRNA in PDA cells and histology score of HNF1A staining in normal and neoplastic ducts..

https://doi.org/10.7554/eLife.33947.011
Figure 2—figure supplement 1
HNF1A expression in PDA cells and patient samples.

(A) qRT-PCR analysis of HNF1A mRNA expression in a panel of conventional and primary PDA lines. HNF1A mRNA expression was normalized to an ACTB internal control and relative to HPNE. Relative HNF1A mRNA expression is indicated as a mean ± SEM, (n = 3). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test using HPNE as the control group. n.s. = non significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (B, C) A pancreatic cancer tissue microarray (TMA) consisting of 41 PDA samples and 18 normal pancreas controls was immunostained for HNF1A. Representative hematoxylin and eosin staining and HNF1A staining of normal pancreas and PDA are shown in (B). Histology scores (HNF1A staining intensity x % HNF1A positive cells) for normal pancreatic ducts (n = 18) versus PDA (n = 41) is shown in (C). Statistical difference was determined by unpaired t test with Welch's correction.

https://doi.org/10.7554/eLife.33947.009
Figure 3 with 1 supplement
Knockdown of HNF1A depletes CSC numbers.

(A) Multiple PDA cells were transfected with HNF1A-targeting siRNA or non-targeting control siRNA for 6 days. Surface expression of CD44 and EPCAM was measured by flow cytometry, and the percentage of CD44High/EPCAMHigh (P2) cells are represented (mean ± SEM, n = 3). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (B) Quantitation of CD24 +cells in multiple primary PDA cells following HNF1A knockdown for 6 days, n = 4. Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (C) NY5, NY8, and NY15 cells expressing LacZ2.1 (L) or two distinct HNF1A-targeting shRNAs (H1 and H2) were lysed and western blotted for HNF1A, CDH17, and Actin, showing effective knockdown of HNF1A and downstream signaling (CDH17). Quantitation of proteins is indicated below the respective blots. (D, E) NY5, NY8, and NY15 cells expressing LacZ2.1 or HNF1A-targeting shRNAs were grown in tumorsphere media on non-adherent plates (1500 cells/well). The number of tumorspheres formed after 6 days were counted (n = 3). Representative images of spheres (100X magnification) are shown in (F) and in Figure 3—figure supplement 1, with quantitation in (E). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. Related data can be found in Figure 3—figure supplement 1.

https://doi.org/10.7554/eLife.33947.012
Figure 3—source data 1

Quantitation of the P2 subpopulation, CD24 expression, and tumorsphere formation following HNF1A knockdown.

https://doi.org/10.7554/eLife.33947.014
Figure 3—figure supplement 1
Knockdown of HNF1A depletes CSC numbers and properties in vitro.

(A) Surface expression of CD44 and EPCAM in multiple primary PDA cells following HNF1A knockdown. Percentages of P2 cells are indicated. (B, C) Surface expression of CD44 and EPCAM was measured by flow cytometry, and the percentage of (B) CD44High/EPCAMLow (P1) cells and (C) CD44Low/EPCAMHigh (P3) cells are represented (mean ± SEM, n = 3). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (D) Representative CD24 and EPCAM surface staining in cells following HNF1A knockdown (6 days). (E) NY5 cells expressing LacZ2.1 or HNF1A-targeting shRNAs were grown in tumorsphere media on non-adherent plates (1500 cells/well). Representative images of spheres (100X magnification) are shown.

https://doi.org/10.7554/eLife.33947.013
Figure 4 with 1 supplement
Overexpression of HNF1A promotes CSC properties in PDA cells and normal pancreatic cell lines.

(A) NY15 and NY53 cells Western blotted for HNF1A and control gene induction following 48 hr ± doxycycline (Dox). Quantitation of proteins is indicated below the respective blots. (B) NY8 cells were treated 48 hr ± Dox to induce ectopic HNF1A. Lysates were western blotted for HNF1A, Actin, and PCSC markers EPCAM and CD44. Quantitation of proteins is indicated below the respective blots. (C) Representative surface expression of CD24 and EPCAM on NY15 cells expressing GFP or HNF1A. (D) Quantitation of CD24 +NY15 GFP and HNF1A cells and NY8 and NY53 LacZ and HNF1A cells by flow cytometry (n = 3). Statistical difference was determined by one-way ANOVA with Tukey’s multiple comparisons test. (E) NY15 GFP and HNF1A, NY8 and NY53 LacZ and HNF1A cells were grown under sphere-forming conditions ± Dox. The number of tumorspheres formed after 7 days were quantitated (n = 4). Statistical difference was determined by one-way ANOVA with Tukey’s multiple comparisons test. Representative images (100X magnification) of spheres are shown in the upper panels. (F, G) HPNE LacZ and HNF1A cells were plated at 200 cells/6 cm dish and treated ±Dox for 2 weeks, fixed, and stained with crystal violet (F). (G) Resultant colonies were quantitated (n = 3). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (H, I) HPDE cells expressing inducible LacZ, LacZ with KRASG12D, HNF1A, or HNF1A with KRASG12D were embedded in soft agar + Dox and monitored for signs of anchorage-independent growth for 21 days. (H) Representative images of resultant colonies (100X magnification) and (I) quantitation of colonies after 21 days (n = 3). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. Related data can be found in Figure 4—figure supplement 1.

https://doi.org/10.7554/eLife.33947.015
Figure 4—source data 1

Quantitation of CD24 expression and tumorsphere formation in PDA cells with HNF1A overexpression, and quantitation of colony formation in HPNE and HPDE cells expressing HNF1A and oncogenic KRAS.

https://doi.org/10.7554/eLife.33947.017
Figure 4—source data 2

Quantitation of CD44+/CD24+ HPDE and HPNE cells overexpressing HNF1A.

https://doi.org/10.7554/eLife.33947.018
Figure 4—figure supplement 1
Overexpression of HNF1A and mutant KRAS in HPDE and HPNE cell.

(A) HPDE and HPNE cells expressing LacZ (L), KRASG12D (K), HNF1A (H), or HNF1A with KRASG12D (HK) were analyzed by western blot to confirm transgene expression and activation of downstream signaling events. Quantitation of proteins is indicated below the respective blots. For doxycycline-inducible HPDE cells, cells were treated ± doxycycline (Dox) (100 ng/ml) for 7 days prior to analysis. (B) HPDE cells expressing inducible LacZ, HNF1A, LacZ with KRASG12D, or HNF1A with KRASG12D were grown to super-confluency (2 weeks) in the presence of 10% FBS + Dox to test for changes in contact-inhibition. Cells were fixed and stained with crystal violet. Representative images (100X magnification) are shown. (C) Flow cytometry analysis of CD44 and CD24 surface expression on HPDE cells expressing LacZ or HNF1A for 7 days or HPNE cells expressing LacZ or HNF1A. The percentage of CD44+/CD24+ cells is indicated as a mean ± SEM (n = 3). Statistical difference between adherent and sphere cells was determined by unpaired t test with Welch's correction, and respective p values are indicated.

https://doi.org/10.7554/eLife.33947.016
Figure 5 with 1 supplement
Knockdown of HNF1A impairs tumor growth and depletes CSCs in vivo.

(A, B) 10,000 firefly luciferase-labeled NY5 and NY15 cells expressing control or HNF1A shRNAs were implanted orthotopically into the pancreata of NOD/SCID mice and monitored by IVIS imaging for 6 weeks (10 mice per group). Representative luminescence image of tumors prior to sacrifice is shown (A). Final tumor volumes determined during necropsy are quantitated in (B). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (C) 103 control or HNF1A-depleted NY5 cells were implanted subcutaneously in NOD/SCID mice (10 mice per shRNA/bilateral injections) for 11 weeks. Tumors were measured by caliper to determine tumor growth (C, upper panel). Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. Red and orange p values indicate LacZ2.1 vs. HNF1A#1 or #2, respectively. ‘n.d.’ indicates that tumors were not detected. Representative tumors excised at sacrifice are shown (C, lower panel). (D, E) NY5 tumors from (A) were dissociated and stained for EPCAM, CD44, and CD24. Representative flow cytometry plots for recovered tumor cells are shown in (D), where the R22 gate denotes EPCAMHigh/CD44High cells, and CD24 +cells are donated in red. Quantitation of EPCAM+/CD44+/CD24 +cells is shown in (E), n = 6 tumors each for shRNA. Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. Related data can be found in Figure 5—figure supplement 1.

https://doi.org/10.7554/eLife.33947.019
Figure 5—source data 1

Quantitation of orthotopic and subcutaneous xenograft tumor volumes, and quantitation of PCSCs following HNF1A knockdown.

https://doi.org/10.7554/eLife.33947.021
Figure 5—source data 2

Quantitation of subcutaneous xenograft tumor volumes following HNF1A knockdown.

https://doi.org/10.7554/eLife.33947.022
Figure 5—figure supplement 1
Effects of HNF1A depletion on PDA xenograft biology.

(A) 103 control or HNF1A-depleted NY15 cells were implanted subcutaneously in NOD/SCID mice (10 mice per shRNA/bilateral injections) for 6 weeks. Tumors were measured by caliper to determine tumor growth. Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. Red and green p values indicate LacZ2.1 vs. HNF1A#1 or #2, respectively. ‘n.d.’ indicates that tumors were not detected. Representative tumors are shown (lower panel). (B) Western blot analysis of HNF1A and Actin from NY5 tumors expressing LacZ2.1 or HNF1A shRNAs (four tumors per group) demonstrating persistent knockdown in vivo. Quantitation of HNF1A is indicated below the blot.

https://doi.org/10.7554/eLife.33947.020
Figure 6 with 2 supplements
HNF1A regulates stemness through POU5F1/OCT4 regulation.

(A) qRT-PCR analysis of POU5F1/OCT4 mRNA in NY5, NY8 and NY15 cells expressing control (LacZ2.1) or HNF1A shRNAs. ACTB was used as an internal control, n = 3. Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (B) LacZ or HNF1A was induced in NY8, NY15 or HPDE cells with doxycycline for 6 days. Levels of POU5F1/OCT4 mRNA were measured by qRT-PCR with ACTB as an internal control, n = 3. Statistical difference was determined by unpaired t test with Welch’s correction. (C) Pearson correlation coefficient of POU5F1/OCT4 and HNF1A mRNA levels from NY PDA cells (n = 22, red) relative to HPNE and HPDE (blue) cells. (D) Western blot of OCT4A and HNF1A protein in NY5 and NY8 cells transfected with POU5F1/OCT4 (labeled OCT4) or HNF1A SMARTpool siRNA for 3 days. Quantitation of proteins is indicated below the respective blots. (E) NY5 and NY8 cells were transfected with HNF1A or POU5F1/OCT4 SMARTpool siRNA for 3 days and then grown in tumorsphere media on non-adherent plates (1500 cells/well). Spheres were quantitated 7 days later, n = 3. Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (F–H) NY15 cells were transfected with POU5F1/OCT4 SMARTpool (SP) siRNA or individual sequences for 3 days and either harvested to assess OCT4A knockdown by Western blot (F) or grown in tumorsphere media on non-adherent plates (1500 cells/well) (G). Spheres were quantitated 7 days later, n = 3. Statistical difference was determined by one-way ANOVA with Dunnett’s multiple comparisons test. Representative spheres are shown in (H). (I) NY8 and NY15 cells transduced with OCT4A (labeled OCT4) or empty vector control (EV) were transiently transfected with control (Ctl) or HNF1A-targeting siRNA for 72 hr, and then grown in tumorsphere media on non-adherent plates (1500 cells/well). Spheres were quantitated 7 days later, n = 3. Statistical difference was determined by one-way ANOVA with Tukey’s multiple comparisons test. Related data can be found in Figure 6—figure supplements 1 and 2.

https://doi.org/10.7554/eLife.33947.023
Figure 6—source data 1

Quantitation of OCT4/POU5F1 mRNA following HNF1A knockdown and overexpression; relative HNF1A and OCT4/POU5F1 mRNA expressions in PDA cells; quantitation of tumorspheres following OCT4/POU5F1 knockdown; and quantitation of tumorsphere formation following OCT4A rescue.

https://doi.org/10.7554/eLife.33947.026
Figure 6—source data 2

Quantitation of ChIP, CLuc activity, annexin V staining, PI staining, and tumorsphere formation.

https://doi.org/10.7554/eLife.33947.027
Figure 6—figure supplement 1
HNF1A binds directly to and activates transcription from the POU5F1/OCT4 upstream LTR region.

(A) Schematic representation of the POU5F1/OCT4 locus, including the canonical exon 1 (E1), distal enhancer region (DE), and the cryptic exon E0 and upstream LTR regions reported in Malakootian et al. (2017). Putative HNF1A half-sites (HS) are indicated by purple triangles. (B) Chromatin immunoprecipitation (ChIP) was performed in NY5, NY8, and NY15 cells using and HNF1A-specific antibody or normal IgG control. Enrichment of the MYOD promoter (negative control), the POU5F1/OCT4 distal enhancer (DE), or the three HNF1A half-sites (HS1-3) was measured by quantitative PCR and normalized to a 2% input control. Statistical difference was determined by one-way ANOVA with Tukey’s multiple comparisons test, and comparisons to enrichment of MYOD by HNF1A are shown. (C) Schematic representation of Cypridina luciferase (CLuc) reporter constructs, including the cloning vector, a reporter containing the LTR from Malakootian et al. (2017) expanded to include the third HNF1A half-site, and a containing the fulllenght canonical POU5F1/OCT4 promoter region. Relative positions of HNF1A half-sites (HS) and the distal enhancer (DE) are indicated. (D) 293FT cells were transfected with 0.5 µg of either the CLuc cloning vector, LTR reporter, or canonical promoter reporter in combination with 0.5 µg of either LacZ or HNF1A expression plasmid and 0.05 µg of a HSV-TK promoter-driven Gaussia-Dura luciferase (GDLuc) expression plasmid for normalization. After 48 hr, conditioned media was collected and secreted CLuc and GDLuc activity was measured. Fold change in CLuc luminescence from HNF1A-transfected cells relative to LacZ-transfected cells is shown (mean ± SEM, n = 3). Statistical difference was determined by one-way ANOVA with Tukey’s multiple comparisons test.

https://doi.org/10.7554/eLife.33947.024
Figure 6—figure supplement 2
Rescue of POU5F1/OCT4 expression in PDA cells.

(A, B) NY8 cells were transiently transfected with control (Ctl) or POU5F1/OCT4-targeting (labeled OCT4) siRNA for 72 hr, followed by (A) annexin V/DAPI-staining or (B) propidium iodide (PI) staining to quantitate apoptotic and cycling cells, respectively. Data is represented as the mean for each group ± SEM, (n = 3). Statistical difference between adherent and sphere cells was determined by unpaired t test with Welch's correction, and respective p values are indicated. (C) NY8 cells transduced with OCT4A or empty vector control (EV) were transiently transfected with control (Ctl) or HNF1A-targeting siRNA for 72 hr, and then grown in tumorsphere media on non-adherent plates (1500 cells/well). Representative images of tumorspheres are shown. (D) Quantitative RT-PCR for POU5F1/OCT4 transcript was measured in NY8 and NY15 empty vector (EV) or OCT4A-transduced cells transfected with control or HNF1A siRNA for 72 hr. ACTB transcript was used as an internal control, n = 3. Statistical difference was determined by one-way ANOVA with Tukey’s multiple comparisons test.

https://doi.org/10.7554/eLife.33947.025
Figure 7 with 1 supplement
HNF1A regulates a transcriptional program associated with poor survival in PDA.

(A) Venn diagrams illustrating overlapping genes with altered transcription (Bru-seq) following HNF1A knockdown in NY8 and NY15 cells. ‘HNF1A upregulated’ genes denote genes that were downregulated by HNF1A shRNAs, while ‘HNF1A downregulated’ genes were upregulated by HNF1A shRNAs. Cells expressed shRNAs constitutively for >14 days prior to Bru-seq analysis. (B) Proportion of HNF1A shRNA-downregulated genes identified in both NY8 and NY15 with HNF1A ChIP-seq peaks. Proximal peaks are ±5 kbp of the transcription start site (TSS) of a given gene and distal peaks are ±100 kbp of a TSS. Peaks are recognized only if they are closer to the TSS of a given gene than to other expressed genes. Peaks overlapping putative enhancer regions (ENCODE) are indicated in dark grey. (C) HNF1A ChIP-seq and HNF1A shRNA Bru-seq traces for the genes CDH17 and EPCAM in NY8 and NY15 cells. Traces represent normalized read coverage (in RPKM) across the indicated genomic ranges. MACS-identified ChIP peaks are represented by bars under the corresponding trace. (D) Transcription factor (TF) motif over-representation analysis of HNF1A upregulated and downregulated genes (±5 kbp of TSS). The top 10 over-represented TF motifs, ranked by z-score, are listed. (E) HNF1A upregulated and bound genes were ranked according to model significance and the direction of survival association using TCGA PDA patient data. Magnitude indicates significance (log10-transformed FDR-adjusted Wald p values for Cox PH models) and sign represents survival direction (determined by hazard ratio). Red bars indicate FDR < 0.1, orange bars indicate FDR < 0.25, and gray bars are not significant. The FDR thresholds are also indicated by dotted horizontal lines. Insets: each histogram represents the null distribution of a permutation test (N = 10,000) for fraction of genes significantly associated with reduced survival (the tests use FDR thresholds of 0.1 or 0.25, as indicated). Vertical lines represent values for the set of HNF1A target genes; red: FDR < 0.1 test; orange: FDR < 0.25 test. * - indicates significant p value estimates for the permutation tests. Related data found in Figure 7—figure supplement 1.

https://doi.org/10.7554/eLife.33947.028
Figure 7—figure supplement 1
Association of HNF1A-responsive genes and survival in PDA.

(A, B) HNF1A-responsive genes were ranked according FDR-adjusted Wald p value and the direction of survival association using TCGA PDA patient data for (A) upregulated genes (including bound and unbound) and (B) downregulated genes. Each bar represents the Cox proportional hazards model for one gene; its magnitude indicates significance (log10-transformed adjusted p values), and its sign represents the hazard ratio (a negative value means that HR >1 and hence reduced survival). Red bars indicate FDR < 0.1, orange bars indicate FDR < 0.25, and gray bars are not significant. The FDR thresholds are also indicated by dotted horizontal lines. Permutation test insets: the histograms represent the null distribution for fraction of genes significantly associated with either increased or reduced survival and at FDR < 0.1 or FDR < 0.25 (as indicated). Vertical lines represent the value for the set of HNF1A upregulated or downregulated genes, respectively; red for the FDR < 0.1 test, orange for the FDR < 0.25 test). * - indicates significant p values for the permutation tests. N = 10,000 for all permutations and the estimated error of p at α = 0.05 is ±0.0034.

https://doi.org/10.7554/eLife.33947.029
Author response image 1
Comparison of TCGA and ICGC survival analyses.
https://doi.org/10.7554/eLife.33947.034
Author response image 2
Comparison of TCGA and ICGC background gene concordance.

Overlap of all genes is on the left, “UP” refers to increased survival genes, “DN” to reduced survival genes.

https://doi.org/10.7554/eLife.33947.035
Author response image 3
Correlation of HNF1A and POU5F1 expression.
https://doi.org/10.7554/eLife.33947.036

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Human)HNF1AThis paperCloned from NY5 cDNA
Gene
(Escherichia coli)
LacZInvitrogenOriginally from Catalog
number: K499000
Subcloned into
pLentipuro3/TO/V5-DEST
Gene
(Aequorea victoria)
PatGFPThis paperVariant of EGFP containing
the following mutations:
S31R, Y40N, S73A, F100S,
N106T, Y146F, N150K, M154T,
V164A, I168T, I172V, A207V
Gene (Human)KRAS G12DThis paperCloned from NY5 cDNA
Gene (Human)POU5F1 (OCT4A)Transomic
Technologies
Catalog number:
BC117435
Subcloned into pLenti6.3
/UbC/V5-DEST
Gene
(Escherichia coli)
LacZ2.1 shRNAThis paperSequence: CACCAAATCGCTGATTT
GTGTAGTCGTTCAAGAGACGACT
ACACAAATCAGCGA
Gene (Human)HNF1A shRNA#1This paperSequence: CACCGCTAGTGGAGGA
GTGCAATTTCAAGAGAATTGCACTC
CTCCACTAGC
Gene (Human)HNF1A shRNA#2This paperSequence: CACCGTCCCTTAGTGA
CAGTGTCTATTCAAGAGATAGA
CACTGTCACTAAGGGAC
Gene
(Escherichia coli)
IVS-TetR-P2A-BsdThis paperIVS-TetR and Bsd were
subcloned from pLenti6/TR
(Invitrogen) with a P2A
peptide linker added by PCR
and Gibson Assembly
Gene
(Aequorea victoria)
PatGFP-Luc2This paperPatGFP and Luc2 (Promega)
were amplified by PCR and
fused by Gibson Assembly
Strain, strain
background
(Mouse)
NOD.CB17-Prkdcscid/JThe Jackson
Laboratory
Catalog number:
001303; RRID:
IMSR_JAX:001303
Cell line (Human)HPDECraig Logsdon,
MD Anderson
Cell line (Human)HPNEATCCCatalog number:
ATCC CRL-4023;
RRID:CVCL_C466
Cell line (Human)Capan-2ATCCCatalog number:
ATCC HTB-80;
RRID:CVCL_0026
Cell line (Human)HPAF-IIATCCCatalog number:
ATCC CRL-1997;
RRID:CVCL_0313
Cell line (Human)BxPC-3ATCCCatalog number:
ATCC CRL-1687;
RRID:CVCL_0186
Cell line (Human)AsPC-1ATCCCatalog number:
ATCC CRL-1682;
RRID:CVCL_0152
Cell line (Human)MiaPaCa-2ATCCCatalog number:
ATCC CRL-1420;
RRID:CVCL_0428
Cell line (Human)Panc-1ATCCCatalog number:
ATCC CRL-1469;
RRID:CVCL_0480
Cell line (Human)NY1This paperLow passage pancreatic
adenocarcinoma patient
primary cell line
established from xenograft
Cell line (Human)NY2This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY3This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY5This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY6This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY8This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY9This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY12This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY15This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY16This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY17This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY19This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY28This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY32This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)NY53This paperLow passage pancreatic
adenocarcinoma patient
primary cell line established
from xenograft
Cell line (Human)293FTInvitrogenCatalog number:
R70007
Transfected
construct (Gaussia)
pTK-GDLucThis paperThe Gaussia coding region
of pTK-Gluc (New England
Biolabs) was replaced with
the Gaussia Dura coding
region (Millipore)
Transfected
construct (Cypridina)
pCLuc-Basic2New England
Biolabs
Catalog number:
N0317S
Transfected
construct (Cypridina)
pCLuc-Basic2/OCT4
LTR promoter
This paper1.7 kbp OCT4 LTR
promoter region from NY5
was subcloned into
pCLuc-Basic2
Transfected
construct (Cypridina)
pCLuc-Basic2/OCT4
canonical promoter
This paper/AddgeneOriginally from
Catalog number:
38776
OCT4 promoter from
phOct4-EGFP (Addgene)
was subcloned into
pCLuc-Basic2
AntibodyCD326 (EpCAM)-FITCMiltenyi BiotecCatalog number:
130-113-263;
RRID:AB_2726064
Application:
flow cytometry
AntibodyBD Pharmingen APC
Mouse Anti-Human
CD44
BD BiosciencesCatalog number:
559942;
RRID:AB_398683
Application:
flow cytometry
AntibodyBD Pharmingen
PE Mouse Anti-Human
CD24
BD BiosciencesCatalog number:
555428;
RRID:AB_395822
Application:
flow cytometry
AntibodyH-2Kd/H-2Dd
clone 34-1-2S
SouthernBiotechCatalog number:
1911–08;
RRID:AB_1085008
Application:
flow cytometry
AntibodyAnti-HNF1 antibody
[GT4110]
AbcamCatalog number:
ab184194;
RRID:AB_2538735
Application:
IHC, Western blot
AntibodyHNF-1 alpha Antibody
(C-19)
Santa Cruz
Biotechnology
Catalog number:
sc-6547;
RRID:AB_648295
ChIP
AntibodyNormal Rabbit IgGCell Signaling
Technology
Catalog number:
2729S;
RRID:AB_1031062
ChIP
AntibodyHNF1α (D7Z2Q)Cell Signaling
Technology
Catalog number:
89670S;
RRID:AB_2728751
Application:
Western blot
Antibodyβ-Actin (clone AC-74)Sigma AldrichCatalog number:
A2228-200UL;
RRID:AB_476697
Application:
Western blot
AntibodyCDH17 antibodyProteintechCatalog number:
50-608-369;
RRID:AB_2728752
Application:
Western blot
AntibodyDPP4/CD26 (D6D8K)Cell Signaling
Technology
Catalog number:
67138S;
RRID:AB_2728750
Application:
Western blot
AntibodyCD44 (156–3 C11)Cell Signaling
Technology
Catalog number:
3570S;
RRID:AB_10693293
Application:
Western blot
AntibodyEpCAM (D1B3)Cell Signaling
Technology
Catalog number:
2626S;
RRID:AB_2728749
Application:
Western blot
AntibodyCleaved Caspase-3
(Asp175) (5A1E)
Cell Signaling
Technology
Catalog number:
9664S;
RRID:AB_2070042
Application:
Western blot
AntibodyCleaved Caspase-6
(Asp162)
Cell Signaling
Technology
Catalog number:
9761S;
RRID:AB_2290879
Application:
Western blot
AntibodyCleaved Caspase-7
(Asp198) (D6H1)
Cell Signaling
Technology
Catalog number:
8438S; R
RID:AB_11178377
Application:
Western blot
AntibodyCleaved Caspase-9
(Asp330) (D2D4)
Cell Signaling
Technology
Catalog number:
7237S;
RRID:AB_10895832
Application:
Western blot
AntibodyCleaved Caspase-9
(Asp315) Antibody
Cell Signaling
Technology
Catalog number:
9505S;
RRID:AB_2290727
Application:
Western blot
AntibodyGFP (D5.1) XPCell Signaling
Technology
Catalog number:
2956S;
RRID:AB_1196615
Application:
Western blot
AntibodyRas (G12D Mutant
Specific) (D8H7)
Cell Signaling
Technology
Catalog number:
14429S;
RRID:AB_2728748
Application:
Western blot
AntibodyPhospho-p44/42 MAPK
(Erk1/2)
(Thr202/Tyr204)
(D13.14.4E) XP
Cell Signaling
Technology
Catalog number:
4370S;
RRID:AB_2315112
Application:
Western blot
AntibodyPhospho-Akt (Ser473)
(D9E) XP
Cell Signaling
Technology
Catalog number:
4060S;
RRID:AB_2315049
Application:
Western blot
AntibodyOct-4A (C52G3)Cell Signaling
Technology
Catalog number:
2890S;
RRID:AB_2167725
Application:
Western blot
AntibodyAnti-KRAS + HRAS +
NRAS antibody
AbcamCatalog number:
ab55391;
RRID:AB_941040
Application:
Western blot
AntibodyAnti-β-GalactosidasePromegaCatalog number:
Z3781;
RRID:AB_430877
Application:
Western blot
AntibodyIRDye 800CW Goat
anti-Mouse IgG
LicorCatalog number:
926–32210;
RRID:AB_621842
Application:
Western blot
AntibodyIRDye 800CW Goat
anti-Rabbit
LicorCatalog number:
926–32211;
RRID:AB_621843
Application:
Western blot
AntibodyIRDye 680LT goat
anti-mouse
LicorCatalog number:
926–68020;
RRID:AB_10706161
Application:
Western blot
AntibodyIRDye 680LT Goat
anti-Rabbit IgG
LicorCatalog number:
926–68021;
RRID:AB_10706309
Application:
Western blot
Recombinant
DNA reagent
pLentipuro3/TO/
V5-DEST
Andrew E. Aplin,
Thomas Jefferson
University
Recombinant
DNA reagent
pLentineo3/TO/
V5-DEST
Andrew E. Aplin,
Thomas Jefferson
University
Recombinant
DNA reagent
pLentihygro3/TO/
V5-DEST
Andrew E. Aplin,
Thomas Jefferson
University
Recombinant
DNA reagent
pLenti0.3/EF/
V5-DEST
This paperHuman EF1-alpha
promoter was substituted
for the CMV promoter of
pLenti6.3/UbC/V5-DEST
and the SV40 promoter/Bsd
cassette was removed
Recombinant
DNA reagent
pLenti6.3/UbC/
V5-DEST
Andrew E. Aplin,
Thomas Jefferson
University
Recombinant
DNA reagent
pLenti6.3/UbC
empty vector
This paperEcoRV digest/re-ligation
to remove Gateway element
Recombinant
DNA reagent
pLentipuro3/
Block-iT-DEST
Andrew E. Aplin,
Thomas Jefferson
University
Recombinant
DNA reagent
pLenti0.3/EF/GW/IVS
-Kozak-TetR-P2A-Bsd
This paperLR recombination of
IVS-TetR-P2A-Bsd cassette
into pLenti0.3/EF/V5-DEST
Recombinant
DNA reagent
pLenti0.3/EF/GW/
PatGFP-Luc2
This paperLR recombination of
PatGFP-Luc2 cassette
into pLenti0.3/EF/V5-DEST
Recombinant
DNA reagent
pLP1Andrew E. Aplin,
Thomas Jefferson
University
Lentivirus packaging
plasmid originally from
Invitrogen
Recombinant
DNA reagent
pLP2Andrew E. Aplin,
Thomas Jefferson
University
Lentivirus packaging
plasmid originally from
Invitrogen
Recombinant
DNA reagent
pLP/VSVGAndrew E. Aplin,
Thomas Jefferson
University
Lentivirus packaging
plasmid originally from
Invitrogen
Sequence-based
reagent
Non-targeting
control siRNA
DharmaconCatalog number:
D-001810-01-20
Sequence-based
reagent
HNF1A siRNA #1DharmaconCatalog number:
D-008215-01-0002
Sequence:
GGAGGAACCGTTTCAAGTG
Sequence-based
reagent
HNF1A siRNA #2DharmaconCatalog number:
D-008215-02-0002
Sequence:
GCAAAGAGGCACTGATCCA
Sequence-based
reagent
POU5F1/OCT4
siRNA #5
DharmaconCatalog number:
D-019591-05-0002
Sequence:
CATCAAAGCTCTGCAGAAA
Sequence-based
reagent
POU5F1/OCT4
siRNA #6
DharmaconCatalog number:
D-019591-06-0002
Sequence:
GATATACACAGGCCGATGT
Sequence-based
reagent
POU5F1/OCT4
siRNA #9
DharmaconCatalog number:
D-019591-09-0002
Sequence:
GCGATCAAGCAGCGACTAT
Sequence-based
reagent
POU5F1/OCT4
siRNA #10
DharmaconCatalog number:
D-019591-10-0002
Sequence:
TCCCATGCATTCAAACTGA
Peptide,
recombinant protein
Recombinant
human EGF
InvitrogenCatalog number:
PHG0311L
Peptide,
recombinant protein
FGF-basic
Recombinant
Human
InvitrogenCatalog number:
PHG0264
Peptide,
recombinant protein
Leukemia
Inhibitory Factor
human
Sigma AldrichCatalog number:
L5283
Peptide,
recombinant protein
Bone Morphogenetic
Protein four human
PeprotechCatalog number:
120–05
Commercial
assay or kit
SimpleChIP Enzymatic
Chromatin IP Kit
(Magnetic Beads)
Cell Signaling
Technology
Catalog number:
9003
Commercial
assay or kit
BioLux Gaussia
Luciferase Assay Kit
New England
Biolabs
Catalog number:
E3300S
Commercial
assay or kit
BioLux
Cypridina Luciferase
Assay Kit
New England
Biolabs
Catalog number:
E3309S
Commercial
assay or kit
RNeasy Plus Mini
Kit coupled with
RNase-free DNase set
QiagenCatalog number:
74136 and 79254
Commercial
assay or kit
High Capacity
RNA-to-cDNA Master
Mix
Applied
Biosystem
Catalog number:
4387406
Commercial
assay or kit
Power SYBR Green
PCR Master Mix
Applied
Biosystem
Catalog number:
4367659
Chemical
compound, drug
APC-Cy7 StreptavidinBD BiosciencesCatalog number:
554063
Chemical
compound, drug
DAPI
(4',6-Diamidino-2-Phenylindole,
Dilactate)
InvitrogenCatalog number:
3571
Chemical
compound, drug
APC Annexin VBD BiosciencesCatalog number:
550474
Chemical
compound, drug
Annexin V Binding
Buffer, 10x concentrate
BD BiosciencesCatalog number:
556454
Chemical
compound, drug
RNase AInvitrogenCatalog number:
12091021
Chemical
compound, drug
Lipofectamine 2000
Reagent
InvitrogenCatalog number:
11668019
Chemical
compound, drug
Lipofectamine RNAiMAX
Reagent
InvitrogenCatalog number:
13778150
Chemical
compound, drug
Propidium iodideInvitrogenCatalog number:
P1304MP
Chemical
compound, drug
GentamicinInvitrogenCatalog number:
15710072
Chemical
compound, drug
Antibiotic-Antimycotic (100X)InvitrogenCatalog number:
15240062
Chemical
compound, drug
N-2 Supplement (100X)InvitrogenCatalog number:
17502–048
Chemical
compound, drug
B-27 Serum-Free
Supplement (50X)
InvitrogenCatalog number:
17504–044
Chemical
compound, drug
DoxycyclineSigma AldrichD9891-100G
Software,
algorithm
GraphPad Prism 6GraphPad Software;
http://www.graphpad.com
RRID:SCR_002798
Software,
algorithm
oPOSSUM 3.0http://opossum.cisreg.ca/oPOSSUM3/;
PMID: 22973536
RRID:SCR_010884
Software,
algorithm
Bowtie v1.1.1PMID: 19261174RRID:SCR_005476
Software,
algorithm
Bowtie v0.12.8PMID: 19261174RRID:SCR_005476
Software,
algorithm
MACS v1.4.2PMID: 18798982RRID:SCR_013291
Software,
algorithm
TopHat v1.4.1PMID: 19289445RRID:SCR_013035
Software,
algorithm
DESeq v1.24.0PMID: 20979621RRID:SCR_000154
Software,
algorithm
bedtools v.2.26.0PMID: 20110278RRID:SCR_006646
Software,
algorithm
survival v2.40–1DOI: 10.1007/978-1-4757-3294-8

Additional files

Supplementary file 1

Cancer stem cell frequencies in PDA subpopulations.

Limiting dilution assay was performed with sorted NY15 cells injected subcutaneously in NOD/SCID mice. The resultant numbers of tumors/injection is tabulated with estimated cancer stem cell frequencies calculated by extreme limiting dilution analysis (ELDA).

https://doi.org/10.7554/eLife.33947.030
Supplementary file 2

Data for generating PDA subpopulation heatmap and HNF1A target gene data (Excel spreadsheet).

Contents of each worksheet are as follows: worksheet 1) notes for summary tables; worksheet 2) Primers for qPCR validation of CSC50 genes; worksheet 3) table includes values represented in the Figure 1E where values are fold changes relative to HL or LH, as indicated; worksheet 4) NY8 and NY15 Bru-seq data related to Figure 7A; worksheet 5) NY8 and NY15 ChIP-seq data related to Figure 7B with information regarding enhancer binding; worksheet 6) summary of selected genes in expression vs survival in TCGA PAAD tumors - related to Figure 7E, S9A, and S9B, worksheet 7) HNF1A-upregulated and -bound genes: association between gene expression and survival in TCGA PAAD tumors - related to Figure 7E; worksheet 8) HNF1A-upregulated genes: association between gene expression and survival in TCGA PAAD tumors - related to Figure 7—figure supplement 1A; worksheet 9) HNF1A-downregulated genes: association between gene expression and survival in TCGA PAAD tumors - related to Figure 7—figure supplement 1B; worksheet 10) TCGA donors used in survival analysis - related to Figure 7E, Figure 7—figure supplement 1A and B; worksheet 11) Overrepresented TF-binding motifs in cancer stem cell gene set (CSC50), oPOSSUM3 results - related to Figure 1; worksheet 12) predicted HNF1A targets - related to Figure 1; worksheet 13) overrepresented TF-binding motifs in HNF1A upregulated genes, oPOSSUM3 results - related to Figure 7D; worksheet 14) overrepresented TF-binding motifs in HNF1A downregulated genes, oPOSSUM3 results - related to Figure 7D; worksheet 15) predicted POU5F1 targets - related to Figure 7D; worksheet 16) HNF1A ChIP-seq peak enhancer overlap, NY15 replicate 1 (rep1) - related to Figure 7B; worksheet 17) A ChIP-seq peak enhancer overlap, NY15 replicate 2 (rep2) - related to Figure 7B; worksheet 18) HNF1A ChIP-seq peak enhancer overlap, NY8 replicate 1 (rep1) - related to Figure 7B; worksheet 19) HNF1A ChIP-seq peak enhancer overlap, NY8 replicate 2 (rep2) - related to Figure 7B.

https://doi.org/10.7554/eLife.33947.031
Transparent reporting form
https://doi.org/10.7554/eLife.33947.032

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