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The basic helix-loop-helix transcription factor E47 reprograms human pancreatic cancer cells to a quiescent acinar state with reduced tumorigenic potential.

Kim S, Lahmy R, Riha C, Yang C, Jakubison BL, van Niekerk J, Staub C, Wu Y, Gates K, Dong DS, Konieczny SF, Itkin-Ansari P - Pancreas (2015)

Bottom Line: Moreover, induction of E47 in human PDA cells in vitro is sufficient to inhibit tumorigenesis.Human PDA cells retain a high degree of plasticity, which can be exploited to induce a quiescent acinar cell state with reduced tumorigenic potential.Moreover, bHLH activity is a critical node coordinately regulating human PDA cell growth versus cell fate.

View Article: PubMed Central - PubMed

Affiliation: From the *Development and Aging Program, Neuroscience, Aging, and Stem Cell Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA; †Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Institute of Women's Life Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea; ‡Department of Pediatrics, University of California, San Diego, La Jolla, CA; §Department of Biological Sciences, Center for Cancer Research, the Bindley Bioscience Center, Purdue University, West Lafayette, IN; and ∥Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA.

ABSTRACT

Objectives: Pancreatic ductal adenocarcinoma (PDA) initiates from quiescent acinar cells that attain a Kras mutation, lose signaling from basic helix-loop-helix (bHLH) transcription factors, undergo acinar-ductal metaplasia, and rapidly acquire increased growth potential. We queried whether PDA cells can be reprogrammed to revert to their original quiescent acinar cell state by shifting key transcription programs.

Methods: Human PDA cell lines were engineered to express an inducible form of the bHLH protein E47. Gene expression, growth, and functional studies were investigated using microarray, quantitative polymerase chain reaction, immunoblots, immunohistochemistry, small interfering RNA, chromatin immunoprecipitation analyses, and cell transplantation into mice.

Results: In human PDA cells, E47 activity triggers stable G0/G1 arrest, which requires the cyclin-dependent kinase inhibitor p21 and the stress response protein TP53INP1. Concurrently, E47 induces high level expression of acinar digestive enzymes and feed forward activation of the acinar maturation network regulated by the bHLH factor MIST1. Moreover, induction of E47 in human PDA cells in vitro is sufficient to inhibit tumorigenesis.

Conclusions: Human PDA cells retain a high degree of plasticity, which can be exploited to induce a quiescent acinar cell state with reduced tumorigenic potential. Moreover, bHLH activity is a critical node coordinately regulating human PDA cell growth versus cell fate.

No MeSH data available.


Related in: MedlinePlus

E47 induces G0/G1 arrest and significantly downregulates cancer-associated genes in PDA cells. A, Immunostaining for the replication marker Ki67 (red) and DAPI (blue), ×200. B, Growth curves (log scale) for PANC-1/E47 cells. *P < 0.05, ***P < 0.001, n = 3 per group. C, Percentage of cells in individual cell cycle phases determined by flow cytometry. D, Venn diagram depicts genes upregulated in human PDA tumors relative to adjacent tissue (GSE16515, red) versus genes downregulated by E47 activity in PANC-1/E47 cells (dataset deposited-GSE55999, blue), overlap P = 1.1E−40. E, Heat map from microarray data showing 50 genes most profoundly downregulated by E47 activity, n = 4 biological replicates each. Arrows depict examples of genes validated by quantitative polymerase chain reaction (qPCR) in 3 PDA/E47 cell lines (Fig. S4, Supplemental Digital Content, http://links.lww.com/MPA/A363). (Note: for simplicity, E47ER cells treated with or without tamoxifen are labeled with the name of the parental cell line +/− E47 activity).
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Figure 1: E47 induces G0/G1 arrest and significantly downregulates cancer-associated genes in PDA cells. A, Immunostaining for the replication marker Ki67 (red) and DAPI (blue), ×200. B, Growth curves (log scale) for PANC-1/E47 cells. *P < 0.05, ***P < 0.001, n = 3 per group. C, Percentage of cells in individual cell cycle phases determined by flow cytometry. D, Venn diagram depicts genes upregulated in human PDA tumors relative to adjacent tissue (GSE16515, red) versus genes downregulated by E47 activity in PANC-1/E47 cells (dataset deposited-GSE55999, blue), overlap P = 1.1E−40. E, Heat map from microarray data showing 50 genes most profoundly downregulated by E47 activity, n = 4 biological replicates each. Arrows depict examples of genes validated by quantitative polymerase chain reaction (qPCR) in 3 PDA/E47 cell lines (Fig. S4, Supplemental Digital Content, http://links.lww.com/MPA/A363). (Note: for simplicity, E47ER cells treated with or without tamoxifen are labeled with the name of the parental cell line +/− E47 activity).

Mentions: Recently, we showed that upon induction of E47 in PANC-1 cells, Ki67 expression was diminished.18 Here, we queried whether this response was universal among different PDA cell lines. As shown in Figure 1A, induction of E47 activity produced rapid loss of Ki67 expression in all cell lines. Importantly, tamoxifen treatment of parental PDA cells lacking ectopic E47 had no effect on Ki67 expression, indicating that cell cycle exit was due to induced E47 activity, not to tamoxifen (Fig. S2, Supplemental Digital Content, http://links.lww.com/MPA/A363). Remarkably, for an 8-day culture period, the number of tamoxifen-induced PANC-1/E47 cells remained static or declined, compared with uninduced PANC-1/E47 cells that expanded 68-fold (Fig. 1B). To determine whether E47 arrested PDA cells in G0/G1, S, or G2/M, flow cytometry was employed to assess DNA content, revealing that E47 induced primarily G0/G1 arrest (Fig. 1C). The observed G0/G1 arrest suggested that E47 might generate stable changes in the cell cycle. To test this hypothesis, E47 was induced with tamoxifen for 8 days, followed by culture for an additional 8 days in the absence of tamoxifen. Notably, tamoxifen-treated cells maintained a nonproliferative state even when switched to normal growth medium (Fig. S3, Supplemental Digital Content, http://links.lww.com/MPA/A363), revealing that temporary induction of E47 activity was sufficient to arrest cells for prolonged periods.


The basic helix-loop-helix transcription factor E47 reprograms human pancreatic cancer cells to a quiescent acinar state with reduced tumorigenic potential.

Kim S, Lahmy R, Riha C, Yang C, Jakubison BL, van Niekerk J, Staub C, Wu Y, Gates K, Dong DS, Konieczny SF, Itkin-Ansari P - Pancreas (2015)

E47 induces G0/G1 arrest and significantly downregulates cancer-associated genes in PDA cells. A, Immunostaining for the replication marker Ki67 (red) and DAPI (blue), ×200. B, Growth curves (log scale) for PANC-1/E47 cells. *P < 0.05, ***P < 0.001, n = 3 per group. C, Percentage of cells in individual cell cycle phases determined by flow cytometry. D, Venn diagram depicts genes upregulated in human PDA tumors relative to adjacent tissue (GSE16515, red) versus genes downregulated by E47 activity in PANC-1/E47 cells (dataset deposited-GSE55999, blue), overlap P = 1.1E−40. E, Heat map from microarray data showing 50 genes most profoundly downregulated by E47 activity, n = 4 biological replicates each. Arrows depict examples of genes validated by quantitative polymerase chain reaction (qPCR) in 3 PDA/E47 cell lines (Fig. S4, Supplemental Digital Content, http://links.lww.com/MPA/A363). (Note: for simplicity, E47ER cells treated with or without tamoxifen are labeled with the name of the parental cell line +/− E47 activity).
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4464938&req=5

Figure 1: E47 induces G0/G1 arrest and significantly downregulates cancer-associated genes in PDA cells. A, Immunostaining for the replication marker Ki67 (red) and DAPI (blue), ×200. B, Growth curves (log scale) for PANC-1/E47 cells. *P < 0.05, ***P < 0.001, n = 3 per group. C, Percentage of cells in individual cell cycle phases determined by flow cytometry. D, Venn diagram depicts genes upregulated in human PDA tumors relative to adjacent tissue (GSE16515, red) versus genes downregulated by E47 activity in PANC-1/E47 cells (dataset deposited-GSE55999, blue), overlap P = 1.1E−40. E, Heat map from microarray data showing 50 genes most profoundly downregulated by E47 activity, n = 4 biological replicates each. Arrows depict examples of genes validated by quantitative polymerase chain reaction (qPCR) in 3 PDA/E47 cell lines (Fig. S4, Supplemental Digital Content, http://links.lww.com/MPA/A363). (Note: for simplicity, E47ER cells treated with or without tamoxifen are labeled with the name of the parental cell line +/− E47 activity).
Mentions: Recently, we showed that upon induction of E47 in PANC-1 cells, Ki67 expression was diminished.18 Here, we queried whether this response was universal among different PDA cell lines. As shown in Figure 1A, induction of E47 activity produced rapid loss of Ki67 expression in all cell lines. Importantly, tamoxifen treatment of parental PDA cells lacking ectopic E47 had no effect on Ki67 expression, indicating that cell cycle exit was due to induced E47 activity, not to tamoxifen (Fig. S2, Supplemental Digital Content, http://links.lww.com/MPA/A363). Remarkably, for an 8-day culture period, the number of tamoxifen-induced PANC-1/E47 cells remained static or declined, compared with uninduced PANC-1/E47 cells that expanded 68-fold (Fig. 1B). To determine whether E47 arrested PDA cells in G0/G1, S, or G2/M, flow cytometry was employed to assess DNA content, revealing that E47 induced primarily G0/G1 arrest (Fig. 1C). The observed G0/G1 arrest suggested that E47 might generate stable changes in the cell cycle. To test this hypothesis, E47 was induced with tamoxifen for 8 days, followed by culture for an additional 8 days in the absence of tamoxifen. Notably, tamoxifen-treated cells maintained a nonproliferative state even when switched to normal growth medium (Fig. S3, Supplemental Digital Content, http://links.lww.com/MPA/A363), revealing that temporary induction of E47 activity was sufficient to arrest cells for prolonged periods.

Bottom Line: Moreover, induction of E47 in human PDA cells in vitro is sufficient to inhibit tumorigenesis.Human PDA cells retain a high degree of plasticity, which can be exploited to induce a quiescent acinar cell state with reduced tumorigenic potential.Moreover, bHLH activity is a critical node coordinately regulating human PDA cell growth versus cell fate.

View Article: PubMed Central - PubMed

Affiliation: From the *Development and Aging Program, Neuroscience, Aging, and Stem Cell Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA; †Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Institute of Women's Life Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea; ‡Department of Pediatrics, University of California, San Diego, La Jolla, CA; §Department of Biological Sciences, Center for Cancer Research, the Bindley Bioscience Center, Purdue University, West Lafayette, IN; and ∥Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA.

ABSTRACT

Objectives: Pancreatic ductal adenocarcinoma (PDA) initiates from quiescent acinar cells that attain a Kras mutation, lose signaling from basic helix-loop-helix (bHLH) transcription factors, undergo acinar-ductal metaplasia, and rapidly acquire increased growth potential. We queried whether PDA cells can be reprogrammed to revert to their original quiescent acinar cell state by shifting key transcription programs.

Methods: Human PDA cell lines were engineered to express an inducible form of the bHLH protein E47. Gene expression, growth, and functional studies were investigated using microarray, quantitative polymerase chain reaction, immunoblots, immunohistochemistry, small interfering RNA, chromatin immunoprecipitation analyses, and cell transplantation into mice.

Results: In human PDA cells, E47 activity triggers stable G0/G1 arrest, which requires the cyclin-dependent kinase inhibitor p21 and the stress response protein TP53INP1. Concurrently, E47 induces high level expression of acinar digestive enzymes and feed forward activation of the acinar maturation network regulated by the bHLH factor MIST1. Moreover, induction of E47 in human PDA cells in vitro is sufficient to inhibit tumorigenesis.

Conclusions: Human PDA cells retain a high degree of plasticity, which can be exploited to induce a quiescent acinar cell state with reduced tumorigenic potential. Moreover, bHLH activity is a critical node coordinately regulating human PDA cell growth versus cell fate.

No MeSH data available.


Related in: MedlinePlus