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Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration.

Thomas ZI, Gibson W, Sexton JZ, Aird KM, Ingram SM, Aldrich A, Lyerly HK, Devi GR, Williams KP - Br. J. Cancer (2011)

Bottom Line: Downregulation of GLI1 expression in SUM149 and rSUM149 by small interfering RNA or a small molecule GLI1 inhibitor resulted in decreased proliferation and increased apoptosis.A novel high-content migration assay allowed us to quantify multiple effects of GLI1 silencing including significant decreases in cell distance travelled and linearity of movement.Our data reveal a role for GLI1 in IBC cell proliferation, survival and migration, which supports the feasibility of targeting GLI1 as a novel therapeutic strategy for IBC patients.

View Article: PubMed Central - PubMed

Affiliation: Biomanufacturing Research Institute and Technology Enterprise, Durham, North Carolina Central University, Durham, NC 27707, USA.

ABSTRACT

Background: Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with distinct molecular profiles. Gene expression profiling previously identified sonic hedgehog (SHH) as part of a gene signature that is differentially regulated in IBC patients.

Methods: The effects of reducing GLI1 levels on protein expression, cell proliferation, apoptosis and migration were determined by immunoblots, MTT assay, Annexin-V/PI assay and conventional and automated cell migration assays.

Results: Evaluation of a panel of breast cancer cell lines revealed elevated GLI1 expression, typically a marker for hedgehog-pathway activation, in a triple-negative, highly invasive IBC cell line, SUM149 and its isogenic-derived counterpart rSUM149 that has acquired resistance to ErbB1/2 targeting strategies. Downregulation of GLI1 expression in SUM149 and rSUM149 by small interfering RNA or a small molecule GLI1 inhibitor resulted in decreased proliferation and increased apoptosis. Further, GLI1 suppression in these cell lines significantly inhibited cell migration as assessed by a wound-healing assay compared with MCF-7, a non-invasive cell line with low GLI1 expression. A novel high-content migration assay allowed us to quantify multiple effects of GLI1 silencing including significant decreases in cell distance travelled and linearity of movement.

Conclusion: Our data reveal a role for GLI1 in IBC cell proliferation, survival and migration, which supports the feasibility of targeting GLI1 as a novel therapeutic strategy for IBC patients.

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Related in: MedlinePlus

High-content imaging and migration analysis of SUM149 H2B-GFP cells demonstrate that cell linearity and distance travelled are decreased in SUM149 cells with GLI1 attenuated. (A) (Left panel) SUM149 cells transduced with BacMam H2B-GFP viral particles. Image was taken with a × 10 Olympus objective. (Right panel) Automated cell detection was performed using CellProfiler by segmenting each nucleus using the Otsu image segmentation. Segmented nuclei are shown by colour (right image). (B) Cell plates were imaged on a BD pathway 855 high-content imaging system with Nikpow spinning disc for live-cell fluorescent imaging. Tiff images (16-bit) were collected every 20 min for 24 h for each well and the data were compiled in an uncompressed avi format to yield one video file per well. CellProfiler software was used for video tracking using the minimum displacement method in the TrackObjects module. Representative trajectories for SUM149 cells treated with a negative control siRNA or GLI1 siRNA are shown. (C) X, Y trajectories were tabulated for each cell observed in the time series and unique object numbers were assigned to cells. Population scatterplot showing distance travelled vs linearity for cells treated with GLI1 siRNA or negative control siRNA. (D) (Left panel) Integrated distance travelled for the negative control siRNA and GLI1 siRNA-treated samples. (Right panel) Average linearity for the negative control siRNA and GLI1 siRNA-treated samples.
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fig6: High-content imaging and migration analysis of SUM149 H2B-GFP cells demonstrate that cell linearity and distance travelled are decreased in SUM149 cells with GLI1 attenuated. (A) (Left panel) SUM149 cells transduced with BacMam H2B-GFP viral particles. Image was taken with a × 10 Olympus objective. (Right panel) Automated cell detection was performed using CellProfiler by segmenting each nucleus using the Otsu image segmentation. Segmented nuclei are shown by colour (right image). (B) Cell plates were imaged on a BD pathway 855 high-content imaging system with Nikpow spinning disc for live-cell fluorescent imaging. Tiff images (16-bit) were collected every 20 min for 24 h for each well and the data were compiled in an uncompressed avi format to yield one video file per well. CellProfiler software was used for video tracking using the minimum displacement method in the TrackObjects module. Representative trajectories for SUM149 cells treated with a negative control siRNA or GLI1 siRNA are shown. (C) X, Y trajectories were tabulated for each cell observed in the time series and unique object numbers were assigned to cells. Population scatterplot showing distance travelled vs linearity for cells treated with GLI1 siRNA or negative control siRNA. (D) (Left panel) Integrated distance travelled for the negative control siRNA and GLI1 siRNA-treated samples. (Right panel) Average linearity for the negative control siRNA and GLI1 siRNA-treated samples.

Mentions: SUM149 cells were transduced with baculoviral particles expressing H2B-GFP to label the nuclei and Figure 6A (left panel) reveals the live-cell tracking as imaged on the BD Imaging workstation. The advantages of H2B-GFP labelling are that this reporter system does not affect cell viability (Quaranta et al, 2009) and cells are brightly labelled by tagging nuclei compared with diffuse labelling as for cytoplasmic markers. As part of our preliminary high-content studies, we observed that dyes such as MitoTracker Red and CellTracker were not suitable for tracking purposes as SUM149 cells treated with these dyes exhibited no motility (data not shown). Further, extended live-cell fluorescent imaging can cause significant phototoxicity so we chose to limit overall light exposure by using a spinning-disc confocal unit, rather than widefield. Although exposure times increase, the spinning disc reduces the field of illumination from the entire well to pinhole sized, dramatically reducing the overall photoexposure per cell.


Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration.

Thomas ZI, Gibson W, Sexton JZ, Aird KM, Ingram SM, Aldrich A, Lyerly HK, Devi GR, Williams KP - Br. J. Cancer (2011)

High-content imaging and migration analysis of SUM149 H2B-GFP cells demonstrate that cell linearity and distance travelled are decreased in SUM149 cells with GLI1 attenuated. (A) (Left panel) SUM149 cells transduced with BacMam H2B-GFP viral particles. Image was taken with a × 10 Olympus objective. (Right panel) Automated cell detection was performed using CellProfiler by segmenting each nucleus using the Otsu image segmentation. Segmented nuclei are shown by colour (right image). (B) Cell plates were imaged on a BD pathway 855 high-content imaging system with Nikpow spinning disc for live-cell fluorescent imaging. Tiff images (16-bit) were collected every 20 min for 24 h for each well and the data were compiled in an uncompressed avi format to yield one video file per well. CellProfiler software was used for video tracking using the minimum displacement method in the TrackObjects module. Representative trajectories for SUM149 cells treated with a negative control siRNA or GLI1 siRNA are shown. (C) X, Y trajectories were tabulated for each cell observed in the time series and unique object numbers were assigned to cells. Population scatterplot showing distance travelled vs linearity for cells treated with GLI1 siRNA or negative control siRNA. (D) (Left panel) Integrated distance travelled for the negative control siRNA and GLI1 siRNA-treated samples. (Right panel) Average linearity for the negative control siRNA and GLI1 siRNA-treated samples.
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Related In: Results  -  Collection

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fig6: High-content imaging and migration analysis of SUM149 H2B-GFP cells demonstrate that cell linearity and distance travelled are decreased in SUM149 cells with GLI1 attenuated. (A) (Left panel) SUM149 cells transduced with BacMam H2B-GFP viral particles. Image was taken with a × 10 Olympus objective. (Right panel) Automated cell detection was performed using CellProfiler by segmenting each nucleus using the Otsu image segmentation. Segmented nuclei are shown by colour (right image). (B) Cell plates were imaged on a BD pathway 855 high-content imaging system with Nikpow spinning disc for live-cell fluorescent imaging. Tiff images (16-bit) were collected every 20 min for 24 h for each well and the data were compiled in an uncompressed avi format to yield one video file per well. CellProfiler software was used for video tracking using the minimum displacement method in the TrackObjects module. Representative trajectories for SUM149 cells treated with a negative control siRNA or GLI1 siRNA are shown. (C) X, Y trajectories were tabulated for each cell observed in the time series and unique object numbers were assigned to cells. Population scatterplot showing distance travelled vs linearity for cells treated with GLI1 siRNA or negative control siRNA. (D) (Left panel) Integrated distance travelled for the negative control siRNA and GLI1 siRNA-treated samples. (Right panel) Average linearity for the negative control siRNA and GLI1 siRNA-treated samples.
Mentions: SUM149 cells were transduced with baculoviral particles expressing H2B-GFP to label the nuclei and Figure 6A (left panel) reveals the live-cell tracking as imaged on the BD Imaging workstation. The advantages of H2B-GFP labelling are that this reporter system does not affect cell viability (Quaranta et al, 2009) and cells are brightly labelled by tagging nuclei compared with diffuse labelling as for cytoplasmic markers. As part of our preliminary high-content studies, we observed that dyes such as MitoTracker Red and CellTracker were not suitable for tracking purposes as SUM149 cells treated with these dyes exhibited no motility (data not shown). Further, extended live-cell fluorescent imaging can cause significant phototoxicity so we chose to limit overall light exposure by using a spinning-disc confocal unit, rather than widefield. Although exposure times increase, the spinning disc reduces the field of illumination from the entire well to pinhole sized, dramatically reducing the overall photoexposure per cell.

Bottom Line: Downregulation of GLI1 expression in SUM149 and rSUM149 by small interfering RNA or a small molecule GLI1 inhibitor resulted in decreased proliferation and increased apoptosis.A novel high-content migration assay allowed us to quantify multiple effects of GLI1 silencing including significant decreases in cell distance travelled and linearity of movement.Our data reveal a role for GLI1 in IBC cell proliferation, survival and migration, which supports the feasibility of targeting GLI1 as a novel therapeutic strategy for IBC patients.

View Article: PubMed Central - PubMed

Affiliation: Biomanufacturing Research Institute and Technology Enterprise, Durham, North Carolina Central University, Durham, NC 27707, USA.

ABSTRACT

Background: Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with distinct molecular profiles. Gene expression profiling previously identified sonic hedgehog (SHH) as part of a gene signature that is differentially regulated in IBC patients.

Methods: The effects of reducing GLI1 levels on protein expression, cell proliferation, apoptosis and migration were determined by immunoblots, MTT assay, Annexin-V/PI assay and conventional and automated cell migration assays.

Results: Evaluation of a panel of breast cancer cell lines revealed elevated GLI1 expression, typically a marker for hedgehog-pathway activation, in a triple-negative, highly invasive IBC cell line, SUM149 and its isogenic-derived counterpart rSUM149 that has acquired resistance to ErbB1/2 targeting strategies. Downregulation of GLI1 expression in SUM149 and rSUM149 by small interfering RNA or a small molecule GLI1 inhibitor resulted in decreased proliferation and increased apoptosis. Further, GLI1 suppression in these cell lines significantly inhibited cell migration as assessed by a wound-healing assay compared with MCF-7, a non-invasive cell line with low GLI1 expression. A novel high-content migration assay allowed us to quantify multiple effects of GLI1 silencing including significant decreases in cell distance travelled and linearity of movement.

Conclusion: Our data reveal a role for GLI1 in IBC cell proliferation, survival and migration, which supports the feasibility of targeting GLI1 as a novel therapeutic strategy for IBC patients.

Show MeSH
Related in: MedlinePlus