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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

Reducing GLI1 expression in SUM149 cells by either siRNA knockdown or small molecule treatment results in decreased proliferation. SUM149 cells (7 × 104 cells per well in six-well plates) were transfected with 100 n of GLI1 siRNA or control siRNA for 72 h. Vehicle is lipofectamine alone. (A) Immunoblot analysis of GLI1 protein expression. The blot was stripped and reprobed for β-actin as an internal control for equal loading. (B) Cell proliferation was measured after 72 h by MTT assay. Experiments were performed in triplicate, P-value <0.05. (C) SUM149 cells were examined for morphology under phase light microscopy using an Olympus IX51 inverted microscope at a × 100 magnification. Bar=100 μm. (D) Immunoblot for GLI1 protein expression in SUM149 cells treated for 72 h with indicated concentrations of GANT58 (left panel) or tomatidine (right panel). β-Actin was used as an internal control for equal loading. (E) Cell proliferation was measured by MTT for SUM149 cells following treatment for 72 h with indicated concentrations of GANT58 or tomatidine as a control. Data are shown as relative to control (%).
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fig3: Reducing GLI1 expression in SUM149 cells by either siRNA knockdown or small molecule treatment results in decreased proliferation. SUM149 cells (7 × 104 cells per well in six-well plates) were transfected with 100 n of GLI1 siRNA or control siRNA for 72 h. Vehicle is lipofectamine alone. (A) Immunoblot analysis of GLI1 protein expression. The blot was stripped and reprobed for β-actin as an internal control for equal loading. (B) Cell proliferation was measured after 72 h by MTT assay. Experiments were performed in triplicate, P-value <0.05. (C) SUM149 cells were examined for morphology under phase light microscopy using an Olympus IX51 inverted microscope at a × 100 magnification. Bar=100 μm. (D) Immunoblot for GLI1 protein expression in SUM149 cells treated for 72 h with indicated concentrations of GANT58 (left panel) or tomatidine (right panel). β-Actin was used as an internal control for equal loading. (E) Cell proliferation was measured by MTT for SUM149 cells following treatment for 72 h with indicated concentrations of GANT58 or tomatidine as a control. Data are shown as relative to control (%).

Mentions: Since SUM149 and rSUM149 cells express high levels of GLI1 but these high levels appear to be Hh-ligand and SMO independent, we evaluated the effects of direct silencing of GLI1 expression on cell behaviour by transfecting GLI1 siRNA and control siRNA for 72 h. Immunoblot analysis in Figure 3A showed decreased GLI1 protein expression by 93% at 100 n GLI1 siRNA compared with control siRNA (P-value <0.05). Further, a significant decrease (P<0.05) in cell proliferation was observed in GLI1 siRNA-treated cells (46–50% decrease compared with controls, Figure 3B) as assessed by the MTT assay. Phase-contrast analysis showed a higher degree of nucleation in the SUM149 cells transfected with GLI1 siRNA compared with the vehicle (lipofectamine only) and control siRNA-transfected cells (Figure 3C).


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)

Reducing GLI1 expression in SUM149 cells by either siRNA knockdown or small molecule treatment results in decreased proliferation. SUM149 cells (7 × 104 cells per well in six-well plates) were transfected with 100 n of GLI1 siRNA or control siRNA for 72 h. Vehicle is lipofectamine alone. (A) Immunoblot analysis of GLI1 protein expression. The blot was stripped and reprobed for β-actin as an internal control for equal loading. (B) Cell proliferation was measured after 72 h by MTT assay. Experiments were performed in triplicate, P-value <0.05. (C) SUM149 cells were examined for morphology under phase light microscopy using an Olympus IX51 inverted microscope at a × 100 magnification. Bar=100 μm. (D) Immunoblot for GLI1 protein expression in SUM149 cells treated for 72 h with indicated concentrations of GANT58 (left panel) or tomatidine (right panel). β-Actin was used as an internal control for equal loading. (E) Cell proliferation was measured by MTT for SUM149 cells following treatment for 72 h with indicated concentrations of GANT58 or tomatidine as a control. Data are shown as relative to control (%).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101910&req=5

fig3: Reducing GLI1 expression in SUM149 cells by either siRNA knockdown or small molecule treatment results in decreased proliferation. SUM149 cells (7 × 104 cells per well in six-well plates) were transfected with 100 n of GLI1 siRNA or control siRNA for 72 h. Vehicle is lipofectamine alone. (A) Immunoblot analysis of GLI1 protein expression. The blot was stripped and reprobed for β-actin as an internal control for equal loading. (B) Cell proliferation was measured after 72 h by MTT assay. Experiments were performed in triplicate, P-value <0.05. (C) SUM149 cells were examined for morphology under phase light microscopy using an Olympus IX51 inverted microscope at a × 100 magnification. Bar=100 μm. (D) Immunoblot for GLI1 protein expression in SUM149 cells treated for 72 h with indicated concentrations of GANT58 (left panel) or tomatidine (right panel). β-Actin was used as an internal control for equal loading. (E) Cell proliferation was measured by MTT for SUM149 cells following treatment for 72 h with indicated concentrations of GANT58 or tomatidine as a control. Data are shown as relative to control (%).
Mentions: Since SUM149 and rSUM149 cells express high levels of GLI1 but these high levels appear to be Hh-ligand and SMO independent, we evaluated the effects of direct silencing of GLI1 expression on cell behaviour by transfecting GLI1 siRNA and control siRNA for 72 h. Immunoblot analysis in Figure 3A showed decreased GLI1 protein expression by 93% at 100 n GLI1 siRNA compared with control siRNA (P-value <0.05). Further, a significant decrease (P<0.05) in cell proliferation was observed in GLI1 siRNA-treated cells (46–50% decrease compared with controls, Figure 3B) as assessed by the MTT assay. Phase-contrast analysis showed a higher degree of nucleation in the SUM149 cells transfected with GLI1 siRNA compared with the vehicle (lipofectamine only) and control siRNA-transfected cells (Figure 3C).

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