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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

GLI1 expression in SUM149 cells is Hh-ligand and SMO independent. (A) RT–PCR analysis of GLI1 mRNA expression in SUM149 and rSUM149 cells treated for 72 h with media or 2 μg ml–1 recombinant ShhN (left panel) or with 100 μg ml–1 5E1 antibody or 100 μg ml–1 isotype control antibody (right panel). β-Actin was used as an internal control. (B) Immunoblot analysis for GLI1 protein expression in SUM149 cells (7 × 104 cells per well in six-well plates) treated for 72 h with media or ShhN (left panel) or with 5E1 antibody or control antibody (right panel). Membranes were stripped and reprobed for β-actin as an internal control for equal loading. (C) Cell proliferation was determined by MTT assay in SUM149 cells (2000 cells per well in 96-well plates) treated for 72 h with media or ShhN (left panel) or with 5E1 antibody or control antibody (right panel). (D) RT–PCR analysis of GLI1 mRNA expression in SUM149 (upper panel) and rSUM149 (lower panel) cells treated for 72 h with the indicated concentrations of KAAD-cyclopamine (KAAD-cyc) or its inactive analogue tomatidine. β-Actin was used as an internal control. (E) Cell proliferation was determined by MTT assay in SUM149 cells (2000 cells per well) plated in 96-well plates following treatment for 72 h with the indicated concentrations of KAAD-cyc and its inactive analogue tomatidine.
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fig2: GLI1 expression in SUM149 cells is Hh-ligand and SMO independent. (A) RT–PCR analysis of GLI1 mRNA expression in SUM149 and rSUM149 cells treated for 72 h with media or 2 μg ml–1 recombinant ShhN (left panel) or with 100 μg ml–1 5E1 antibody or 100 μg ml–1 isotype control antibody (right panel). β-Actin was used as an internal control. (B) Immunoblot analysis for GLI1 protein expression in SUM149 cells (7 × 104 cells per well in six-well plates) treated for 72 h with media or ShhN (left panel) or with 5E1 antibody or control antibody (right panel). Membranes were stripped and reprobed for β-actin as an internal control for equal loading. (C) Cell proliferation was determined by MTT assay in SUM149 cells (2000 cells per well in 96-well plates) treated for 72 h with media or ShhN (left panel) or with 5E1 antibody or control antibody (right panel). (D) RT–PCR analysis of GLI1 mRNA expression in SUM149 (upper panel) and rSUM149 (lower panel) cells treated for 72 h with the indicated concentrations of KAAD-cyclopamine (KAAD-cyc) or its inactive analogue tomatidine. β-Actin was used as an internal control. (E) Cell proliferation was determined by MTT assay in SUM149 cells (2000 cells per well) plated in 96-well plates following treatment for 72 h with the indicated concentrations of KAAD-cyc and its inactive analogue tomatidine.

Mentions: We next tested the responsiveness of these IBC cell lines to exogenous ShhN (active N-terminal form of Shh) ligand. Using RT–PCR of GLI1 mRNA levels as a sensitive readout of Hh-pathway activity, we observed that the addition of exogenous recombinant ShhN ligand to SUM149 or rSUM149 cells compared with media alone did not increase the expression of GLI1 (Figure 2A, left panel).


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)

GLI1 expression in SUM149 cells is Hh-ligand and SMO independent. (A) RT–PCR analysis of GLI1 mRNA expression in SUM149 and rSUM149 cells treated for 72 h with media or 2 μg ml–1 recombinant ShhN (left panel) or with 100 μg ml–1 5E1 antibody or 100 μg ml–1 isotype control antibody (right panel). β-Actin was used as an internal control. (B) Immunoblot analysis for GLI1 protein expression in SUM149 cells (7 × 104 cells per well in six-well plates) treated for 72 h with media or ShhN (left panel) or with 5E1 antibody or control antibody (right panel). Membranes were stripped and reprobed for β-actin as an internal control for equal loading. (C) Cell proliferation was determined by MTT assay in SUM149 cells (2000 cells per well in 96-well plates) treated for 72 h with media or ShhN (left panel) or with 5E1 antibody or control antibody (right panel). (D) RT–PCR analysis of GLI1 mRNA expression in SUM149 (upper panel) and rSUM149 (lower panel) cells treated for 72 h with the indicated concentrations of KAAD-cyclopamine (KAAD-cyc) or its inactive analogue tomatidine. β-Actin was used as an internal control. (E) Cell proliferation was determined by MTT assay in SUM149 cells (2000 cells per well) plated in 96-well plates following treatment for 72 h with the indicated concentrations of KAAD-cyc and its inactive analogue tomatidine.
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Related In: Results  -  Collection

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fig2: GLI1 expression in SUM149 cells is Hh-ligand and SMO independent. (A) RT–PCR analysis of GLI1 mRNA expression in SUM149 and rSUM149 cells treated for 72 h with media or 2 μg ml–1 recombinant ShhN (left panel) or with 100 μg ml–1 5E1 antibody or 100 μg ml–1 isotype control antibody (right panel). β-Actin was used as an internal control. (B) Immunoblot analysis for GLI1 protein expression in SUM149 cells (7 × 104 cells per well in six-well plates) treated for 72 h with media or ShhN (left panel) or with 5E1 antibody or control antibody (right panel). Membranes were stripped and reprobed for β-actin as an internal control for equal loading. (C) Cell proliferation was determined by MTT assay in SUM149 cells (2000 cells per well in 96-well plates) treated for 72 h with media or ShhN (left panel) or with 5E1 antibody or control antibody (right panel). (D) RT–PCR analysis of GLI1 mRNA expression in SUM149 (upper panel) and rSUM149 (lower panel) cells treated for 72 h with the indicated concentrations of KAAD-cyclopamine (KAAD-cyc) or its inactive analogue tomatidine. β-Actin was used as an internal control. (E) Cell proliferation was determined by MTT assay in SUM149 cells (2000 cells per well) plated in 96-well plates following treatment for 72 h with the indicated concentrations of KAAD-cyc and its inactive analogue tomatidine.
Mentions: We next tested the responsiveness of these IBC cell lines to exogenous ShhN (active N-terminal form of Shh) ligand. Using RT–PCR of GLI1 mRNA levels as a sensitive readout of Hh-pathway activity, we observed that the addition of exogenous recombinant ShhN ligand to SUM149 or rSUM149 cells compared with media alone did not increase the expression of GLI1 (Figure 2A, left panel).

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