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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 and SHH expression in IBC and non-IBC cell lines. Total RNA (1 μg) was reverse transcribed and assessed for expression of GLI1 (A) and SHH (B) mRNA using real-time PCR. β-Actin was used as an internal control. Data indicate values performed in triplicate and the fold difference relative to HMEC.
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fig1: GLI1 and SHH expression in IBC and non-IBC cell lines. Total RNA (1 μg) was reverse transcribed and assessed for expression of GLI1 (A) and SHH (B) mRNA using real-time PCR. β-Actin was used as an internal control. Data indicate values performed in triplicate and the fold difference relative to HMEC.

Mentions: Notably, qRT–PCR analysis of basal GLI1 expression revealed that IBC cell lines, SUM149 and rSUM149, had a 19.4-fold and 28.7-fold higher level of GLI1 expression, respectively, relative to HMEC, and GLI1 expression was significantly higher compared with the other non-IBC cells tested (Figure 1A). The low expression levels of GLI1 mRNA observed in the HMEC and MCF-7 cells are consistent with previous reports for these cell lines (Kubo et al, 2004; Mukherjee et al, 2006; Zhang et al, 2008). GLI1 protein levels, as assessed by immunoblot analysis, reflected GLI1 mRNA levels in that GLI1 protein was expressed at higher levels in SUM149 (two-fold) and rSUM149 (1.5-fold) compared with MCF-7 cells and higher than in the other IBC cell lines tested, SUM190 and rSUM190 (data not shown).


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 and SHH expression in IBC and non-IBC cell lines. Total RNA (1 μg) was reverse transcribed and assessed for expression of GLI1 (A) and SHH (B) mRNA using real-time PCR. β-Actin was used as an internal control. Data indicate values performed in triplicate and the fold difference relative to HMEC.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101910&req=5

fig1: GLI1 and SHH expression in IBC and non-IBC cell lines. Total RNA (1 μg) was reverse transcribed and assessed for expression of GLI1 (A) and SHH (B) mRNA using real-time PCR. β-Actin was used as an internal control. Data indicate values performed in triplicate and the fold difference relative to HMEC.
Mentions: Notably, qRT–PCR analysis of basal GLI1 expression revealed that IBC cell lines, SUM149 and rSUM149, had a 19.4-fold and 28.7-fold higher level of GLI1 expression, respectively, relative to HMEC, and GLI1 expression was significantly higher compared with the other non-IBC cells tested (Figure 1A). The low expression levels of GLI1 mRNA observed in the HMEC and MCF-7 cells are consistent with previous reports for these cell lines (Kubo et al, 2004; Mukherjee et al, 2006; Zhang et al, 2008). GLI1 protein levels, as assessed by immunoblot analysis, reflected GLI1 mRNA levels in that GLI1 protein was expressed at higher levels in SUM149 (two-fold) and rSUM149 (1.5-fold) compared with MCF-7 cells and higher than in the other IBC cell lines tested, SUM190 and rSUM190 (data not shown).

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