We report herein a role for the transcription factor GLI1 in the growth, survival and migration of two IBC cell lines, SUM149 cells, a triple-negative IBC cell line with activated EGFR, isolated from a primary tumour of an IBC patient (Forozan et al, 1999
; Ignatoski and Ethier, 1999
) and rSUM149, an isogenic cell line with acquired resistance to ErbB1/2 inhibitors (Aird et al, 2010
). Both SUM149 and rSUM149 expressed significantly higher levels of GLI1 compared with other IBC cell lines SUM190 and rSUM190, non-IBC cell lines MCF-7, SK-BR-3, BT474 MDA-MB-231 and normal mammary epithelial cells evaluated in this study. To validate the role of elevated GLI1 expression in SUM149 and rSUM149 cell growth, we investigated direct blocking of GLI1 expression by RNA interference. The decrease in cell proliferation and increase in apoptosis in SUM149 cells treated with GLI1 siRNA corresponded with decreased levels of the most potent anti-apoptotic proteins XIAP and its target procaspase 9. The role of apoptotic dysregulation as mediated by the inhibitor of apoptosis protein XIAP has been recently reported by our group as a mechanism of therapeutic resistance to apoptosis in IBC cells (Aird et al, 2008
). Further, herein we observed that the migration ability of the SUM149 and rSUM149 cells when GLI1 was silenced was dramatically reduced compared with control-transfected cells as assessed in both a conventional scratch assay and a novel automated wound-healing assay utilising H2B-GFP-tagged cells. The present study, therefore suggests that upregulation of GLI1 is important for survival and migration in these IBC cells.
The only previous reports connecting the Hh-Gli pathway with IBC was a gene array study linking a three-gene signature including the Sonic hedgehog
) gene with poor outcome in IBC patients (Bieche et al, 2004
), and another gene expression study demonstrating that IBC is characterised by Hh-pathway hyperactivation (Van Laere et al, 2010
). GLI1 has been reported extensively as a universal marker for Hh-pathway activation. Elevated GLI1 expression and its role in a number of cancers has been widely reported, including a recent study that the conditional expression of GLI1 in mouse mammary glands results in mammary tumours (Fiaschi et al, 2009
). Aberrant activation of GLI1 expression might arise from a number of possible mechanisms with both canonical Hh-pathway-dependent (Rubin and de Sauvage, 2006
; Yauch et al, 2008
) and non-canonical Hh-independent (Lauth and Toftgard, 2007
; Jenkins, 2009
) activation of GLI1 reported for different cancers. We observed that neither the addition of exogenous Shh ligand nor a blocking Hh antibody was able to affect the growth or levels of GLI1 in SUM149 or rSUM149 IBC cell lines. Lack of responsiveness to added Hh-ligand is expected considering the very low levels of SMO expression observed in SUM149 cells (data not shown). Similarly, most non-IBC breast cancer cell lines do not respond to exogenous ShhN to activate Hh signalling or increase GLI (Zhang et al, 2008
). Taken together with our observation of the high concentration of KAAD-cyc required to elicit growth effects, the present study suggests that in SUM149 cells GLI1 activation is not Hh-ligand or SMO dependent.
The data from GLI1 siRNA silencing studies suggest a more effective approach to targeting SUM149 cells would be by directly inhibiting GLI1 transcription. To further probe the molecular basis for GLI1 activation in SUM149 cells, we tested the effects of a direct GLI inhibitor, GANT58, which has been reported to block GLI1-induced transcription in vitro
and prevent additional tumour growth in xenograft tumour models (Lauth et al, 2007
). We observed that GANT58 also reduced cell proliferation of SUM149 cells and reduced GLI1 protein expression. Although its mechanism of action is unclear, GANT58 has been reported to act on nuclear localised GLI (Lauth et al, 2007
) and it appears to be highly selective for GLI as it has no effect on other signalling pathways such as TNF/NFκB and Ras/MAPK or on protein folding or nuclear transport (Lauth et al, 2007
). Targeting GLI1 in the clinic with siRNA is not currently feasible and the availability of more potent GLI inhibitors would be beneficial. Other GLI1 inhibitors have been recently described (Hosoya et al, 2008
; Hyman et al, 2009
; Mahindroo et al, 2009
) that target multiple steps in GLI regulation, although with micromolar potencies comparable to the GANT compound.
IBC is an aggressive clinical subtype of breast cancer that is an extremely invasive and metastatic disease (Cristofanilli et al, 2007
) that falls into two major subtypes; Basal-like or ErbB2 overexpressing (Van Laere et al, 2007
). The cellular models that were used for this study are the only well-established IBC cellular models available; SUM149, an ErbB1 activated, triple-negative (ErbB2-, ER- and PR-) cell line and SUM190, an ErbB2 overexpressed, ER- cell line, both of which have been derived from primary IBC tumours (Forozan et al, 1999
). Both these cell lines are sensitive to ErbB1/2 targeting agents (Aird et al, 2008
). SUM149 cells were generated from an aggressive IBC (Forozan et al, 1999
) and have been characterised as a Basal B subtype of breast cancer cell (Neve et al, 2006
), which is frequently more aggressive and invasive than other breast cancer cell types. As GLI1 has been observed to promote cell migration and invasion in other cancer cell types (Das et al, 2009
; Lo et al, 2009
), including non-IBC breast cancer cell lines (Kameda et al, 2009
; Souzaki et al, 2011
), we were interested to determine if it has a role in the invasive potential of SUM149 and rSUM149 cells. Further, IBC is unusual in that it is an aggressive type of breast cancer that maintains strong E-cadherin expression (Kleer et al, 2001
; Colpaert et al, 2003
; Dong et al, 2007
). We wanted to investigate whether the high levels of GLI1 in SUM149 cells have a role in its unique migratory phenotype. Silencing of GLI1 reduced the level of E-cadherin but not of Snail, a transcription factor that promotes epithelial–mesenchymal transitions and is a key target of GLI1 (Li et al, 2006
). The molecular mechanisms linking GLI1 expression to E-cadherin and the invasive phenotype of SUM149 warrants further investigation.
In our study, we used the in vitro
wound-healing assay as a measure of migration and found that silencing of GLI1 in SUM149 and rSUM149 cells dramatically reduces cell migration over 24
h. From a previous study looking at the invasive potential of 30 breast cancer cell lines across subtypes, SUM149 falls within the Basal B subtype of highly invasive cells as assessed in 24
h Boyden chamber assays (Neve et al, 2006
). In contrast, other reports have suggested a less migratory phenotype for SUM149 (Nieman et al, 1999
; Hoffmeyer et al, 2005
) and that migration occurs by a passive dissemination process (Hoffmeyer et al, 2005
). These differences may be attributable to differences in format and time assessed as one report used the wound-healing assay over a shorter (7.5
h) time frame (Hoffmeyer et al, 2005
Studies in non-IBC breast cancer cell lines have suggested a link between ER status, GLI1 levels and survival, and shown that GLI1 silencing in ER negative breast cancer markedly reduces survival (Zhao et al, 2009
; Xu et al, 2010
) and invasiveness (Kameda et al, 2009
; Souzaki et al, 2011
). Although both SUM149 and SUM190 are ER- we only observed high GLI1 levels in SUM149. Presumably additional factors contribute to the elevated GLI1 status in the triple-negative Basal B-like SUM149 cells.
In addition, we were interested in developing high-content imaging assays to not only glean more quantitative information from the motility wound-healing assay, but to also extract other critical parameters defining migration behaviour. The advantages of the wound-healing assay are that it can be miniaturised and is compatible with microscopy, making it adaptable to high-throughput imaging. Such a HCAM system allows the analysis of both population and single-cell data on a heterogeneous population of cells and can potentially link phenotypic variability to genetic differences (Quaranta et al, 2009
). To obtain a more quantitative assessment and further assess the role of GLI1 in the migratory phenotype, we tagged SUM149 cells with H2B-GFP to fluorescently label nuclei of living cells and using a high-content live-cell fluorescence-based imaging system demonstrated that we can track not only cell migration but additional parameters of motility such as linearity of movement and integrated distance travelled. Both average linearity and distance travelled were reduced in SUM149 cells with GLI1 silenced. Further, our proof-of-principle study described here allowed us to distinguish differences in phenotype. GLI1 knockdown cells continue to move although with much reduced linearity and distance travelled. Indeed, the mean velocity between controls and GLI1 knockdown did not change significantly. In comparison, we observed that dyes such as MitoTracker Red affect the motility phenotype of SUM149 cells with the result that these cells have no motility. We expect this type of multi-parameter analysis of cell migration after H2B-GFP tagging to be generally applicable to many cell types to assess effects on migration.
Our data suggest a Hh-ligand-independent/SMO-independent mechanism of GLI1 gene overexpression operates in SUM149 and rSUM149 cells although we cannot rule out a role for other Hh-pathway components such as changes in SUFU (suppressor of FUSED) in activating GLI1. A number of other pathways have been reported to influence the cellular levels of GLI1 (Riobo et al, 2006b
) including TGF-β
(Dennler et al, 2007
), PI3K/AKT (Riobo et al, 2006a
), Ras/Mek (Stecca et al, 2007
), and a recently described novel GLI1-CDK2-dependent mechanism (Rizvi et al, 2010
). Further analyses using a range of pharmacological inhibitors to assess the role of other pathways in activating GLI1 and the phenotype of SUM149 and rSUM149 cells are planned in future studies. The understanding of those pathways converging on GLI in SUM149 will guide drug development strategies. It has been suggested that due to the complexity of signalling inputs into GLI (Ruiz et al, 2007
; Stecca and Ruiz, 2010
), targeting GLI in cancers may provide a more comprehensive strategy for treating both canonical and non-canonical Hh-pathway-dependent cancers. Further, it has been proposed that GLI-directed compounds that block growth in one cell type may be ineffective in others (Hyman et al, 2009
) and indeed, it may be necessary to directly screen specific cancer cell lines for compounds that specifically inhibit GLI1 expression in a particular cellular context.
Few studies have examined the molecular biology of IBC due to its relative low incidence; however, due to its high aggressiveness, mortality rate, and resistance to chemotherapeutic drugs (Rouesse et al, 1986
), additional investigations are warranted. In light of our observation that GLI1 has a role in proliferation, survival and migration of a subset of IBC cells, we propose that direct targeting of GLI1 transcription may be a novel and promising strategy for targeting triple-negative/Basal B IBC modelled by SUM149 cells.