Significance of glioma-associated oncogene homolog 1 (GLI1) expression in claudin-low breast cancer and crosstalk with the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway

Abstract

Introduction: The recently identified claudin-low subtype of breast cancer is enriched for cells with stem-like and mesenchymal-like characteristics. This subtype is most often triple-negative (lacking the estrogen and progesterone receptors (ER, PR) as well as lacking epidermal growth factor 2 (HER2) amplification) and has a poor prognosis. There are few targeted treatment options available for patients with this highly aggressive type of cancer.

Methods: Using a high throughput inhibitor screen, we identified high expression of glioma-associated oncogene homolog 1 (GLI1), the effector molecule of the hedgehog (Hh) pathway, as a critical determinant of cell lines that have undergone an epithelial to mesenchymal transition (EMT).

Results: High GLI1 expression is a property of claudin-low cells and tumors and correlates with markers of EMT and breast cancer stem cells. Knockdown of GLI1 expression in claudin-low cell lines resulted in reduced cell viability, motility, clonogenicity, self-renewal, and reduced tumor growth of orthotopic xenografts. We observed non-canonical activation of GLI1 in claudin-low and EMT cell lines, and identified crosstalk with the NFκB pathway.

Conclusions: This work highlights the importance of GLI1 in the maintenance of characteristics of metastatic breast cancer stem cells. Remarkably, treatment with an inhibitor of the NFκB pathway reproducibly reduces GLI1 expression and protein levels. We further provide direct evidence for the binding of the NFκB subunit p65 to the GLI1 promoter in both EMT and claudin-low cell lines. Our results uncover crosstalk between NFκB and GLI1 signals and suggest that targeting these pathways may be effective against the claudin-low breast cancer subtype.

Figure 1

JK184 is more effective at inhibiting EMT cell proliferation. (A) Clustering of Spearman ranked data of inhibition concentration (IC) values for the inhibitor screen. Rank values are as follows for μM concentrations: 1: no IC values are between 0 and 10; 2: IC₁₀ between 0 and 10, and IC₂₅ not between 0 and 10, and IC₅₀ not between 0 and 10; 3: IC₂₅ between 0 and 10, and IC₅₀ not between 0 and 10; 4: IC₅₀ between 0.1 and 10; 5: IC₅₀ between 0.01 and 0.1; 6: IC₅₀ between 0.001 and 0.01; 7: IC₅₀ < 0.001. Asterisks mark the agents mentioned in the text. (B) Dose-response curve for JK184 treatment of HMLE-shEcad cells (blue) and HMLE-shGFP cells (green). Error bars indicate coefficient of variance between triplicate treatment. (C) Dose-response curve for JK184 treatment of HMLE-shEcad cells (blue), HMLE-shGFP cells (green), HMLE-Snail cells (purple), and HMLE-pBP cells (gray). Treatment was conducted in 96-well plates. (D) Immunoblot showing response of GLI1 expression in HMLE cells with JK184 drug treatment. Doses were 0, 0.002, and 0.004 μM JK184 for 72 h. GAPDH serves as a loading control. GLI1 quantification levels were normalized to HMLE-shEcad untreated levels, and appear below the lane numbers in the figure. (E) Real-time PCR data showing relative GLI1 mRNA transcript levels from cells treated as in D). Error bars represent standard error between two independent experiments. * = P ≤0.05, t test. ** = P ≤0.005, t test. GAPDH, glyceraldehyde phosphate dehydrogenase; GLI1, glioma-associated oncogene 1; HMLE, human mammary epithelial cells, transformed with large T and small t antigens.

Figure 2

Claudin-low cell lines express higher transcript and protein levels of GLI1 . (A) Expression of CD44, CD24, CDH1, GLI, and CLDN3, 4, and 7, transcripts across a panel of breast cancer cell lines [34]. Multiple probes for each gene were extracted from microarray data and their expression values were Z transformed and plotted as a heat map. BaA = basal A, BaB = basal B, Lu = luminal. Asterisks denote cell lines originally identified as claudin-low [5]. Yellow box surrounds the expression of Gli family members in claudin-low cell lines. (B) Graph of relative Gli family expression levels in claudin-low (I) and EMT (II) cell lines, relative to controls. Error bars represent standard error, and n.s. indicates that no signal was achieved under the parameters used. (C) Immunoblot of GLI1 levels in the indicated claudin-low cell lines. GAPDH serves as a loading control. (D) Plot of GLI1 expression data from UNC337 mammary tumor and tissue dataset, based on subtype. 99% confidence interval is shown, with outliers plotted as single data points. Significance was calculated using one-way ANOVA using multiple comparisons, and all significant comparisons between the claudin low data set and others are indicated. ** = P ≤0.005. ANOVA, analysis of variance; EMT, epithelial-to-mesenchymal transition; GAPDH, glyceraldehyde phosphate dehydrogenase; GLI1, glioma-associated oncogene 1.

Figure 3

JK184 inhibits growth of claudin-low cell lines. (A) Real-time RT-PCR analysis of Gli1 RNA expression in JK184-treated claudin-low cell lines. Doses were 0, 0.002, and 0.004 μM JK184 for 72 h. Values were normalized to DMSO vehicle control, error bars are the standard error between two independent experiments. * = P ≤0.05, t test. ** = P ≤0.005, t test. (B) Western blot showing protein expression levels in claudin-low cells treated as in A). Lanes 19 to 21 are from a separate gel. (C) Dose-response curve for 72 h JK184 treatment of claudin-low and MCF10a, HMLE-shGFP, HMLE-pBP, and MTSV1-7 cell lines. (D) Flow cytometric analyses of cells treated with JK184 (0.02 μM) for four days followed by staining with Annexin-V and propidium iodide (PI). Images representative of three independent experiments. (E) Light microscopy images of cells treated as in A). Scale bar indicates 100 μm.

Figure 4

Knockdown of GLI1 decreases proliferation of claudin-low cell lines. (A) Western blot of cells infected with retrovirus expressing either non-targeting shRNA (NT), or shRNA targeted against GLI1 (#1, #2). Cells were infected, and selected for three days prior to blotting. GAPDH serves as a loading control. Quantification is relative to NT for each cell line. Blot is representative of three independent experiments. (B) Plot of GLI1 transcript levels in response to the shRNAs and treatment described in A). Error bars represent the standard error between three independent experiments. (C) Light microscopy images of MDA.MB.157 cells infected with either non-targeting (NT) or GLI1 -directed shRNA. Scale bars indicate 100 μm. (D, E) Plot detailing the proliferation of MDA.MB.436 cells (D) or BT549 cells (E) infected with the indicated retroviruses and grown in selection media. GAPDH, glyceraldehyde phosphate dehydrogenase; GLI1, glioma-associated oncogene 1; sh, short hairpin.

Figure 5

Decrease in GLI1 expression inhibits cell migration and anchorage-independent growth. (A) Panel I: Cartoon depicting experimental setup. Cells (yellow) were plated in the top of a Boyden chamber in 1% FBS above a lower-chamber of media containing 10% FBS. After 16 hours the number of cells that have migrated to the bottom side of the filter were stained and counted. II: Light microscopy images of the bottom side of the migration filter after staining. BT549 cells were infected with the indicated retroviruses and selected prior to experimentation. Scale bar indicates 100 μm. III: Migration of BT549 and MDA.MB.436 cells infected with the indicated retroviruses and assayed as described in I. (B) Clonogenic colony formation assay in BT549 (I) and MDA.MB.436 (II) cells. (C) Panel I: Representative 12 day spheres formed in MDA.MB.436 cells infected with the indicated retroviruses. Scale bar indicates 100 μm. II: Relative sphere formation by MDA.MB.436 cells. (D) Panel I: GLI1 mRNA expression obtained from real-time RT-PCR analysis of mRNA from adherent cells incubated in mammosphere medium (white bars) or sphere cells grown under non-adherent conditions (gray bars). II: Western blot detailing GLI1 levels in HMLE-shEcad or HMLE-Snail adherent and sphere cells. For all: * = P ≤0.05, t test. ** = P ≤0.005, t test. (E) (I) Plot of tumor volume over time arising from orthotopic injection of MDA.MB.436 cells infected with either non-targeting (NT) or GLI1 knockdown constructs. n = six animals. (II) Final averaged tumor weights at seven weeks post-injection for n = six animals. (III) Final averaged tumor weights from an independent biological replicate of the orthotopic xenograft study, conducted on four animals. FBS, fetal bovine serum; GLI1, glioma-associated oncogene 1.

Figure 6

EMT and claudin-low cells are insensitive to Hedgehog (Hh) pathway inhibitors. (A) Inhibition concentration (IC)₅₀ values obtained with other Hh pathway antagonists from the 384-well screen. NF indicates not determined, either because no curve fit could be obtained, or the IC₅₀ was outside the experimental concentrations tested. (B) GLI1 transcript levels in HMLE-shEcad and HMLE-shGFP cells treated with cyclopamine (0, 5, or 10 μM for 72 hours). (C) Immunoblot of GLI1 in HMLE cells treated with cyclopamine as in B). (D) Gli1 transcript levels after 16 h treatment with 1 μM JK184 or triptolide. Error bars indicate the standard error of two independent experiments. * = P <0.05, t test. ** = P <0.005, t test. (E) Anti-GLI1 immunoblot following treatment described in A). Lanes 1 to 3 are from a separate gel. Quantification is normalized to no treatment condition. EMT, epithelial-to-mesenchymal transition; GLI1, glioma-associated oncogene 1; HMLE, human mammary epithelial cells, transformed with large T and small t antigens.

Figure 7

Crosstalk between NFκB and GLI1 signaling pathways. (A) Immunofluorescence images showing localization of NFκB p50 (Panel I) or p65 (Panel II) subunits in HMLE-shEcad (top row) or HMLE-shGFP (lower row) cells. Scale bar indicates 50 μm. (B) Map of putative NFκB binding sites surrounding the GLI1 promoter. Red arrow marks transcription start site. Green lines indicate sequences matching the consensus κB binding site; putative sites are numbered 1 to 6. Blue arrows indicate primer sets used for ChIP. (C) ChIP of HMLE-shEcad cells with indicated antibodies. Error bars indicate standard error among three independent experiments. (D) NFκB p65 ChIP to the GLI1 promoter (site 1) following six-hour treatment with 1 μM triptolide. Graph was normalized to binding with vehicle control (DMSO). Error bar indicates standard error among three independent experiments. (E) Real-time PCR data showing RELA, NFKB1, and GLI1 transcript levels after infection of HMLE-shEcad cells with non-targeting (NT) or shRELA and shNFKB1 virus (shNFκB) virus. (F) Western blot of Gli1, p65, and p50 levels in HMLE-shEcad cells infected with either non-targeting (NT) or shRELA and shNFKB1 virus (shNFκB). GAPDH serves as a loading control. For all: * = P ≤0.05, t test. ** = P ≤0.005, t test. ChIP, chromatin immunoprecipitation; GLI1, glioma-associated oncogene 1; RELA, v-rel reticuloendotheliosis viral oncogene homolog A; NFKB1, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; sh, short hairpin.