Involvement of hedgehog pathway in early onset, aggressive molecular subtypes and metastatic potential of breast cancer

Abstract

Background: Dysregulation of hedgehog pathway is observed in numerous cancers. Relevance of hedgehog pathway genes in cancer cohort and inhibition of its downstream effector (GLI1) towards metastasis in cell lines are explored in the study.

Method: One hundred fifty fresh tumours of breast cancer patients were collected for the study. Based on differential expression, panel of 6 key regulators of the pathway (SHH, DHH, IHH, PTCH1, SMO and GLI1) in microarray datasets were identified. Expressional profiles of aforementioned genes were later correlated with clinico-pathological parameters in Pakistani breast cancer cohort at transcript and protein levels. In addition, GLI1 over expressing breast cancer cell lines (MDA-MB-231 and MCF-7) were treated with GANT61 to explore its probable effects on metastasis.

Result: SHH, DHH, PTCH1 and GLI1 were significantly over-expressed in tumours as compared with respective normal mammary tissues. A significant correlation of SHH, DHH and GLI1 expression with advanced tumour size, stages, grades, nodal involvement and distant metastasis was observed (p < 0.05). Over-expression of SHH, DHH and GLI1 was significantly related with patients having early onset and pre-menopausal status. Of note, hedgehog pathway was frequently up regulated in luminal B and triple negative breast cancer affected women. In addition, positive correlations were observed among aforementioned members of pathway and Ki67 (r-value: 0.63-0.78) emphasizing their role towards disease progression. Exposure of GANT61 (inhibitor for GLI1) significantly restricted cell proliferation, reduced cell motility and invasion.

Conclusion: Role of activated hedgehog pathway in breast cancer metastasis provides a novel target for cancer therapy against aggressive cancer subtypes.

Keywords: Breast cancer; DHH; GANT61; GLI1; Hedgehog pathway; SHH.

Fig. 1

Expression Profiling of Hedgehog Pathway Members in Pakistani Cohort. a. Scatter plots showing mean relative mRNA expression of SHH, DHH, IHH, PTCH1, SMO and GLI1 in tumor samples as compared to normal tissues. b. Heatmap showing differential expression of analyzed molecules in tumors on left side and adjacent normal mammary tissues on right side. c. Immunostaining of SHH (A, B), DHH (C, D) and GLI1 (E, F) in normal tissues (A, C, E) and tumor tissues (B, D, F). (Wilcoxon Signed Rank Test, p < 0.0001)

Fig. 2

Transcript Profiling of Hedgehog Pathway using Box Whisker Plots. Association of relative expression of hedgehog pathway components with a. Nuclear grades, b. Tumor size, c. Nodal involvement, d. Metastasis, e. Age at disease onset, f. Menopausal status. (Kruskal-Wallis Anova (a, b and c) and Mann-Whitney U test (d, e and f), *p < 0.05, **p < 0.001, ***p < 0.0001)

Fig. 3

Correlation of Hedgehog molecules with Intrinsic Molecular Subtypes and Overall Survival of patients. a. Expression variation of hedgehog pathway in molecular subtypes (Luminal A, Luminal B, HER2 and Triple negative) of breast cancer patients evaluated using qPCR. (Kruskal-Wallis Anova, *p < 0.05, ***p < 0.0001). b. Kaplan Meier plots showing association of SHH, DHH and GLI1 with Overall survival in Pakistani population (red = high expression, blue = low expression, significant p < 0.05)

Fig. 4

Effect of GANT61 on proliferation, apoptosis and hedgehog pathway. Inhibitory effect of GANT61 on proliferation and apoptosis of breast cancer cells was tested using in vitro models. Cell viability assays were conducted using CCK-8, to observe the effect of GANT61 on cell proliferation in MDA-MB-231 and MCF-7 in a. Dose dependent (5, 10, 15, 20 μM) and b. Time dependent manner (24, 48, 72, 96 h). Apoptosis was assessed using Annexin V at variable concentrations (5, 10, 15, 20 μM) of GANT61 in c.MDA-MB-231 and d.MCF-7 (Unpaired t test, *p < 0.05, **p < 0.001, ***p < 0.0001). IC50 of GANT61 was determined to be 10 μM. Inhibition of expression of hedgehog pathway after treatment with GANT61 (10 μM) for 48 h. Relative mRNA expression of SHH, PTCH1 and GLI1 using qPCR in e. MDA-MB-231, f. MCF-7 (Mann Whitney U test, *p < 0.05, **p < 0.001, ***p < 0.0001). g. Protein expression of SHH, PTCH1 and GLI1 using western blot having β-actin as internal control in both cell lines. All experiments were conducted in triplicates, performed thrice and values are represented as mean ± S.D.

Fig. 5

Effect of GANT61 on motility and invasion of breast cancer cells. MDA-MB-231 and MCF-7 were treated with the conditional medium containing 10 μM GANT61 and the control medium. a. Migration was assessed using scratch assay and readings were taken after every twelve hour for 48 h, upper panel of both cell lines represent 0 h and the lower panel represents 48 h. b. Graph showing difference of distance migrated between untreated and treated cells at each time point of scratch assay. c. Invasion was assessed using Boyden chamber transwell assays and difference between treated and untreated cells was evaluated after 24 h. d. Graphical representation of number of cells invaded as mean ± S.D. between untreated and treated cells in both cell lines. (Unpaired t test, ***, ###p < 0.0001). All experiments were conducted in triplicates and were performed thrice