Analysis of Primary Cilium Expression and Hedgehog Pathway Activation in Mesothelioma Throws Back Its Complex Biology

Abstract

The primary cilium (PC) is a sensory organelle present on the cell surface, modulating the activity of many pathways. Dysfunctions in the PC lead to different pathologic conditions including cancer. Hedgehog signaling (Hh) is regulated by PC and the loss of its control has been observed in many cancers, including mesothelioma. Malignant pleural mesothelioma (MPM) is a fatal cancer of the pleural membranes with poor therapeutic options. Recently, overexpression of the Hh transcriptional activator GL1 has been demonstrated to be associated with poor overall survival (OS) in MPM. However, unlike other cancers, the response to G-protein-coupled receptor smoothened (SMO)/Hh inhibitors is poor, mainly attributable to the lack of markers for patient stratification. For all these reasons, and in particular for the role of PC in the regulation of Hh, we investigated for the first time the status of PC in MPM tissues, demonstrating intra- and inter-heterogeneity in its expression. We also correlated the presence of PC with the activation of the Hh pathway, providing uncovered evidence of a PC-independent regulation of the Hh signaling in MPM. Our study contributes to the understanding MPM heterogeneity, thus helping to identify patients who might benefit from Hh inhibitors.

Keywords: GLI1; hedgehog signaling; mesothelioma; pleural; primary cilium.

Figure 1

Primary cilium expression in Malignant Pleural Mesothelioma FFPE. (A) Representative images of PC positivity from two different epithelioid MPM. (B) Representative images of two different PC-negative tissues; upper panel, a biphasic MPM; lower panel, epithelioid MPM. (C,D) Representative images of PC intra-heterogeneity from two different epithelioid MPM. From left to right: A and B, haematoxylin and eosin staining (magnification 5×), arl13b immunostaining (magnification 5× and 20×); (C,D), haematoxylin and eosin staining (magnification 5×), arl13b immunostaining (magnification 5× and 40×). Ciliated cells are indicated with arrows.

Figure 2

IF analysis of PC expression in selected primary MPM and non-tumoral mesothelial cell lines. PC-positive cell lines: HMC7 normal mesothelial, MMP21, MMP23; PC-negative cell lines: MMP1, MMP4, MMP14. blue: dapi; green: Arl13b. Magnification 20×. Primary cilia are indicated with arrows.

Figure 3

qRT-PCR analysis of GLI1, PTCH1 and c-MYC. The expression of Hh-related genes is upregulated in MPM cells loosing PC (gray bars), compared to normal mesothelial cells HMC7. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 4

IF analysis of nuclear GLI1 in MPM cell lines and HMC7 normal mesothelial cells. Hh/GLI1 signaling is activated in PC-negative cells MMP1 and MMP4, and in PC-positive cell lines MMP18 and MMP23. Red: GLI1; green: Arl13b; blue: DAPI. The arrows indicate the cells with nuclear GLI1. Magnification 20×.

Figure 5

IHC analysis of nuclear GLI1 in MPM FFPE. Magnification 10X. From left to right: haematoxylin and eosin staining, Gli1 immunostaining. (A) Epithelioid MPM, negative staining scored 0; (B) Epithelioid MPM, faint staining scored 1; (C) Biphasic MPM, moderate staining scored 2; (D) Epithelioid MPM, strong staining scored 3.

Figure 6

Differential expression analysis of GLI1 gene between biphasic and epithelioid MPM. The analysis was drawn using the Deseq2-package, PlotCounts. The GLI1 gene has Log2FoldChange < 0, indicating that the gene is down-regulated in epithelial tissues, but it is not significantly differentially expressed since padj = 0.81. Y axis: normalized count; X axis: Histological subtype. Epithelial = yes; non-epithelial = no.