Loss of Primary Cilia Drives Switching from Hedgehog to Ras/MAPK Pathway in Resistant Basal Cell Carcinoma

Abstract

Basal cell carcinomas (BCCs) rely on Hedgehog (HH) pathway growth signal amplification by the microtubule-based organelle, the primary cilium. Despite naive tumor responsiveness to Smoothened inhibitors (Smoⁱ), resistance in advanced tumors remains common. Although the resistant BCCs usually maintain HH pathway activation, squamous cell carcinomas with Ras/MAPK pathway activation also arise, and the molecular basis of tumor type and pathway selection are still obscure. Here, we identify the primary cilium as a critical determinant controlling tumor pathway switching. Strikingly, Smoothened inhibitor-resistant BCCs have an increased mutational load in ciliome genes, resulting in reduced primary cilia and HH pathway activation compared with naive or Gorlin syndrome patient BCCs. Gene set enrichment analysis of resistant BCCs with a low HH pathway signature showed increased Ras/MAPK pathway activation. Tissue analysis confirmed an inverse relationship between primary cilia presence and Ras/MAPK activation, and primary cilia removal in BCCs potentiated Ras/MAPK pathway activation. Moreover, activating Ras in HH-responsive cell lines conferred resistance to both canonical (vismodegib) and noncanonical (atypical protein kinase C and MRTF inhibitors) HH pathway inhibitors and conferred sensitivity to MAPK inhibitors. Our results provide insights into BCC treatment and identify the primary cilium as an important lineage gatekeeper, preventing HH-to-Ras/MAPK pathway switching.

Figure 1.. Human resistant BCC harbor reduced primary cilia.

(a) Top-lists of GO cellular components and molecular functions associated with genes commonly mutated in resistant BCCs. (b) Quantification of the mutations found in the ciliome of Gorlin patients, sporadic naïve and resistant human BCCs using whole exome sequencing. Numbers indicate the mean values for each category. (c) Acetylated tubulin (cilia shaft), pericentrin (cilia body) and DAPI immunostainings of Gorlin patient, sporadic naïve and resistant human BCCs. White arrowheads indicate cilia. (d) Quantification of cilia in the tumor compartment shown in (c). Dots represent microscope fields, distributed across n>3 tumors. FE stands for fold enrichment. FDR stands for false-discovery rate. Scale bars indicate 25μm. Horizontal bars and error bars in (b) and (d) represent the mean ± SD. *p < 0.05, ***p < 0.001.

Figure 2.. Loss of primary cilia goes with HH pathway inactivation and Ras/MAPK pathway activation in a subset of resistant BCCs.

(a) Algorithm for the analysis of RNA-sequencing obtained from vismodegib-sensitive and - resistant human BCCs. (b) GSEA for HH and Ras pathways activation in “HH-high” versus “HH-low” human resistant BCCs. NES stands for normalized enrichment score. (c) Adjacent immunostainings for cilia (acetylated tubulin and pericentrin), Gli1 (as a readout for HH pathway activation) and p-MEK (as readout for Ras/MAPK pathway activation) in resistant human BCCs. White arrowheads indicate cilia. Higher magnifications are shown in framed pictures. (d) Correlation between nuclear Gli1 and p-MEK relative intensity in resistant human BCCs, annotated for cilia density. Dots represent microscope fields, distributed across 4 different resistant BCC tumors. Scale bars indicate 25μm. **p < 0.01. r stands for Spearman correlation factor.

Figure 3.. Loss of primary cilia potentiates Ras/MAPK pathway activation.

(a) GSEA for HH pathway activation in RNA-sequencing datas obtained from ASZ_PA upon DMSO (control) versus CBD (10μM) treatment. (b) GSEA for Ras pathway activation in RNA-sequencing datas obtained from ASZ_PA or ASZ_ERRasV12/Dox-4OHT upon DMSO (control) versus CBD (10μM) treatment. (c) Western blot for p-ERK1/2 (as readout for Ras/MAPK pathway activation), total ERK1/2 and β tubulin (loading control) in ASZ_ERRasV12 and UWBCC_ERRasV12 upon doxycycline/4OHT and DMSO (control) versus CBD treatment. (d) p-MEK immunostaining (as readout for Ras/MAPK pathway activation) in ASZ_ERRasV12 and UWBCC_ERRasV12 upon doxycycline/4OHT and DMSO (control) versus CBD treatment. (e) Quantification of p-MEK immunostaining in ASZ_ERRasV12 and UWBCC_ERRasV12 upon doxycycline/4OHT and DMSO (control) versus CBD treatment. (f) Western blot for p-ERK1/2, total ERK1/2 and β tubulin (loading control) in ASZ_ERRasV12 and UWBCC_ERRasV12 upon doxycycline/4OHT after transfection with ns_siRNA versus OFD1_siRNA. Scale bars indicate 25μm. Horizontal and error bars in (e) represent the mean ± SD. *p < 0.05, **p < 0.01 ***p < 0.001.

Figure 4.. HH to Ras pathway switching confers resistance to canonical and non-canonical HH pathway inhibitors and sensitivity to MEK inhibitor UO126 in vitro.

(a) Vismodegib sensitivity assessed by MTT assay in 3T3_ERRasV12 upon stimulation with Smoothened agonist (SAG, 100nM) and Doxycycline/4OHT. (b) PSI (aPKC inhibitor) sensitivity assessed by MTT assay in ASZ_ERRasV12 and UWBCC_ERRasV12 upon Doxycycline/4OHT. (c) CCG1423 (MRTF inhibitor) sensitivity assessed by MTT assay in ASZ_ERRasV12 or UWBCC_ERRasV12 upon Doxycycline/4OHT. (d) UO126 (MEK inhibitor) sensitivity assessed by MTT assay in ASZ_ERRasV12 or UWBCC_ERRasV12 upon Doxycycline/4OHT. When reached, IC50 (half-maximal inhibitory concentration) are indicated in red lines, both in the absence (full line) or presence (dashed line) of Doxycycline/4OHT. Dots and error bars represent the mean ± SD for n ≥ 6 per group. *p < 0.05, **p < 0.01 ***p < 0.001.