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 microtubulebased organelle, the primary cilium. Despite naive tumor responsiveness to Smoothened inhibitors (Smo i ), 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.

Isoform-specific aPKC renders primary cilia dispensable for Hedgehog signaling and basal cell carcinoma growth

“…We previously reported that the 303 cilia-specific genes that comprise the SYSCILIA list (van Dam et al 2013) had significantly lower mutation rates in human BCCs compared to genome-wide mutation rates (Bonilla et al 2016;Kuonen et al 2019), an expected result given the importance of the HH pathway for BCC initiation and progression. However, many SMO antagonist-resistant tumors displayed clonal loss of primary cilia (Kuonen et al 2019), suggesting that some of the cilia-specific genes may be mutated more than normal.…”

“…We previously reported that the 303 cilia-specific genes that comprise the SYSCILIA list (van Dam et al 2013) had significantly lower mutation rates in human BCCs compared to genome-wide mutation rates (Bonilla et al 2016;Kuonen et al 2019), an expected result given the importance of the HH pathway for BCC initiation and progression. However, many SMO antagonist-resistant tumors displayed clonal loss of primary cilia (Kuonen et al 2019), suggesting that some of the cilia-specific genes may be mutated more than normal. When we analyzed 161 cilia-specific genes that contained at least one mutation in our previously identified list of genes commonly mutated in SMO antagonist-resistant human BCCs , we found a subset of highly mutated genes in drug-resistant BCCs compared to drug-sensitive and normal aged skin ( Figure 1A, Supplementary Table 1).…”

“…Despite clonal loss of primary cilia in many advanced and drug-resistant human BCC, the vast majority of tumors maintained HH target gene induction Kuonen et al 2019;Sharpe et al 2015). To explore the possibility that disrupting expression of Alms1 and Usher genes positively influences primary ciliogenesis and HH signaling, we used shRNA to knock down (KD) expression of each gene in HH-responsive NIH3T3 murine fibroblasts.…”

“…Alterations in the signaling or structural components have been implicated in multiple ciliopathies and cancer (Hildebrandt et al, 2011), such as basal cell carcinoma (BCC). BCCs require high sustained HH signaling for growth but paradoxically lose primary cilia during later stages while maintaining HH pathway activation Kuonen et al, 2019). Loss of primary cilia also occurs in advanced or metastatic breast cancer (Yuan et al, 2010), renal cancer (Schraml et al, 2009), prostate cancer (Hassounah et al, 2013), pancreatic cancer (Seeley et al, 2009), and melanoma (Kim et al, 2011), indicating a wide-spread issue in late-stage cancers that are not well understood.…”

“…Roughly half of drug-resistant BCC patients harbor SMO mutations that either block drug binding or render SMO constitutively active Sharpe et al, 2015), indicating a dire need to identify downstream therapeutic targets to combat resistance to SMO antagonists. Recently, we observed an increase in mutations within ciliaassociated genes that resulted in disrupted primary cilia and allowed BCCs to convert to squamous cell carcinomas (SCCs) in a small percentage of tumors by shutting down the HH pathway and increasing the RAS/MAPK pathway to confer resistance to SMO antagonists (Kuonen et al, 2019). These converted SCCs are sensitive to MAPK inhibitors but represent a small percentage of the total resistant BCCs which largely maintain HH pathway activation .…”