Hippo tumour suppressor and Hedgehog pathway crosstalk at the primary cilia

Thesis

Inappropriate activation of the Hedgehog signalling pathway is implicated in broad range of cancers, particularly basal cell carcinoma and medulloblastoma. Recent attempts to target Hedgehog signalling clinically have yielded mixed patient responses, highlighting the need for further study into Hedgehog pathway regulation. Vertebrate Hedgehog signalling occurs within, and is dependent on, the primary cilia. The function of a critical negative regulator of Hedgehog signalling, Suppressor of Fused (Sufu), is regulated by post-translational modification within its intrinsically disordered region, spanning amino acids 279-360. Sufu has previously been shown to be stabilised by phosphorylation at Ser342/Ser346 and Ser352, where S342 phosphorylated Sufu appears to localise to the cilium tip. Here, S342 of Sufu was identified as a potential substrate of the Large Tumour Suppressor (LATS) kinases, LATS1 and LATS2, of the Hippo pathway. This project aimed to determine whether Sufu is regulated by LATS1/2 phosphorylation. LATS1/2 were found to interact with and phosphorylate Sufu at S342. This phosphorylation did not appear to affect the levels of Sufu or of its S352 phosphorylated form, indicating that LATS1/2 does not regulate Sufu stability. Immunofluorescent staining of endogenous Sufu confirmed that Sufu localises to the primary cilium and accumulates at the cilium tip. Reduction of LATS1/2 lead to removal of Sufu from primary cilia, suggesting that S342 phosphorylation of Sufu promotes the ciliary localisation of Sufu. Additionally, S352 phosphorylated Sufu localises to the basal body of primary cilia, confirming that Nek2A phosphorylation of Sufu inhibits its entry into the cilium. Comparison of the mRNA expression of two well-known Hedgehog target genes, GLI1 and PTCH1, between wild type and LATS1/2^-/- MEFs revealed that loss of LATS1/2 phosphorylation diminishes the repressive activity of Sufu on Hedgehog target gene transcription in response to Hedgehog pathway activation. Thus, LATS1/2 phosphorylation of Sufu at S342 appears to maintain its ability to repress Hedgehog target gene transcription. This adds to the growing body of literature showing that the Hippo and Hedgehog signalling pathways interact and coordinate to regulate cell proliferation. The data presented here unveils a novel role for LATS1/2 in restricting Hedgehog signalling via Sufu, and highlights the importance of LATS1/2 as tumour suppressors. This may identify LATS1/2 as potential targets in the treatment of Hedgehog signalling-dependent tumorigenesis.

Authors: Traynor, E

• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: Primary Cilia; Hippo pathway; Hedgehog signalling pathway
• Subjects: Cell Biology; Cell signalling; Cancer; Oncology; Protein phosphorylation; Protein regulation