Dottoressa di ricerca

ciclo: XXXIV


The canonical Hedgehog (Hh) signalling pathway, driving activation of SMO and GLI1, has been associated with tumorigenesis and progression of different cancer types; however, its role in colorectal (CRC) remains controversial. The C-terminal domain (CTD) of the Hh receptor PTCH1 is known to have SMO and GLI-independent functions, including regulation of the autophagic flux, apoptosis, and MAPK signalling, known as “non-canonical” functions, in addition to regulate the protein’s turnover. Given that those novel functions are implicated in tumorigenesis, we explored the existence of PTCH1 CTD mutations in cancer specimens. Analysis of the TCGA Cancer Atlas database revealed relatively frequent somatic mutations in the CTD of PTCH1 in CRC: S1203(fs) and R1308(fs) and Y1316 (fs), which result in premature truncations of the CTD. To determine the pathogenic role of those mutations, we engineered the human metastatic CRC cell line SW620 using CRISPR/Cas9 to create two clones (C9 and C15) with frameshift-causing indel mutations in PTCH1 between S1203 and R1308. Truncation of the CTD increased cell proliferation, anchorage-independent growth and invasion in vitro compared to parental PTCH1 WT cells. Xenograft studies allowed us to confirm a strong increase in tumour growth in masses derived from one of the mutant CTD cells. Using molecular and RNAseq analysis we showed that PTCH1 CTD truncations induce EGFR-MAPK, PI3K-AKT and TGFb signalling pathways, known to crosstalk with Hh signalling in cancer cells. In this study, we showed that PTCH1 CTD truncations result in non-canonical GLI upregulation, having a pivotal role in the more aggressive phenotype of these cells. Moreover, we suggest GLI as a possible therapeutic target for inhibiting proliferation of PTCH1 CTD mutant cells. Complementary studies showed that both clones displayed an enhanced autophagic flux, impaired apoptosis and show evidence of metabolic reprogramming, which provides a proliferative advantage in this cancer CRC context. Altogether, our study highlights the role of PTCH1 CTD mutations in cancer progression through dysregulation of non-canonical Hh signalling.

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