Autophagic flux regulation by the cholesterol/H+ antiporter PTCH1

Lead Research Organisation: University of Leeds
Department Name: Inst of Molecular & Cellular Biology

Abstract

Development of a fertilized egg into a properly formed embryo requires a number of signals that perfectly orchestrate the formation of tissues and organs. One of those important signalling events is the so-called "Hedgehog" pathway that when absent provokes very serious congenital defects incompatible with life. Conversely, excessive activity of the Hedgehog pathway after birth is a common cause of childhood brain tumours and adult brain, skin, lung, prostate, and gut cancers. My group has contributed to the understanding of how the Hedgehog signals are perceived by cells and transmitted into different outcomes, depending on the tissue type and the context. Here, we propose to investigate a novel function of the Hedgehog pathway in regulating autophagy, the "self-eating" behaviour of cells. We found that the Hedgehog receptor PTCH1, a tumour suppressor, inhibits autophagy. Cells normally feed themselves from available nutrients; however, in conditions of starvation like those encountered by tumour cells that grow faster that the vasculature that nourishes them, cells degrade part of their contents to survive. Autophagy is essential for cancer cells survival. We will investigate how the Hedgehog pathway regulates autophagy at the molecular level. We will build onto our recent publication that shows that interaction between the C-terminal domain of PTCH1 and ATG101 is necessary for inhibition of autophagy, characterised by reduced number of acidic vesicles where degradation occurs. We propose that PTCH1 acts as a transporter, dissipating the proton gradient of those vesicles (autolysosomes) by a mechanism coupled to cholesterol transport. We will test this hypothesis and will investigate if this function of PTCH1 is lost by mutations of the C-terminal domain frequently found in cancer. We hope that our findings will increase the knowledge on new targets for cancer therapy and will reveal new ways in which cells adapt and survive to different stressors.

Technical Summary

The Hedgehog signalling pathway is a master regulator of growth and differentiation. Binding of any of three Hh isoforms to their receptor Patched1 (PTCH1) activates both canonical and non-canonical signals. PTCH1 is a 12-transmembrane protein with homology to bacterial RND permeates and to some cholesterol transporters. Recent the cryo-EM structures of PTCH1 revealed that it contains a tunnel with aligned cholesterol-like densities, which together with functional studies support the notion that PTCH1 is a cholesterol transporter. We have recently reported that PTCH1 interaction with ATG101 through its C-terminal domain (CTD) reduces autophagic flux and acidification of autolysosomes. This is a novel non-canonical function of the tumour suppressor PTCH1, since autophagy is essential for detoxification of protein aggregates and damaged organelles and for survival of cancer cells under conditions of scarce nutrient availability. Here, we aim to define the mechanism by which PTCH1 inhibits autophagic flux. We propose that interaction with ATG101 is required to localise PTCH1 to the autophagome/autolysosome membrane, where it causes alkalisation of the autolysosomal lumen by means of its cholesterol/H+ antiporter activity. We will determine if PTCH1 impairs autolysosome acidification by its RND domain and if cholesterol transport is necessary for inhibition of the autophagic flux by PTCH1. We also investigate if the Niemann-Pick C1 (NPC1)/NPC2 cholesterol handling system is necessary to stimulate PTCH1's translocation of cholesterol inside autolysosomes and dissipation of the proton gradient. We have also identified three frequent mutations in the CTD of PTCH1 in some epithelial cancers and will test if these mutations abolish the interaction of PTCH1 with ATG101 and, therefore, cannot inhibit autophagy, giving a growth advantage to cancer cells.

Planned Impact

This research project will generate fundamental knowledge about the tumour suppressor PTCH1 that will be useful to both increase its anti-autophagic activity (in cancer) and decrease it (in regenerative medicine, tissue engineering). Because of the tumour suppressor role of PTCH1, applications that mimic its function and increase its stability will be useful in the oncology field. Conversely, strategies that reduce PTCH1 activity will be of value for applications in stem cell-based therapies for neurodegenerative diseases, regenerative medicine (spinal cord injury), and skin replacement tissue engineering, which will benefit of transient controlled activation of canonical Hedgehog signalling.
The outcomes of our research will directly benefit the commercial private sector, since it will offer a platform for analysis, validation, and development of small inhibitors and tools for research of commercial value. We are interested in working with Avacta, a mid-size biotechnology company based in the UK (Wheterby). We will pursue a partnership, a formal collaboration to test lead inhibitors in canonical and non-canonical Hedgehog signalling at the University of Leeds with a small budget for pilot experiments and exchange of personnel for short periods of 2-3 months. At the end of the grant life, we will pursue an application for a CASE studentship with the industrial partner.
Avacta has strong links with the University of Leeds and the Astbury Biostructure Laboratory to screen and refine affimers (adhirons) that target proteins with affinities equivalent to antibodies, but which are cheap to generate in large quantities in bacteria. We will seek to identify adhirons that compete for binding of some regulators of PTCH1 that will serve for many purposes: 1) as research tools to investigate the effect of blocking single protein-protein interactions in the academic setting (academic beneficiaries), 2) as potential diagnostic tools to investigate the presence of common truncation in PTCH1 in cancer (leading toward personalised medicine), and 3) as the basis for modifications to increase their cell permeability, including specific targeting (therapeutics). This avenue of impact will increase research tools and therapeutic development, enrich the already fruitful collaboration between Avacta and many investigators at the University of Leeds, increase PDRAs training in a highly competitive technology and increase PDRAs employment attractiveness.
Dr. Riobo-Del Galdo will further reach to public beneficiaries through ideation and delivery of lectures targeted to high school student audience (KS4). She has established connections with a group of Science teachers at Allerton High School in Leeds, Mr. Jonathan Allcock and Dr. Kirsty Bryant, and has already been a guest lecturer at the school. Dr. Riobo-Del Galdo will undertake a training session of People Like Me, which disarms stereotypes in STEM careers for girls aged 11-14 to deliver an activity following the lectures at the school to inspire girls to opt in STEM-related A-level subjects. She will also continue her participation in University Open Days to educate prospective Sixth Form students and their parents in the development of impact that starts from basic cellular and structural biology research.

Publications

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Timmis AJ (2020) Another twist to the GLI code. in The Biochemical journal

 
Description While this award is 1 year and 3 months in, our timeline was severely impacted by the COVID pandemic, resulting in a temporary change of focus from mechanistic investigation of how the tumour suppressor PTCH1 suppresses autophagy to investigating what happens when PTCH1 cannot exert this function. For this, we pushed forward a new collaboration with the University of Rome, from which we had generated cells carrying a mutation in PTCH1 described in colon cancer, which we predicted to abolish its function on autophagy. We found that the PTCH1 mutation prevented its interaction with the ATG101 protein, impaired suppression of autophagy and increased cell division and survival (manuscript in preparation). Comparison of the genes expressed in those mutant cells with normal cells was done by bioinformatic analysis of RNA-seq- which we could carry out remotely. We found a profound change in many signalling pathways, in addition to those regulating autophagy: metabolic pathways in cancer, MAPK signalling, and ribosomal proteins, which will be further investigated in future projects for which we are submitting applications.
After the labs re-opened at low occupancy levels, we could move on with the proposed project. We successfully generated PTCH1, NPC1 and NPC2-deficient cells using CRISPR/Cas9. We optimised fluorescent cholesterol staining in cells using filipin. We generated PTCH1 mutants that prevent cholesterol mobilisation and we are currently testing their capacity to block autophagy completion. Overall, we have had a good progress considering that the report represents the work of 8 months.
Exploitation Route The findings of this project at the moment point to existence of somatic mutations in PTCH1 which do not perturb canonical signalling but cannot restrain autophagy, leading to a proliferative advantage of the cells carrying the mutation. Indeed, we confirmed that around 5% of cancers of the colon and endometrium carry these mutations, making autophagy an attractive process to target in a group of patients. Our RNA-seq analysis, however, reveal unexpected roles of the PTCH1 C-terminal domain beyond autophagy in the regulation of cell growth, receptor tyrosine kinase signalling and even ribosomal paralogy usage that will bring new knowledge to the academic community and provide mechanistic links to other growth regulatory pathways that are of interest to the Pharma and biotech industries.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Collaboration with Prof. Gianluca Canettieri 
Organisation Sapienza University of Rome
Country Italy 
Sector Academic/University 
PI Contribution Our study of PTCH1 in regulation of autophagy generated an interest of studying its role in cancer development. This was formalised by joint supervision of a PGR student Ms Begoña Caballero Ruiz, funded by the University of Rome. The PGR has already generated a large amount of data from colon cancer cells engineered to carry a truncation in the C-terminal domain of PTCH1, which is necessary for its regulatory role on autophagy. We corroborated that the mutant cells show enhanced basal autophagy and generated RNA-seq data with very interesting leads. The findings are currently being drafted for 2 publications.
Collaborator Contribution The partner contributes to the full stipend and tuition support of the PRG, as well as to consumables related to about 50% of the PhD duration.
Impact Articles in preparation: 1- Somatic mutations in the C-terminal domain of PTCH1 increase autophagic flux in normal and cancer cells. Cintli C. Morales-Alcala, M. Begoña Caballero Ruiz, Gianluca Canettieri and Natalia Riobo-Del Galdo (in preparation for Cancers) 2- Mutations in the C-terminal domain of PTCH1 promote colorectal cancer cell growth. M. Begoña Caballero Ruiz, Gianluca Canettieri and Natalia Riobo-Del Galdo
Start Year 2019