Autophagic flux regulation by the cholesterol/H+ antiporter PTCH1

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.

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.

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.