Proteomic-based approach to investigate the role of iRhoms in cancer

ADAM17 (A Disintegrin and Metalloprotease 17), also known as tumour necrosis factor-alpha converting enzyme (TACE), is a cell surface enzyme that belongs to the ADAM family of proteases. It is involved in shedding of over 80 different substrates, including growth factors (e.g. the epidermal growth factor receptor EGFR–ligands), cytokines (e.g. tumour necrosis factor-α –TNF), adhesion molecules, endocytic receptors and others. ADAM17 plays a crucial role in numerous biological processes but if dysregulated, leads to several pathologies, such as auto-inflammatory disorders and cancer. ADAM17 facilitates cancer progression by promoting cell proliferation, survival, migration and invasion. For instance, its action on EGFR ligands can activate downstream signalling pathways like MAPK and PI3K, which are often impaired in cancer. Additionally, the cleavage of TNF contributes to inflammation, creating a pro-tumour microenvironment. The maturation, trafficking and function of ADAM17 are controlled by the seven-membrane-spanning proteins iRhom1 and iRhom2. At the cell surface, iRhoms can integrate the stimuli leading to ADAM17 activation, functioning as the regulatory subunit of an iRhom/ADAM17 catalytic complex. While iRhom1 and iRhom2 share largely redundant roles in facilitating ADAM17 maturation, they differ significantly in their regulation of substrate selectivity. Evidence suggests that iRhom2 can support the stimulated shedding of most ADAM17 substrates, whereas iRhom1 appears to enable stimulated shedding for only a limited subset of substrates, such as TGFα.On these premises, I conducted an unbiased mass spectrometry-based analysis to investigate in a systematic manner the sheddome resulting from the activity of iRhom1/ADAM17 or iRhom2/ADAM17 catalytic complexes. I found that the shedding of most ADAM17 substrates was supported by both iRhoms in murine and human fibroblasts. However, in the human fibroblast system, the efficiency of substrate shedding varied depending on whether ADAM17 was associated with iRhom1 or iRhom2, resulting in a continuum ranging from substrates preferentially cleaved by the iRhom1/ADAM17 complex to those preferentially cleaved by the iRhom2/ADAM17 complex. In addition to enabling a systematic investigation of substrate selectivity, this approach identified several novel ADAM17 substrates, including SIRPα, MXRA8 and the major histocompatibility complex class I (MHC-I). Validation of MHC-I shedding by orthogonal methods, such as Western blotting and flow cytometry, demonstrated that iRhom2 regulated its surface levels through mechanisms beyond shedding. The loss of iRhom2 impaired MHC-I stability and trafficking to the cell surface in various cancer cell lines, including leukaemia and chondrosarcoma. MHC-I, a transmembrane protein expressed in all eukaryotic nucleated cells, plays a key role in the presentation of foreign antigens, such as mutated oncogenic proteins, to the immune system. Its loss from the cell surface is of significant relevance in cancer, as it represents one of the immune evasion mechanisms adopted by several cancers, such as pancreatic ductal adenocarcinoma (PDAC). In conclusion, this PhD project offered useful prompts for future investigation into iRhom/ADAM17 biology, particularly in the context of cancer. It identified novel ADAM17 substrates with established roles in cancer progression, such as SIRPα and MHC-I. Notably, it uncovered a key regulatory mechanism of MHC-I that modulates its surface levels in cancer cells. This mechanism may play a significant role in how PDAC evades immune surveillance during cancer progression. The finding that iRhom2 enhances MHC-I levels at the cell surface highlights its potential as a therapeutic target to improve the efficacy of immunotherapy, particularly in poorly immunogenic cancers (cold tumours) like PDAC. Preliminary results on three PDAC cell lines with increasing aggressiveness – PaTU8902, Panc1, AsPC1 – showed that surface levels of MHC-I decreased lacking iRhom2 and rescued when iRhom2 is present. Investigating iRhom2 regulation of MHC-I, along with its counterpart iRhom1, in PDAC is my follow-up project.