Chondroitin sulfate proteoglycan 4 increases invasion of recessive dystrophic epidermolysis bullosa-associated cutaneous squamous cell carcinoma by modifying transforming growth factor-β signalling

Background Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic skin-blistering disorder that often progresses to metastatic cutaneous squamous cell carcinoma (cSCC) at chronic wound sites. Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell-surface proteoglycan that is an oncoantigen in multiple malignancies, where it modulates oncogenic signalling, drives epithelial-to-mesenchymal transition (EMT) and enables cell motility. Objectives To evaluate CSPG4 expression and function in RDEB cSCC. Methods RDEB cSCC cell lines were used to assess CSPG4-dependent changes in invasive potential, transforming growth factor (TGF)-beta 1-stimulated signal activation and clinically relevant cytopathology metrics in an in vitro full-thickness tumour model. CSPG4 expression in RDEB cSCC and non-RDEB cSCC tumours was analysed via immunohistochemistry and single-cell RNA sequencing (scRNA-Seq), respectively. Results Inhibiting CSPG4 expression reduced invasive potential in multiple RDEB cSCC cell lines and altered membrane-proximal TGF-beta signal activation via changes in SMAD3 phosphorylation. CSPG4 expression was uniformly localized to basal layer keratinocytes in fibrotic RDEB skin and tumour cells at the tumour-stroma interface at the invasive front in RDEB cSCC tumours in vivo. Analysis of published scRNA-Seq data revealed that CSPG4 expression was correlated with an enhanced EMT transcriptomic signature in cells at the tumour-stroma interface of non-RDEB cSCC tumours. Cytopathological metrics, for example nucleus : cell area ratio, were influenced by CSPG4 expression in in vitro tumour models. Conclusions We determined that CSPG4 expression in RDEB cSCC cell lines enhanced the invasive potential of tumours. Mechanistically, CSPG4 was found to enhance membrane-proximal TGF-beta-stimulated signalling via SMAD3, which is a key mediator of EMT in RDEB cSCC. The implication of these studies is that CSPG4 may represent a therapeutic target that can be leveraged for the clinical management of patients with RDEB cSCC. Recessive dystrophic epidermolysis bullosa (or 'RDEB') is a rare skin disease that affects 3 in 1 million children worldwide. People with RDEB lack an essential protein that allows the skin to resist tearing. This results in widespread blistering on the body and chronic wounds. People with RDEB can develop a lethal form of 'metastatic' skin cancer (where cancer cells spread to other parts of the body). However, before a cancer becomes metastatic, a cell must 'invade' its original tissue for it to access other parts of the body. This USA-based study investigated a protein called 'CSPG4'. This protein is present on the surfaces of cancer cells but not in most normal tissues. Our aim was to look at whether CSPG4 enhances the invasion of skin cancer associated with RDEB. This mechanism could be targeted by anti-cancer treatments. We used skin cancer cells from people with RDEB to demonstrate that CSPG4 increases the invasive ability of cancer cells. It does this by changing how the cells respond to a signal (called 'TGF-beta') that is present in high levels in RDEB skin. We used cancer tissue from people with RDEB to show that CSPG4 is produced by cells at the edges of tumours. These cells are responsible for invading the surrounding normal tissue. Our findings suggest that CSPG4 could be a therapeutic target. Targeting this protein may limit the ability of RDEB-associated skin cancer to invade and metastasize.