RSPO1-mutated fibroblasts from non-tumoural areas of palmoplantar keratoderma display a cancer-associated phenotype.

R-spondin (RSPO)1 is a fibroblast-secreted protein that belongs to the R-spondin protein family which is essential for reproductive organ development, epithelial stem cell renewal and cancer induction or suppression. RSPO1 gene mutations cause palmoplantar hyperkeratosis with squamous cell carcinoma (SCC) of the skin, 46XX sex reversal and true hermaphroditism. To characterize RSPO1-deficient skin fibroblasts derived from two patients with mutations in RSPO1, with palmoplantar hyperkeratosis, recurrent SCC and 46XX sex reversal, to provide further insight into disease-related skin tumourigenesis. Fibroblast cultures from non-tumoural palmoplantar skin biopsies were established to evaluate features and properties that may be altered at cancer onset, i.e. proliferation, extracellular matrix contraction and invasion, as well as TGF-β and matrix metalloproteinase (MMP) secretion. Fibroblasts demonstrated increased proliferative potential in vitro, a high level of collagen contraction and invasion by SCC cells, release of high levels of pro-inflammatory and pro-fibrotic TGF-β, and increased expression of MMP1 and MMP3. Analysis of the expression of selected proteins associated with RSPO1-activated pathways confirmed sustained activation of the TGF-β signalling pathway and indicated a loss of TGF-β inhibitory feedback. Also, treatment of fibroblasts with a recombinant RSPO1 protein aggravated this pro-inflammatory phenotype, suggesting caution in designing therapeutic strategies based on restoration of protein function. Our findings indicate that fibroblasts from RSPO1-mutated patients behave similarly to cancer-associated fibroblasts. Chronic inflammation and fibrotic changes in palmoplantar skin may play a role in SCC development and recurrence, possibly by irreversibly activating the tumourigenic phenotype of fibroblasts.