RSPO1-mutated keratinocytes from palmoplantar keratoderma display impaired differentiation, alteration of cell-cell adhesion, EMT-like phenotype and invasiveness properties: implications for squamous cell carcinoma susceptibility in patients with 46XX disorder of sexual development.

BACKGROUND: Secreted R-spondin (RSPO) proteins play a key role in reproductive organ development, epithelial stem cell renewal and cancer induction by reinforcing canonical Wnt signaling. We have previously reported that palmoplantar keratoderma (PPK), predisposition to cutaneous squamous cell carcinoma (SCC) development and sex reversal segregate as autosomal recessive trait in patients carrying RSPO1-mutations. Although our previous findings suggested that RSPO1 secreted from fibroblasts regulates keratinocyte growth or differentiation, the role of this protein in the epidermis remains largely unexplored. Our study was aimed at expanding the phenotypic, molecular and functional characterization of RSPO1-mutated skin and keratinocytes. RESULTS: Cultured primary keratinocytes from PPK skin of a RSPO1-mutated XX-sex reversed patient displayed highly impaired differentiation and epithelial-mesenchymal transition (EMT)-like phenotype. Interestingly, RSPO1-mutated PPK skin expressed markers of increased proliferation, dedifferentiation and altered cell-cell adhesion. Furthermore, all these signs were more evident in SCC specimens of the patient. Cultured PPK patient's keratinocytes exhibited increased expression of cell‒matrix adhesion proteins and extracellular matrix remodeling enzymes. Moreover, they showed invasiveness properties in an organotypic skin model in presence of PPK fibroblasts, which behave like cancer-associated fibroblasts. However, the co-culture with normal fibroblasts or treatment with the recombinant RSPO1 protein did not revert or reduce the EMT-like phenotype and invasion capability of PPK keratinocytes. Notably, RSPO1-mutated PPK fibroblasts induced a hyperproliferative and dedifferentiated phenotype of age-matched normal control plantar keratinocytes. Wnt signaling has a key role in both PPK promotion and SCC development. Accordingly, Wnt mediators were differentially expressed in both PPK keratinocytes and skin specimens of RSPO1-mutated patient compared to control. CONCLUSIONS: Altogether our data indicate that the absence of RSPO1 in patients with 46XX disorder of sexual development affects the skin microenvironment and epidermal integrity, thus contributing to the risk of SCC tumorigenesis in palmoplantar regions exposed to major frictional stresses.

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-ß 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-ß, 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-ß signalling pathway and indicated a loss of TGF-ß 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.

Letter to the Editor: "Ion Channels in Brain Metastasis"-Ion Channels in Cancer Set up and Metastatic Progression.

The review by Klumpp, L. et al. entitled Ion Channels in Brain Metastasis [1] discusses the role of ion channels in breast cancer, lung cancer and melanoma in metastatic tropism to the brain [...].

Ion channels expression and function are strongly modified in solid tumors and vascular malformations.

BACKGROUND: Several cellular functions relate to ion-channels activity. Physiologically relevant chains of events leading to angiogenesis, cell cycle and different forms of cell death, require transmembrane voltage control. We hypothesized that the unordered angiogenesis occurring in solid cancers and vascular malformations might associate, at least in part, to ion-transport alteration. METHODS: The expression level of several ion-channels was analyzed in human solid tumor biopsies. Expression of 90 genes coding for ion-channels related proteins was investigated within the Oncomine database, in 25 independent patients-datasets referring to five histologically-different solid tumors (namely, bladder cancer, glioblastoma, melanoma, breast invasive-ductal cancer, lung carcinoma), in a total of 3673 patients (674 control-samples and 2999 cancer-samples). Furthermore, the ion-channel activity was directly assessed by measuring in vivo the electrical sympathetic skin responses (SSR) on the skin of 14 patients affected by the flat port-wine stains vascular malformation, i.e., a non-tumor vascular malformation clinical model. RESULTS: Several ion-channels showed significantly increased expression in tumors (p < 0.0005); nine genes (namely, CACNA1D, FXYD3, FXYD5, HTR3A, KCNE3, KCNE4, KCNN4, CLIC1, TRPM3) showed such significant modification in at least half of datasets investigated for each cancer type. Moreover, in vivo analyses in flat port-wine stains patients showed a significantly reduced SSR in the affected skin as compared to the contralateral healthy skin (p < 0.05), in both latency and amplitude measurements. CONCLUSIONS: All together these data identify ion-channel genes showing significantly modified expression in different tumors and cancer-vessels, and indicate a relevant electrophysiological alteration in human vascular malformations. Such data suggest a possible role and a potential diagnostic application of the ion-electron transport in vascular disorders underlying tumor neo-angiogenesis and vascular malformations.

Identification of serum regression signs in infantile hemangioma.

Vessel proliferation underlies a number of serious pathological conditions. Infantile Hemangioma (IH) is a low-aggressive vascular tumor, interesting as an in vivo model of spontaneous tumor regression. Identifying mechanisms underlying IH spontaneous regression may then help to elucidate vessel-growth control, strongly deregulated in other serious conditions such as sarcoma, melanoma, diabetic retinopathy. The present study was aimed at identifying early regression indicators within hematological parameters. Thirty-four blood samples were collected from IH diagnosed babies (20-months median age), spontaneously regressing with age. Nineteen serum standard blood-tests were carried out using diagnostic reagents; in addition, serum-expression of 27 cytokine/chemokines was measured. Samples were divided in three age-groups, namely ≤ 12, 13 to 24 and >24 months-age, respectively. Red-cells count, Hemoglobin, Hematocrit, Neutrophils, Lymphocytes, MCP-1 and MIP-1beta were significantly different in the three age-groups, according to one-way ANOVA analysis. The same parameters showed a significant Pearson-correlation with age, supporting the direct link of age with IH-regression. ROC analysis showed that red-cells count, Hemoglobin, Hematocrit, MCP-1 and MIP-1beta levels significantly discriminate IH in the proliferating-phase from IH in the regressing-phase. Such data indicate for the first time that standard hematological tests and cytokine serum-expression values may effectively discriminate proliferating- from regressing-IH, unrevealing early regression signs, and demonstrate that standard blood-tests may have novel unsuspected diagnostic/prognostic relevance in altered vessel-growth conditions.