The role of stromal Wnt/Beta-catenin and epidermal Ras-Raf-MEK-ERK MAPK signaling in human squamous cell carcinoma

Abstract

The prevalence of cutaneous squamous cell carcinoma (cSCC) is constantly increasing worldwide, however, the mechanisms driving tumorigenesis are not yet understood. The aim of this study was to understand the contribution of the Wnt/Beta-catenin and the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathways in cSCC development and progression using 2D keratinocyte monolayer cultures, 3D organotypic cultures of the skin (OTCs) as well as primary human cSCC samples. Deregulated canonical Wnt signaling in tumor cells, as detected by nuclear translocation of Beta- catenin, is well described for a number of human cSCCs. Here, we found that aberrant Wnt/Beta- catenin activation is not restricted to tumor cells, but was additionally or exclusively detected in stromal fibroblasts. This suggested a second role of aberrant Wnt signaling in the stroma of developing cSCCs. Gene expression analysis of fibroblasts identified Interleukin-8 (IL-8), C-C motif chemokine 2 (CCL-2) and Matrix metalloproteinase-1 (MMP-1) as targets of Wnt/Beta-catenin. In agreement, IL-8 and CCL-2 were secreted by Wnt-3a-stimulated fibroblasts in OTCs and could also be detected in human cSCCs in situ. As consequence, IL-8 and CCL-2 caused hyper-proliferation and early invasion of non-tumorigenic HaCaT keratinocytes. Importantly, neutralizing antibodies against IL-8 and CCL-2 abolished these Wnt- dependent effects in OTCs. MMP-1 was also highly expressed in Wnt-3a-stimulated OTCs as well as in human cSCCs and was shown to be active. Furthermore, degradation of the basement membrane correlated with invasive growth of benign-tumorigenic H-Ras G12V -transfected HaCaT-RAS A-5 cells, demonstrating the importance of the Wnt-dependent fibroblast-keratinocyte cross-talk for skin cancer progression. Ras-Raf-MEK-ERK MAPK signaling is constitutively active in most melanomas due to a B-Raf V600E - mutation leading to increased proliferation and survival. Clinical trials targeting oncogenic B-Raf in melanoma by mutation-specific inhibitors like Vemurafenib displayed high response rates, but caused at the same time development of cSCCs. This suggested a direct involvement of Vemurafenib in the progression of pre-existing lesions. Vemurafenib stimulation of B-Raf V600E -mutant A375 melanoma cells confirmed a block in MEK-ERK signaling, while all B-Raf wild-type cells, such as dermal fibroblasts and different keratinocytes, revealed MEK-ERK hyper-activation. Despite MAPK-activation, neither proliferation nor survival was altered in the keratinocytes. Analyses of continuously Vemurafenib treated HaCaT cells did not show genomic instability, but rather led to a selective growth advantage of genetic subpopulations. To study the role of aberrant B-Raf signaling in the tissue context, OTCs were treated with Vemurafenib. As result, Vemurafenib directly increased differentiation of the normal and transformed keratinocytes, resembling the hyperkeratotic skin phenotype of many melanoma patients. Remarkably, Vemurafenib stimulation of HaCaT-RAS A-5 cells caused high MMP-1 and MMP-3 expression, which correlated with Collagen remodeling and invasion into the stroma. Since both, differentiation and invasion were abolished by co-treatment with the MEK-inhibitor Cobimetinib and as patients receiving the same therapy showed highly decreased cSCC numbers, this strongly suggests that the phenotypes in in vitro as well as the rapidly developing well-differentiated cSCCs in vivo are direct results of Vemurafenib-induced ERK-activation. Taken together, this work unraveled two novel aspects, namely Wnt/Beta-catenin deregulation in the tumor-stroma and aberrant Raf-MEK-ERK MAPK signaling in keratinocytes as oncogenic events driving cSCC progression.