Abstract A76: Vemurafenib induces differentiation, invasion, and stromal activation in a 3D human skin carcinoma model

Abstract

Abstract Adverse effects of melanoma therapy by the BRAFV600E-targeting small-molecule inhibitor Vemurafenib lead to the development of skin keratoacanthomas (KAs) and squamous cell carcinomas (SCCs) in a significant number of patients. Since KAs and SCCs are observed within a few weeks after onset of therapy, we hypothesize that Vemurafenib treatment is directly involved in the progression of preexisting oncogenic keratinocyte lesions. Using monolayer cultures of normal human dermal and epidermal cells as well as 3D organotypic co-cultures of the skin, we investigated direct and indirect effects of Vemurafenib (1 µM and 5 µM) on cutaneous cells. By treating BRAFV600E-mutant A375 melanoma cells, we confirmed that Vemurafenib blocks RAF/MEK/ERK-mitogen activated protein kinase (MAPK) signaling. Treatment of all BRAFwild type cells, including dermal fibroblasts, normal keratinocytes as well as premalignant HaCaT and benign-tumorigenic HaCaT-HRAS A-5 cells resulted in MAPK pathway hyper-activation, while Akt and p38 pathways were not affected. Despite MEK/ERK-activation, neither proliferation, nor apoptosis were altered in HaCaT keratinocytes. Moreover, genetic analyses of HaCaT cells treated for five weeks with Vemurafenib did not show genomic instability, but rather led to a selective growth advantage of genetic subpopulations. In order to investigate long-term effects of increased BRAF signaling, normal keratinocytes, HaCaT and HaCaT-HRAS A-5 cells were treated with Vemurafenib in organotypic co-cultures. After one week of Vemurafenib treatment all analyzed keratinocytes showed a dose-dependent increase in differentiation, resembling hyperkeratosis observed in Vemurafenib-treated melanoma patients. This was maintained after more than five weeks of continuous treatment, while we did not observe Vemurafenib-related toxicity. However, only HaCaT-HRAS A-5 cells invaded the dermal equivalent after three to five weeks of treatment in a dose-dependent manner. Thus, our findings strongly correlate with the in vivo data of rapidly developing hyperkeratosis and KAs/SCCs in Vemurafenib-treated melanoma patients. It yet has to be determined, whether the increased differentiation and the invasive growth are direct effects by Vemurafenib on the epidermal cells or if these are mediated indirectly through an altered microenvironment induced by MAPK-activated fibroblasts. In conclusion, we could demonstrate that the selective BRAFV600E inhibitor Vemurafenib directly triggers RAF/MEK/ERK-MAPK pathway hyper-activation in cutaneous BRAFwild type cells. Moreover, we observed increased differentiation of all analyzed keratinocytes and invasive growth of benign HaCaT-HRAS A-5 cells in organotypic cultures. This reflects the clinical observation of well-differentiated KAs and SCCs from preexisting lesions after Vemurafenib treatment, providing evidence for the hypothesis of a selective pressure for premalignant cells. Citation Format: Marius Tham, Manuel Berning, Petra Boukamp. Vemurafenib induces differentiation, invasion, and stromal activation in a 3D human skin carcinoma model. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A76. doi:10.1158/1538-7445.CHTME14-A76