German Cancer Research Center (DKFZ) Heidelberg, Baden-Wurt
L. Li1,2, K. Shehzad2, R. L. Perez1, R. Jennemann3, P. J. Nelson2, R. Sandhoff3, and P. E. Huber1; 1German Cancer Research Center (dkfz) and University Hospital Center, Molecular and Radiation Oncology, Heidelberg, Germany, 2Medical Clinic and Polyclinic IV LMU, Munich, Germany, 3German Cancer Research Center (dkfz), Lipid Pathobiochemistry, Heidelberg, Germany
Purpose/Objective(s): Dermal inflammation and fibrosis are common and potentially severe acute and chronic side effects of cancer radiotherapy in various anatomical sites. The Dickkopf protein family plays a role in regulating Wnt signaling, known for its significance in fibrogenesis across multiple organs. However, the role of Dickkopf–Wnt axis signaling in the context of radiation-induced fibrosis, particularly in the skin, remains largely unknown. This study aims to investigate the involvement of Dickkopf-3 (DKK3) - Wnt pathway in radiation-induced dermal inflammation and fibrosis both in vitro and in a mouse model in vivo. Materials/
Methods: In vivo, global DKK3 knockout C57BL/6 mice, wildtype littermate controls, and DKK3/Wnt reporter mice were exposed to a single dose of 20 Gy X-rays to the thorax. Skin biopsy specimens were systematically collected for histology (H&E, aSMA, collagen [Goldner], Ki67) and immunostaining. Quantitative analysis focused on hyperplasia, fibrosis development, and immune responses. In vitro, specific DKK3 knockout/overexpressing immortalized human keratinocytes were compared to wild type. Radiosensitivity was assessed through clonogenicity and proliferation assays; apoptosis, DNA-DSB damage, and repair dynamics via ?H2AX FACS. Changes on the protein and RNA level were measured, as well as DKK3 status-induced modifications of the interplay between keratinocytes and human macrophages using a coculture model. Results: In vitro and in vivo IR induced DKK3 in keratinocytes. In vivo, DKK3-/- mice exhibited partial protection against clinical radiation damage, presenting reduced ulceration and hair loss. Histologically, DKK3-/- mice displayed less hyperplasia, reduced cell proliferation, and notably diminished fibrosis, evidenced by decreased collagen and aSMA deposition. Intriguingly, irradiated DKK3-/- mice showed an increase, not a decrease, in macrophages (F4/80) and monocytes (HR3) in the skin compared to WT mice, which exhibited a macrophage polarization shift towards M2, in line with the coculture data in vitro. The global radiosensitivity and DSB repair dynamics differences between DKK+/+ and DKK3-/- keratinocytes were relatively small. However, IR-induced reactive oxygen species (ROS) led to DKK3 overexpression and activated the canonical Wnt pathway via TGF-ß, resulting in upregulated fibrosis signaling on fibroblasts. Conclusion: Our in vitro and in vivo findings suggest that DKK3 and Wnt play regulatory roles in radiation-induced skin damage and fibrosis, with keratinocytes being crucial in this process. Downregulation of DKK3 may mitigate radiation-induced skin damage and fibrosis. The attenuated fibrosis in DKK3-/- mice is associated with reduced ROS, TGF-ß, and canonical Wnt signaling, as well as an increase in repolarized macrophages. Overall, the data suggest that DKK3 could be a potential therapeutic target and diagnostic marker for radiation-induced skin fibrosis.
