Deciphering the Functional Role of RIPK4 in Melanoma

Abstract

The receptor-interacting protein kinase 4 (RIPK4) plays an important role in the development and maintenance of various tissues including skin, but its role in melanoma has not been reported. Using patient-derived cell lines and clinical samples, we show that RIPK4 is expressed in melanomas at different levels. This heterogenous expression, together with very low level of RIPK4 in melanocytes, indicates that the role of this kinase in melanoma is context-dependent. While the analysis of microarray data has revealed no straightforward correlation between the stage of melanoma progression and RIPK4 expression in vivo, relatively high levels of RIPK4 are in metastatic melanoma cell lines. RIPK4 down-regulation by siRNA resulted in the attenuation of invasive potential as assessed by time-lapse video microscopy, wound-healing and transmigration assays. These effects were accompanied by reduced level of pro-invasive proteins such as MMP9, MMP2, and N-cadherin. Incubation of melanoma cells with phorbol ester (PMA) increased PKC-1β level and hyperphosphorylation of RIPK4 resulting in degradation of RIPK4. Interestingly, incubation of cells with PMA for short and long durations revealed that cell migration is controlled by the NF-κB signaling in a RIPK4-dependent (RIPK4high) or independent (RIPK4low) manner depending on cell origin (distant or lymph node metastasis) or phenotype (mesenchymal or epithelial).

The involvement of RIPK4 in TNF-α-stimulated IL-6 and IL-8 production by melanoma cells

“…Several studies indicate that RIPK4 can act as both a tumor suppressor and a tumor oncogene, depending on the type of cancer (Xu et al 2020 ). While it has been reported that RIPK4 is involved in skin tumorigenesis by regulating DLV2 or Pkp1 phosphorylation (Huang et al 2013 ; Lee et al 2017 ), our recent study shows an important role of this kinase in melanoma progression by involvement in NFκB and Wnt/β-catenin transduction signaling leading to increased migration, invasive potential, and cell proliferation as well tumor growth in vivo (Madej et al 2021 ; Wronski et al 2024 ).…”

“…Formalin-fixed paraffin-embedded 4 µm melanoma sections were used for both IHC (for RIPK4) and IF (for p65 NFκB subunit), as previously described (Janjetovic et al 2011 ; Madej et al 2021 ). The characteristic of melanoma patients is presented in Supplementary materials Table S1 .…”

“…IHC sections were assessed under the BX41 (Olympus Optical Co., Tokyo, Japan) microscope, ColorView III camera (Soft Imaging System, Hanover, Germany), and analySIS 3.2 software (Soft Imaging System). The sections were evaluated semiquantitatively, as previously described (Madej et al 2021 ). Since we observed heterogeneous agranular and granular staining, the assessment was performed separately for each staining pattern.…”

“…In this study, we confirmed using RNA-seq analyses that RIPK4 is potentially involved in key biological processes such as cell adhesion, migration, proliferation, differentiation, and inflammatory processes in melanoma. We focused on RIPK4-dependent differentially expressed genes (DEG) associated with inflammation in WM266.4 melanoma cells which exhibit high levels of RIPK4 expression (Madej et al 2021 ; Wronski et al 2024 ). We suggest that RIPK4 may play a significant role in modulating the expression of BIRC3, IL-8, and IL-6 in response to TNF-α stimulation via the NFκB or/and p38/MAPK signaling pathways.…”

Necroptosis-associated classification combined with tumor microenvironment characteristic analysis of cutaneous melanoma

“…Melanin pigmentation regulates the expression of multiple genes and plays a critical role in many cellular processes in melanoma cells 10 – 12 . It can affect the behavior of melanoma cells, their surrounding environment, susceptibility to the therapy, and viability of immune cells 10 , 13 , 14 .…”