Vemurafenib and Dabrafenib Downregulates RIPK4 Level

Abstract

Vemurafenib and dabrafenib are BRAF kinase inhibitors (BRAFi) used for the treatment of patients with melanoma carrying the V600E BRAF mutation. However, melanoma cells develop resistance to both drugs when used as monotherapy. Therefore, mechanisms of drug resistance are investigated, and new molecular targets are sought that could completely inhibit melanoma progression. Since receptor-interacting protein kinase (RIPK4) probably functions as an oncogene in melanoma and its structure is similar to the BRAF protein, we analyzed the impact of vemurafenib and dabrafenib on RIPK4 in melanomas. The in silico study confirmed the high similarity of BRAF kinase domains to the RIPK4 protein at both the sequence and structural levels and suggests that BRAFi could directly bind to RIPK4 even more strongly than to ATP. Furthermore, BRAFi inhibited ERK1/2 activity and lowered RIPK4 protein levels in BRAF-mutated melanoma cells (A375 and WM266.4), while in wild-type BRAF cells (BLM and LoVo), both inhibitors decreased the level of RIPK4 and enhanced ERK1/2 activity. The phosphorylation of phosphatidylethanolamine binding protein 1 (PEBP1)—a suppressor of the BRAF/MEK/ERK pathway—via RIPK4 observed in pancreatic cancer did not occur in melanoma. Neither downregulation nor upregulation of RIPK4 in BRAF- mutated cells affected PEBP1 levels or the BRAF/MEK/ERK pathway. The downregulation of RIPK4 inhibited cell proliferation and the FAK/AKT pathway, and increased BRAFi efficiency in WM266.4 cells. However, the silencing of RIPK4 did not induce apoptosis or necroptosis. Our study suggests that RIPK4 may be an off-target for BRAF inhibitors.

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

“…This upregulation of RGS2 inhibits MAPK and AKT signaling, thereby preventing melanoma cell growth, as demonstrated by Lin et al (Lin et al 2021 ). Consistent with our previous findings, we noted a significant reduction in the activation of the AKT/FAK signaling axis in WM266.4 cells with a decreased level of RIPK4 (Madej et al 2023 ), which may be a result of induced RGS2 expression.…”

“…1 A). The tested WM266.4 cell line exhibited high expression of this kinase, as indicated by our previous data (Madej et al 2021 , 2023 ). After 48 h of cell transfection, with siRNA specific for RIPK4 or negative siRNA as a control, isolated RNA was sequenced using the BGISEQ500 platform.…”

“…Interestingly, when analyzing GO terms describing biological processes, DEGs were enriched in transcripts involved in “cellular metabolism” and “regulation of gene expression,” as well as terms related to cancer progression such as “motility/adhesion,” “immune system process,” “cell differentiation,” “regulation of apoptosis,” and “proliferation” clusters. We previously observed that RIPK4 downregulation inhibits proliferation, migration, and tumor growth in WM266.4 and A375 cells (Madej et al 2021 , 2023 ; Wronski et al 2024 ). In the current study, we identified DEGs associated with these key biological processes: adhesion/migration (41 genes), differentiation (52 genes), cell proliferation (45 genes), and inflammation-related genes (42 genes).…”

“…In addition, the analysis confirms our previous findings that silencing RIPK4 in WM266.4 cells significantly reduces their proliferative potential. This effect is attributed to the inhibition of the cyclin-dependent kinase pathway CDK2/CDK6, suggesting impaired proliferation rather than increased cell death as the underlying mechanism (Madej et al 2021 , 2023 ). Interestingly, proteins responsible for regulating cell adhesion, such as NOTCH3 (Pekkonen et al 2018 ) and ADAM12 (Roy et al 2017 ), are known to also play a role in controlling cell proliferation.…”

“…The structural similarity of the RIPK4 kinase domain to both BRAF wild-type and V600 mutant proteins suggests its potential as a target for BRAF inhibitors (BRAFi). In the presence of vemurafenib or dabrafenib, the level of RIPK4 decreases regardless of the presence of BRAF mutations and independently of BRAF/ERK/MEK signaling (Madej et al 2023 ). Reducing the level of a potential oncogene such as RIPK4, although it seems desirable, may result in other side effects.…”