Abstract
RAS mutations occur in more than 30% of all human cancers but efforts to directly target mutant RAS signaling as a cancer therapy have yet to succeed. As alternative strategies, RAF and MEK inhibitors have been developed to block oncogenic signaling downstream of RAS. As might be expected, studies of these inhibitors have indicated that tumors with RAS or BRAF mutations display resistance RAF or MEK inhibitors. In order to better understand the mechanistic basis for this resistance, we conducted a RNAi-based screen to identify genes that mediated chemoresistance to the RAF kinase inhibitor RAF265 in a BRAF (V600E) mutant melanoma cell line that is resistant to this drug. In this way, we found that knockdown of protein kinase D3 (PRKD3) could enhance cell killing of RAF and MEK inhibitors across multiple melanoma cell lines of various genotypes and sensitivities to RAF265. PRKD3 blockade cooperated with RAF265 to prevent reactivation of the MAPK signaling pathway, interrupt cell cycle progression, trigger apoptosis, and inhibit colony formation growth. Our findings offer initial proof-of-concept that PRKD3 is a valid target to overcome drug resistance being encountered widely in the clinic with RAF or MEK inhibitors.
Highlights
RAS–RAF mitogen-activated protein kinase (MAPK) signaling cascade plays a central role in the regulation of cell proliferation and survival, whereas the deregulation of this pathway frequently occurs in human cancers [1,2,3]
By using A2058 and A375 (RAF265 sensitive cell line) as melanoma cellular models, we showed that protein kinase D3 (PRKD3) inhibition cooperates with RAF265 to prevent the reactivation of MAPK signaling pathway, induce PARP cleavage and caspase activity, interrupt cell-cycle progression, and inhibit colony formation
As other RAF or MAP/ERK kinase (MEK) inhibitors, RAF265 showed less efficacy in tumors with RAS mutations compared with tumors with a BRAF mutation [31]
Summary
RAS–RAF mitogen-activated protein kinase (MAPK) signaling cascade plays a central role in the regulation of cell proliferation and survival, whereas the deregulation of this pathway frequently occurs in human cancers [1,2,3]. As mutations in RAS or BRAF occur in more than 30% of human cancers, these proteins are very attractive therapeutic targets in many cancer types. BRAF mutations occur in about 7% of human cancers, with highest prevalence in melanomas (66%) and thyroid (35%–70%) tumors [4, 5]. 80% of all BRAF mutations are concentrated on a single substitution of glutamic acid for valine (V600E) within the kinase domain [4]. Compared with BRAF, mutations in the 2 RAF isoforms, ARAF and CRAF, are rarely found in human cancers, which is likely due to lower basal kinase activities [6, 7].
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