Proteomic Profiling of BRAFV600E Mutant Colon Cancer Cells Reveals the Involvement of Nucleophosmin/c-Myc Axis in Modulating the Response and Resistance to BRAF Inhibition by Vemurafenib

Abstract

BRAFV600E mutations are found in approximately 10% of colorectal cancer patients and are associated with worse prognosis and poor outcomes with systemic therapies. The aim of this study was to identify novel druggable features of BRAFV600E-mutated colon cancer (CC) cells associated with the response and resistance to BRAFV600E inhibitor vemurafenib. Towards this aim, we carried out global proteomic profiling of BRAFV600E mutant vs. KRAS mutant/BRAF wild-type and double wild-type KRAS/BRAF CC cells followed by bioinformatics analyses. Validation of selected proteomic features was performed by immunohistochemistry and in silico using the TCGA database. We reveal an increased abundance and activity of nucleophosmin (NPM1) in BRAFV600E-mutated CC in vitro, in silico and in tumor tissues from colon adenocarcinoma patients and demonstrate the roles of NPM1 and its interaction partner c-Myc in conveying the resistance to vemurafenib. Pharmacological inhibition of NPM1 effectively restored the sensitivity of vemurafenib-resistant BRAF-mutated CC cells by down-regulating c-Myc expression and activity and consequently suppressing its transcriptional targets RanBP1 and phosphoserine phosphatase that regulate centrosome duplication and serine biosynthesis, respectively. Altogether, findings from this study suggest that the NPM1/c-Myc axis could represent a promising therapeutic target to thwart resistance to vemurafenib in BRAF-mutated CC.

The Sphingosine Kinase 2 Inhibitor ABC294640 Restores the Sensitivity of BRAFV600E Mutant Colon Cancer Cells to Vemurafenib by Reducing AKT-Mediated Expression of Nucleophosmin and Translationally-Controlled Tumour Protein

“…Specifically, nucleophosmin associates with unduplicated centrosomes; however, CDK2/cyclin E-mediated phosphorylation of nucleophosmin on threonine 199 promotes its dissociation from the centrosomes and enables initiation of centrosome duplication [ 23 ]. Previously, we found significantly increased abundance of cytoplasmic p -NPM1 (Thr199) in tumour tissue from BRAF-mutated in comparison with wild-type BRAF colon adenocarcinoma patients and demonstrated the role of p-NPM1 (Thr199) in mediating the resistance to vemurafenib in BRAF mutant colon cancer cells [ 16 ]. In the present study, we showed a significant decline in the expression levels of phospho-NPM1 (Thr199) in resistant RKO cells grown in the presence of ABC294640 and vemurafenib in comparison to either drug alone.…”

“…However, the non-responsiveness of colon cancer cells to vemurafenib also seems to include the mechanisms that regulate de novo ceramide synthesis pathway, as indicated by a marked rise in the baseline levels of sphinganine in resistant cells. This pathway can be metabolically triggered by metabolic loading with serine, whose increased biosynthesis pathway at the enzyme level has previously been shown to be a distinctive metabolic feature of BRAFV600E mutant colon cancer cells associated with the development of resistance to vemurafenib [ 15 , 16 ]. Our data suggest that the acquired resistance to vemurafenib could be, at least partially, associated with the induction of distinct pathways of ceramide formation whose differential activation is possible due to spatial separation of the enzymes regulating ceramide generation [ 17 ].…”

Association between Altered Oncogenic Signaling Pathways and Overall Survival of Patients with Metastatic Colorectal Cancer

“…In this study, the survival of patients with BRAF and MYC mutation co-occurrence was shorter than those with either BRAF or MYC mutation. Moreover, some in vitro studies demonstrated that MYC is a key element in tumor progression and treatment resistance of BRAF -mutated CRC [ 54 , 55 ]. Studies investigating effective treatments are warranted for this aggressive subtype of CRC carrying both the BRAF and MYC mutation.…”

Emerging actionable targets to treat therapy-resistant colorectal cancers

“…At variance co-targeting MEK and MCL-1 (either via silencing or via a smallmolecule antagonist) significantly increased cytotoxicity in vitro and reduced tumor growth in vivo [121] . Chen et al [122] demonstrated that in BRAF V600E CRC cell lines BRAF inhibitors upregulated the WNT/betacatenin pathway through activating FAK independently of EGFR and MEK signaling [Figure 2]. Accordingly, combined inhibition of BRAF/WNT pathways or BRAF/FAK pathways exerted strong synergistic antitumor both in vitro and in vivo [122] .…”

“…Chen et al [122] demonstrated that in BRAF V600E CRC cell lines BRAF inhibitors upregulated the WNT/betacatenin pathway through activating FAK independently of EGFR and MEK signaling [Figure 2]. Accordingly, combined inhibition of BRAF/WNT pathways or BRAF/FAK pathways exerted strong synergistic antitumor both in vitro and in vivo [122] . Recently, another mechanism of resistance to vemurafenib has been identified in the increased abundance and activity of nucleophosmin (NPM1), a protein that regulate centrosome duplication and histone assembly and that is induced by the inhibitor in resistant cells.…”