Disruption of TWIST1-RELA binding by mutation and competitive inhibition to validate the TWIST1 WR domain as a therapeutic target

Abstract

BackgroundMost cancer deaths result from tumor cells that have metastasized beyond their tissue of origin, or have developed drug resistance. Across many cancer types, patients with advanced stage disease would benefit from a novel therapy preventing or reversing these changes. To this end, we have investigated the unique WR domain of the transcription factor TWIST1, which has been shown to play a role in driving metastasis and drug resistance.MethodsIn this study, we identified evolutionarily well-conserved residues within the TWIST1 WR domain and used alanine substitution to determine their role in WR domain-mediated protein binding. Co-immunoprecipitation was used to assay binding affinity between TWIST1 and the NFκB subunit p65 (RELA). Biological activity of this complex was assayed using a dual luciferase assay system in which firefly luciferase was driven by the interleukin-8 (IL-8) promoter, which is upregulated by the TWIST1-RELA complex. Finally, in order to inhibit the TWIST1-RELA interaction, we created a fusion protein comprising GFP and the WR domain. Cell fractionation and proteasome inhibition experiments were utilized to elucidate the mechanism of action of the GFP-WR fusion.ResultsWe found that the central residues of the WR domain (W190, R191, E193) were important for TWIST1 binding to RELA, and for increased activation of the IL-8 promoter. We also found that the C-terminal 245 residues of RELA are important for TWIST1 binding and IL-8 promoter activation. Finally, we found the GFP-WR fusion protein antagonized TWIST1-RELA binding and downstream signaling. Co-expression of GFP-WR with TWIST1 and RELA led to proteasomal degradation of TWIST1, which could be inhibited by MG132 treatment.ConclusionsThese data provide evidence that mutation or inhibition of the WR domain reduces TWIST1 activity, and may represent a potential therapeutic modality.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3169-9) contains supplementary material, which is available to authorized users.

Hyaluronic acid conjugated nanoparticle delivery of siRNA against TWIST reduces tumor burden and enhances sensitivity to cisplatin in ovarian cancer

“…Moreover, MSN-HA-siQ control mice developed great amounts of tumor, metastasis, and ascites as compared to MSN-HA-siTWIST + cisplatin group (Figure 5 A-C) which showed no large dispersed lesions. It is important to note that cisplatin + MSN-siTWIST hindered cancer growth by almost 50%, cumulatively, (Figure 5, A-C), as shown previously 32 . However, MSN-HA-siTWIST + cisplatin combination therapy inhibited tumor growth to an even more significant degree, with almost a 75% drop in number of tumor in comparison to CISPLATIN alone and 90% compared to controls (Figure 5, A-C).…”

“…The chemotherapy only treatment mouse group (CISPLATIN only) was left with 60% less tumor than the siQ control. MSN-siTWIST with cisplatin, without HA, provided similar results to our previous studies 32 , reducing tumor volume an additional 20% compared to cisplatin alone; whereas the MSN-HA-siTWIST with cisplatin chemotherapy treatment group (MSN-HA-siTWIST + cisplatin) exhibited the most promising outcome, with almost 90% decrease in tumor burden as compared to the siQ control and measured by bioluminescence (Figure 4).…”

“…TWIST has been shown to be significantly associated with metastasis, EMT, drug resistance, and poor prognosis 12, 44-47 , thus making it an optimal target for ovarian cancer therapy. We and others have shown that the effective knockdown of TWIST leads to a decrease in motility, followed by an increase in chemotherapy sensitivity in ovarian cancer 22, 32, 47 . The delivery of our siRNA by MSN-HA was shown to have a profound effect on both TWIST silencing and chemoresistance in the EOC model (Figure 3 C-D).…”

Cyclophilin A regulates the apoptosis of A549 cells by stabilizing Twist1 protein

“…It has been reported that p65 increased the nuclear expression of Twist1 and interacted with Twist1 after TNF-α stimulation (Roberts et al, 2017;Yu et al, 2014). In addition, previous studies have pointed out that CypA interacts with p65 (Sun et al, 2014).…”

“…It has been reported that Twist1 interacts with p65 to regulate TNF-α-mediated EMT (Li et al, 2012;Roberts et al, 2017;Yu et al, 2014). We performed co-immunoprecipitation assays to explore the effect of CypA on the interaction between Twist1 and p65.…”