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.
Highlights
Most cancer deaths result from tumor cells that have metastasized beyond their tissue of origin, or have developed drug resistance
We have previously shown that the WR domain mediates a binding interaction between TWIST1 and the NF-κB subunit RELA, and that this interaction leads to transcriptional upregulation of the inflammatory cytokine interleukin 8 (IL-8) in a manner independent of TWIST1-DNA binding [18]
In order to determine the contribution of individual amino acids in the WR domain to TWIST1-RELA binding, we transiently expressed RELA and all TWIST1 alleles in HEK293 cells and performed co-immunoprecipitation (CoIP)
Summary
Most cancer deaths result from tumor cells that have metastasized beyond their tissue of origin, or have developed drug resistance. 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. The majority of cancer deaths are the result of tumor cells metastasizing beyond their original niche [1]. In ovarian cancer in particular, late stage at discovery and drug resistance are major challenges [3, 4], resulting in five year survival rates of approximately 25% [3, 5]. TWIST1 has been implicated in number of pro-progression phenotypes in cancers, including angiogenesis [9], increased cancer cell stemness [10,11,12,13], and cell survival signaling [14, 15] (Fig. 1a)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
