Design and Characterization of a Cell-Penetrating Peptide Derived from the SOX2 Transcription Factor.

Neha S Gandhi,Adil Malik,Pilar Blancafort,Wan Jun Tie,Jyotsna Batra,Ricardo L Mancera,Kimberly A Young,Yu Jie Kan,Edina Wang,Anabel Sorolla

doi:10.3390/ijms22179354

Abstract

SOX2 is an oncogenic transcription factor overexpressed in nearly half of the basal-like triple-negative breast cancers associated with very poor outcomes. Targeting and inhibiting SOX2 is clinically relevant as high SOX2 mRNA levels are positively correlated with decreased overall survival and progression-free survival in patients affected with breast cancer. Given its key role as a master regulator of cell proliferation, SOX2 represents an important scaffold for the engineering of dominant-negative synthetic DNA-binding domains (DBDs) that act by blocking or interfering with the oncogenic activity of the endogenous transcription factor in cancer cells. We have synthesized an interference peptide (iPep) encompassing a truncated 24 amino acid long C-terminus of SOX2 containing a potential SOX-specific nuclear localization sequence, and the determinants of the binding of SOX2 to the DNA and to its transcription factor binding partners. We found that the resulting peptide (SOX2-iPep) possessed intrinsic cell penetration and promising nuclear localization into breast cancer cells, and decreased cellular proliferation of SOX2 overexpressing cell lines. The novel SOX2-iPep was found to exhibit a random coil conformation predominantly in solution. Molecular dynamics simulations were used to characterize the interactions of both the SOX2 transcription factor and the SOX2-iPep with FGF4-enhancer DNA in the presence of the POU domain of the partner transcription factor OCT4. Predictions of the free energy of binding revealed that the iPep largely retained the binding affinity for DNA of parental SOX2. This work will enable the future engineering of novel dominant interference peptides to transport different therapeutic cargo molecules such as anti-cancer drugs into cells.

Highlights

IntroductionTranscription factors (TFs) are proteins capable of binding onto specific DNA sequences and affect the regulation of subsequent transcription by interacting either through creativecommons.org/licenses/by/ 4.0/)
Targeting and inhibiting SOX2 is clinically relevant as high SOX2 mRNA levels positively correlate with decreased overall survival and progression-free survival before 270 months in patients affected with breast cancer (Figure 1)
We found that the SOX2-Interference Peptide (iPep) was internalized very rapidly and effectively in 4.9% of T11 cells within two hours and co-localized with Hoechst staining into the nucleus of the breast cancer cells (Supplementary Figure S1)

Summary

Introduction

Transcription factors (TFs) are proteins capable of binding onto specific DNA sequences and affect the regulation of subsequent transcription by interacting either through creativecommons.org/licenses/by/ 4.0/) Their DNA-binding domains (DBDs) or with other TFs and chromatin cofactors and modifiers [1]. The SOX (SRY-related High Mobility Group (HMG)-box) family of proteins are related to the SRY (Sex determining Region Y; a sex-determining gene on the Y chromosome in both marsupial and placental mammals) and comprise nearly half of the known HMGbox proteins. These TFs play important roles in a variety of developmental processes, in organogenesis [7,8]. The HMG TF SOX2 is highly expressed in embryonic stem cells (ESCs), in neural progenitor cells [9] and is one of the TFs necessary for the formation of induced pluripotent stem cells from somatic cells [10]

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Conclusion