Pavan Kumar Puvvula, Anne M. Moon
Abstract
We performed a forward genetic screen to discover peptides that specifically target breast cancer cells using a Penetratin tagged, random 15mer peptide library. We identified a group of novel peptides that specifically inhibited the proliferation and survival of breast cancer cells without affecting normal primary mammary epithelial cells or fibroblasts. The intrinsic apoptotic pathway is activated by these peptides in the face of abnormal expression of numerous cell cycle regulatory genes. Associated alterations in histone marks, nuclear structure, and levels of critical RNA binding proteins vary in a peptide specific manner. This study demonstrates a novel method for the discovery of new potential therapeutic peptides.
Introduction
Cell-penetrating peptides (CPPs) are comprised of amino acids that function as carrier molecules to transduce various cargo molecules across cell membranes [1–4]. In the recent past, several potential anti-cancer therapeutic peptides have come to light utilizing the advantages of CPPs. The main principle underlying these discoveries is the identification of dominant-negative domains of proteins with anti-cancer properties. By implementing this methodology, MYC and ATF5-derived CPPs were shown to restrict tumor growth and have now progressed to clinical trials [5–11]. In line with the above studies, dominant-negative domains of ANXA1, p50, connexin-43, and Grb-7 were shown to inhibit gastric and colon cancers [12], breast cancer [13], glioma [14], and human epidermoid carcinoma [15]. The U.S. Food Drug Administration has authorized 15 peptide-based drugs in recent years [16]. One of the main challenges in developing an effective peptide is identifying a domain that can disrupt cancer-specific cellular functions but not harm normal cell types. The next challenge is to enhance the anti-cancer potential of the effective region by chemical alterations or addition of other moieties. Such modifications have been shown to generate peptides that function more effectively than the wild-type domain for MYC and ATM [7]. The time- and effort- and resource- intensive nature of these challenges hinders preclinical studies and progress toward clinical implementation.
Results
Generation of the random peptide library
Our recent investigations on RBM39-RRM3-, SAFA-RGG- and hnRNPK-RGG- [3, 22, 23] derived cell-penetrating peptides revealed that treating cells with a penetratin-tagged peptide from these functional domains inhibited cancer cell growth and survival. This mode of action prompted us to develop a new unbiased technique to discover novel cell-penetrating peptides that could be developed therapeutically for use in various disease models.
Discussion
We recently demonstrated that RNA-binding domains of hnRNPU, RBM39 and hnRNPK [3, 22, 23] inhibit the survival of a range of cancer cells. Here we established an unbiased method to discover peptides with anti-cancer properties. Previously, several studies have explored the benefits of random peptide libraries with the phage display technique [19, 38–41] to identify cancer cell-specific ligands [42–45]. The main drawback in these studies was the lack of a tag or peptide to facilitate cargo penetration and intracellular distribution; here we employed the Penetratin tag to overcome this issue. Our results show that a subset of the random peptides we generated disrupt breast cancer cell proliferation and survival without affecting normal cells. Our data indicate that these effects stem from impacts on multiple critical cellular processes including dysregulation of histone marks, altered cell cycle and cell death regulatory gene expression, disrupted nuclear membrane structure, and abnormalities in the amount and location of a subset of RNA binding proteins with known effects on cancer cell survival.
Acknowledgments
We thank the Weis Center for Research at Geisinger Clinic for supporting this project.
Citation: Puvvula PK, Moon AM (2024) Discovery and characterization of anti-cancer peptides from a random peptide library. PLoS ONE 19(2): e0293072. https://doi.org/10.1371/journal.pone.0293072
Editor: Arunava Roy, University of South Florida, UNITED STATES
Received: July 20, 2023; Accepted: October 3, 2023; Published: February 13, 2024
Copyright: © 2024 Puvvula, Moon. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The authors received no specific funding for this work.
Competing interests: THe authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0293072
