Inhibiting breast cancer progression by targeting SND1 using a novel peptide identified from phage display

Abstract

Breast cancer is the most common cancer in women, causing 626,679 deaths worldwide in 2018. SND1 is a multifunctional oncoprotein overexpressed in almost all cancers, especially in advanced and metastatic cases. SND1-MTDH interaction contributes to the initiation and progression of breast cancer, making it a promising target for breast cancer treatment. In this study, recombinant SN1/2 domain of SND1 was expressed in E.coli and purified with HisTrap affinity column. SN1/2 was used as bait in a phage display screening. After 4 rounds of screening, 20 phages were randomly picked and sequenced. Amino acids W and Y were found to be highly enriched in these phages. The most repeated peptide 4-2 was demonstrated to have high binding affinity towards SN1/2. Peptide 4-2 could disrupt 22-mer MTDH peptide from interacting with SN1/2 in ELISA assay and could also disrupt SND1-MTDH full-length protein interaction in co-immunoprecipitation assay. An RR-TAT cell penetrating peptide was attached to the N-terminus of peptide 4-2 to generate CPP-4-2 peptide. CPP-4-2 peptide could penetrate and preferentially kill breast cancer cells by inducing apoptosis compared to other cancer types or normal cells. Mechanistic investigation suggested that peptide 4-2 could interact with SND1 and disrupt SND1-MTDH interaction, which probably lead to the degradation of SND1. Overexpression of SND1 could reduce the cytotoxicity of peptide 4-2 to breast cancer cells, indicating that the disruption of SND1-MTDH interaction and subsequent degradation of SND1 by peptide 4-2 was the possible reason for breast cancer cell death. It was found that peptide 4-2 could regulate Akt pathway by upregulating p-Akt S473 and degrading Akt. The degradation of Akt by peptide 4-2 was proteasome-dependent and was partially dependent on the phosphorylation of Akt at S473. On the other hand, peptide 4-2 could also regulate another SND1 downstream target NF-κB2 by enhancing the transcription of pro-apoptotic NF-κB2. The degradation of Akt and the enhanced transcription of NF-κB2 induced by peptide 4-2 might be the reasons for breast cancer cell death. Mutational analysis suggested that W10 but not Y4 or Y11 was essential in the activities of peptide 4-2, including cytotoxicity, SND1-interaction, SND1 downregulation, Akt degradation and NF-κB2 activation. In summary, peptide 4-2 selectively killed breast cancer cells by inducing apoptosis possibly through interacting with SND1, disrupting SND1-MTDH interaction and inducing SND1 degradation. Peptide 4-2 could also affect SND1 downstream targets, Akt and NF-κB2 by degrading Akt and enhancing the transcription of pro-apoptotic NF-κB2, which possibly lead to breast cancer cell death. W10 rather than Y4 or Y11 was the essential amino acid in the activities of peptide 4-2.