Stapled BIG3 helical peptide ERAP potentiates anti-tumour activity for breast cancer therapeutics

Abstract

Estradiol (E2) and the oestrogen receptor-alpha (ERα) signalling pathway play pivotal roles in the proliferative activity of breast cancer cells. Recent findings show that the brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex plays a crucial role in E2/ERα signalling modulation in breast cancer cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using the ERα activity-regulator synthetic peptide (ERAP: 165–177 amino acids), derived from α-helical BIG3 sequence, resulted in a significant anti-tumour effect. However, the duration of this effect was very short for viable clinical application. We developed the chemically modified ERAP using stapling methods (stapledERAP) to improve the duration of its antitumour effects. The stapledERAP specifically inhibited the BIG3-PHB2 interaction and exhibited long-lasting suppressive activity. Its intracellular localization without the membrane-permeable polyarginine sequence was possible via the formation of a stable α-helix structure by stapling. Tumour bearing-mice treated daily or weekly with stapledERAP effectively prevented the BIG3-PHB2 interaction, leading to complete regression of E2-dependent tumours in vivo. Most importantly, combination of stapledERAP with tamoxifen, fulvestrant, and everolimus caused synergistic inhibitory effects on growth of breast cancer cells. Our findings suggested that the stapled ERAP may be a promising anti-tumour drug to suppress luminal-type breast cancer growth.

Functional genomics for breast cancer drug target discovery

“…Phosphorylated PHB2 translocates to nuclear ERα and plasma membrane-type ERα in an E2-dependent manner, resulting in multiple suppression of E2-dependent transcriptional activity and membrane-type ERα-mediated nongenomic signaling (various phosphorylation cascades) in ERα-positive breast cancer [ 89 , 95 , 96 ]. Similar findings have also been reported for orthotopic breast cancer xenografts in nude mice [ 88 , 89 , 97 ]. The most notable aspects of this therapeutic strategy are that (1) PHB2 reactivated by ERAP suppresses various E2-ERα signaling networks, which are responsible for resistance to endocrine therapy, and (2) ERAP has antitumor effect on endocrine-resistant breast cancer xenografts [ 89 ].…”

“…Considering its clinical use, it is essential to improve the proteolytic stability of ERAP to better maintain its inhibitory activity. Focusing on the fact that the relevant amino acid sequences of ERAP form an alpha-helix structure, we developed chemically modified ERAP (hereafter referred to as stapled ERAP: stERAP) via cross-linking of specific amino acids, with amino acids essential for BIG3-PHB2 interactions (Gln165, Asp169, and Gln173) spatially arranged at appropriate positions [ 97 ]. stERAP led to enhanced stabilization of the alpha-helical structure, potential protease resistance, and enhanced cell permeability, without the membrane-permeable polyarginine sequence of conventional ERAP [ 97 ].…”

“…Focusing on the fact that the relevant amino acid sequences of ERAP form an alpha-helix structure, we developed chemically modified ERAP (hereafter referred to as stapled ERAP: stERAP) via cross-linking of specific amino acids, with amino acids essential for BIG3-PHB2 interactions (Gln165, Asp169, and Gln173) spatially arranged at appropriate positions [ 97 ]. stERAP led to enhanced stabilization of the alpha-helical structure, potential protease resistance, and enhanced cell permeability, without the membrane-permeable polyarginine sequence of conventional ERAP [ 97 ]. As a result of these properties, stERAP achieved E2-dependent responsiveness with long-term stability, exhibiting antitumor effect after weekly intravenous administration (10 mg/kg body weight) in clinical application (Table 2 ) [ 97 ].…”

“…stERAP led to enhanced stabilization of the alpha-helical structure, potential protease resistance, and enhanced cell permeability, without the membrane-permeable polyarginine sequence of conventional ERAP [ 97 ]. As a result of these properties, stERAP achieved E2-dependent responsiveness with long-term stability, exhibiting antitumor effect after weekly intravenous administration (10 mg/kg body weight) in clinical application (Table 2 ) [ 97 ]. We further demonstrated that stERAP potentiates antitumor activity in clinically problematic endocrine-resistant breast cancer.…”

Significance of prohibitin domain family in tumorigenesis and its implication in cancer diagnosis and treatment

“…Therefore, inhibition of the interaction of BIG3-PHB2 is a feasible strategy in estrogen-dependent cancer therapy. In fact, researchers isolated the stable ERα activity-regulator synthetic peptide (ERAP: 165-177 amino acids), a short peptide derived from α-helical BIG3 sequence that specifically binds to PHB2 and competitively prevents the BIG3/PHB2 interaction, and then showed that ERAP promotes PHB2/REA complex nuclear translocation and suppresses cancer cell proliferation in vitro and in vivo 133 . Moreover, PHB2 released from the BIG3-PHB2 complex by ERAP treatment reduces phosphorylation levels of AKT and MAPK, resulting in significant suppression of proliferation in ERα-positive breast cancer cells.…”