Targeting the leptin receptor: a potential new mode of treatment for breast cancer

Keywords::

• adipokine
• antagonist
• pluripotent target
• peptide-based drugs
• triple-negative breast cancer

According to recent worldwide statistics, breast cancer (BC) is the most frequently diagnosed malignancy and the leading cause of cancer-related death among females, accounting for 23% of cancer cases and 14% of cancer deaths [1]. While the majority of BC cases are reported in developed regions, BC death rates have been decreasing in North America and several European countries over the past 25 years, largely as a result of early detection and improved treatment [1]. In addition to surgery and radiation therapy, conventional chemotherapy measures include anthracyclines, taxanes and platinum compounds [2]. A large proportion of BCs (60–80%) express steroid hormone (particularly estrogen) receptors and these malignancies respond to endocrine therapy with various antiestrogens or aromatase inhibitors [3]. Furthermore, approximately 20% of BC cases are HER2-positive and can be effectively treated with anti-HER2 antibodies, such as trastuzumab [4]. However, the aforementioned therapies are not applicable to BC that does not express HER2 as well as estrogen and progesterone receptors. These triple-negative BCs (TNBCs) represent approximately 15% of all cases and are more prevalent in younger women, especially African–Americans and Hispanics [5–9]. TNBC is characterized by an aggressive phenotype and although TNBC is originally usually sensitive to chemotherapy, patients experience earlier relapse than in other BC types and develop visceral metastasis, frequently brain metastasis [5]. Despite a great number of potential biotargets, a clearly dominant pathway implicated in this malignancy has not yet been characterized. Contemporary measures, mostly investigational, attempt to inhibit DNA repair (poly-ADP-ribose polymerase 1 enzyme inhibitors such as iniparib), angiogenesis (VEGF inhibitors) signaling pathways induced by the EGF receptor (e.g., small-molecule inhibitors such as lapanitib and erlotinib), TNFRSF10B, trail receptor 2 (e.g., tigatuzumab antibody) or different oncogenic kinases (e.g., dasatinib multi-tyrosine kinase inhibitor) [6,10]. These biologic agents are being explored in over 100 BC clinical trials, usually in combination with traditional chemotherapy [101]. From these, currently over 50 clinical trials are underway to identify satisfactory therapies only directed at TNBC [6]. Unfortunately, many trials fail for various reasons [7], including inappropriate patient selection for iniparib or poor accrual for erlotinib. Clearly, the repertoire of validated biotargets and potential drugs for BC, especially for tumors that develop secondary resistance to initial therapies and TNBC, has to be expanded to include compounds that can interfere with cancer growth and metastasis at multiple points and stages of the disease. Multikinase small-molecule inhibitors and their combination with established targeted and traditional therapies would certainly fit the bill, but discussion of these options is beyond the scope of this article. Here, we wish to focus on peptide-based compounds acting as antagonists to the receptor of the obesity hormone leptin (ObR). We believe these modified peptides have the potential to fill the gap.

Obesity significantly (30–50%) increases the risk of BC in postmenopausal women and decreases the efficacy of BC therapies in all BC patients [11]. Importantly, excess bodyweight has been associated with the development of TNBC [8,12]. Among obesity-related factors that are known to impact BC development and progression, a prominent place is occupied by the fat tissue-derived adipokine leptin [13]. In addition to its primary anorexigenic functions in the brain, peripherally circulating leptin affects numerous organs, including the mammary glands. Examination of BC tissue samples reveals that ObR is overexpressed in BC versus nonmalignant mammary epithelium. Notably, ObR is found in approximately 90% of all BCs, including TNBC [13,14]. In vitro studies demonstrate that leptin stimulates breast cancer cell growth and survival. The hormone can also increase the invasiveness and metastatic potential of BC cells [15], and promote angiogenesis while maintaining a microenvironment favorable for BC development [16]. Furthermore, leptin is known to crosstalk with and transactivate several pathways that are targets for BC therapy, including the estrogen, EGF receptor and HER2 pathways [13,17,18]. Consequently, in recent years the leptin/ObR system has emerged as a new and promising target in BC therapy [13,19]. Owing to the pluripotent actions of leptin, ObR antagonists have the potential to exert multimodal therapeutic benefits in the clinical setting.

Antagonists to the receptor of the obesity hormone leptin are being developed both as mutants of the full protein and peptide fragments representing single receptor-binding sites. Superposition of leptin’s sequence with other cytokines, such as human IL-6, bovine granulocyte–colony-stimulating factor or human oncostatin M reveals three potential bivalent ObR binding sites (sites I–III), mostly around the pairwise helices that characterize the secondary structure of the protein ligand [20]. When a triple alanine-substituted mutant of leptin at site I, attached to a polyethylene glycol carrier, was injected into mice, the animals exhibited a rapid and dramatic increase in food intake, indicating true ObR-antagonistic activities [21]. Further improvement in this pegylated ObR antagonist, including an additional mutation at site II, arm a, resulted in a 14-fold improved ObR-binding ability [22]. However, these protein variants have not been tested for their efficacy in BC animal models. What was tested was a pegylated analog of a site II, arm b, peptide fragment called LPrA2 [23]. Pegylated LPrA2 strongly inhibits the growth of estrogen receptor-positive and somewhat of estrogen receptor-negative cells xenotransplanted into immunocompromised mice and reduces the level of proangiogenic and pro-proliferative markers providing support for the potential use of leptin signaling inhibition in BC. Having said this, our results indicate that leptin fragments and their analogs that antagonize ObR in the presence of leptin exert either lower activity than the native protein or display agonistic activities in the absence of leptin in vitro[24]. Frequently, these postulated ObR antagonists exhibit differential activities in vitro and in vivo including a site II, arm b fragment [25]. Thus, these leptin fragments are not true ObR antagonists; they are rather partial agonists depending upon the presence of other ObR ligands, and if developed as drugs will face considerable regulatory scrutiny.

Our ultimate goal has always been to develop ObR ligands that retain the antagonist properties in a wide concentration range (at least 1000-fold) irrespective of other ObR-interacting molecules in the microenvironment. As leptin stimulates the growth of BC cells at low nanomolar concentrations, we attempted to generate ObR antagonists acting at picomolar concentrations in vitro in order to efficiently compete with the natural ligand in vivo. Finally, we aimed at equipping our ObR antagonists with favorable pharmacological properties. These include stabilization of the termini with non-natural amino acid residues to avoid exopeptidase cleavage in biological fluids and reaching acceptable absorption and biodistribution properties without any nonleptin-related attachment modules included. Every single element in combination therapy carries its own toxic properties and delivery enhancers (covalently or noncovalently linked alike) are not exceptions to this rule. Based on this design strategy, we developed a nine-amino acid-long peptide analogs of ObR binding site III of leptin that act as selective ObR inhibitors without any partial agonistic activity [14,25].

The lead peptide-based therapeutic, Allo-aca (with the sequence H–alloThr–Glu–Nva–Val–Ala–Leu–Ser–Arg–Aca–NH₂), reduces leptin-dependent growth and signaling in hormone-positive and -negative BC cell lines with IC₅₀ values of 50–200 pM [14,25]. In immunocompromised mice Allo-aca suppresses the growth of established hormone-sensitive orthotopic human BC xenografts by 45–51% when administered either intraperitoneally or subcutaneously for 38 days at a dose as low as 0.1 mg/kg/day [25]. The ObR antagonist peptides penetrate through the blood–brain barrier, probably via short-form ObR-mediated transport that is resident at the blood–brain barrier, and interfere with CNS functions [14,25]. When added to normal mice intraperitoneally, subcutaneously or even orally, the lead antagonists accelerate normal weight increase for 10–12 days; however, reduced efficacy is observed at later time points or higher doses [14,25]. In normal growing rats, intranasal Allo-aca administration at 0.1 mg/kg/day for 20 days results in a 2% net total bodyweight gain without signs of resistance induction. In immunocompromised mice carrying human TNBC xenografts, Allo-aca administered subcutaneously significantly extends the average survival time from 15.4 days (untreated controls) to 24 and 28.1 days at 0.1- and 1-mg/kg/day doses, respectively. In parallel, conventional treatment with 1 mg/kg/day intraperitoneal cisplatin prolongs the average survival time to 18.6 days only, while administration of 20 mg/kg/day oral tamoxifen (negative control) has no significant survival effects relative to controls.

What is truly attractive in Allo-aca, and probably other ObR antagonists, is its ability to interfere with BC development at multiple stages. In the TNBC model, the survival figures are much more impressive than the primary tumor growth inhibition data. This can be due to at least partial inhibition of metastasis and angiogenesis, and perhaps delaying systemic inflammatory processes. Our ObR antagonist peptides are highly active against leptin-induced proliferation of glioma cells in vitro and with their great biodistribution to the brain – based on both weight gain measurements and direct imaging studies [14,25] – they could interfere with brain metastasis, a process that is typical for aggressive BC progression. Our most recent studies indicate that the ObR antagonist peptides inhibit leptin-induced angiogenesis of tumor cells [26] and leptin-induced inflammatory signal transduction events in vitro, as well as autoimmunity-derived inflammation in animal models [27]. Similar to many other peptide drugs [28], our modified leptin fragments can be taken up by olfactory neurons and can be applied intranasally for brain penetration, if needed. Not only can Allo-aca be administered in a less invasive way than current chemotherapy measures that have to be added intravenously, it also shows remarkable low toxicity parameters. In normal mice Allo-aca produces no signs of systemic toxicity up to the highest studied subcutaneous bolus dose of 50 mg/kg while, as expected, it induces a modest bodyweight increase of 6–10% [14].

Taken together, Allo-aca or its analogs under development with their multimodal cellular activities, advantageous administration route and safety profile can be useful additions to the existing oncology drug repertoire against various tumor forms characterized by ObR overexpression such as breast, brain, prostate and colon cancers. While not likely a sole solution for TNBC management, a selective, specific and pharmacologically acceptable ObR antagonist such as Allo-aca may become an attractive addition to current treatment protocols for these malignancies.

Financial & competing interests disclosure

The authors are inventors on a patent application covering peptide Allo-aca and analogs for the treatment of various cancer, arthritis and autoimmune disease forms. The patent is owned by Temple University and currently not licensed to any entity, although Peptherx, Inc., a biotechnology company founded by the authors, has a temporary option for the technology. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.