Flubendazole overcomes trastuzumab resistance by targeting cancer stem-like properties and HER2 signaling in HER2-positive breast cancer

Highlights

• Flubendazole induces apoptosis in HER2-positive breast cancer cells.

• Trastuzumab-resistant cells are sensitive to flubendazole.

• Flubendazole effectively targets cancer stem-like properties.

• Flubendazole downregulates truncated p95HER2, phospho-HER2, and phospho-HER3.

• Flubendazole suppresses tumor growth in trastuzumab-resistant xenografts.

Abstract

Although trastuzumab provides significant clinical benefit for HER2-positive breast cancers, responses are limited by the emergence of resistance. Trastuzumab resistance is a multi-factorial phenomenon thought to arise from the presence of cancer stem cells and interactions between truncated p95HER2 and HER family members. Flubendazole (FLU) is a potent anthelmintic agent with an exceptional safety profile. Evidence also suggests that it can act as an anticancer agent in several cancer cell types. We sought to investigate the effect of FLU on apoptosis, HER2/Akt signaling, breast cancer stem cell (BCSC)-like properties and trastuzumab resistance in HER2-positive breast cancer cells. FLU treatment induced apoptosis, associated with a significant downregulation of truncated p95HER2, phospho-HER2, phospho-HER3 and phospho-Akt levels, as well as suppression of HER2/HER3 hetero-dimerization in both trastuzumab-sensitive and –resistant lines. FLU effectively targeted BCSC-like properties including aldehyde dehydrogenase 1 (ALDH1) expression and the CD44^high/CD24^low phenotype, concomitant with a suppression of mammosphere-forming ability. FLU administration also caused significant tumor suppression in trastuzumab-resistant xenografts, coinciding with the downregulation of BCSC-related markers and intracellular HER2. These findings highlight the mechanisms of action of FLU in overcoming trastuzumab resistance in breast cancer.

Introduction

The human epidermal growth factor receptor 2 (HER2) is amplified in 20–30% of all breast cancers, and its overexpression is linked to aggressive tumor behavior and a poorer prognosis [1], [2]. Although HER2 has no known direct activating ligand, it can be activated through homo- and hetero-dimerization with other HER family receptors including EGFR (HER1), HER3 and HER4 [3], [4]. The HER2/HER3 dimer is the most potent oncogenic pair, and dimerization leads to activation of their kinase domains and subsequent activation of downstream signaling pathways, including phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) [3], [4], [5], [6]. As a consequence, these signaling cascades promote the expression of genes that orchestrate cell survival, proliferation and differentiation, which fosters tumor progression [5], [7].

Trastuzumab is a humanized monoclonal antibody that disrupts HER2 activation by binding to its extracellular domain IV, leading to antibody-dependent cell-mediated cytotoxicity (ADCC) [8], [9], [10]. It has a significant clinical impact on disease-free and overall survival in patients with HER2-amplified metastatic and early breast cancer [11], [12], [13]. However, despite these outcomes in some cases, nearly 70% of patients experience primary or acquired resistance [12], and this issue remains a significant unmet medical need. Several potential mechanisms of resistance to trastuzumab include dimerization between members of the HER family, hyperactivation of PI3K/Akt signaling, mutation or loss of PTEN, and the upregulation of other receptor tyrosine kinases such as insulin-like growth factor 1 receptor (IGF-1R) and c-MET [14]. Of particular note, oncogenic p95HER2 is a major factor in trastuzumab resistance and is frequently found in 30% of HER2-positive breast cancers [15]. p95HER2 is a C-terminal fragment of HER2 that lacks the extracellular trastuzumab-binding domain but retains constitutive kinase activity, activating downstream signaling [16].

Emerging evidence suggests that there is a significant correlation between HER2 overexpression and breast cancer stem cell (BCSC)-like populations [17], [18]. BCSCs constitute a small subset of tumor cells with tumor-initiating potential, characterized by the CD44^high/CD24^low phenotype and high aldehyde dehydrogenase 1 (ALDH1) activity [17], [19]. These cells give rise to tumorigenesis, tumor growth and propagation, and are also associated with tumor recurrence and resistance to treatment [20], [21]. Preclinical studies have demonstrated that BCSCs are sensitive to trastuzumab treatment in HER2-positive breast cancer cells, but trastuzumab fails to eliminate trastuzumab-resistant BCSCs, driving recurrence and metastatic spread [18], [22]. Therefore, new treatment options are needed for the targeting of BCSC-like properties for a sustainable therapeutic approach to HER2-positive breast cancers.

Flubendazole (FLU) belongs to a large group of benzimidazole derivatives and is currently available in a number of countries worldwide for human use to treat gastrointestinal nematodes [23], [24], [25], [26]. Recent preclinical studies have shown that FLU exerts anti-proliferative activity in various types of cancers including leukemia, myeloma, neuroblastoma, colorectal and triple-negative breast cancer in vitro and in vivo [27], [28], [29], [30]. FLU is an inhibitor of tubulin polymerization that disrupts microtubule structure [27], however, its effects on HER2-positive breast cancers have not been elucidated. In the present study, we sought to characterize the mechanism of action responsible for FLU's novel effects in targeting BCSC and the HER2/Akt signaling pathway in trastuzumab-resistant HER2-positive breast cancer in vitro and in vivo.