In vivo dual targeting of the oncogenic Ether-à-go-go-1 potassium channel by calcitriol and astemizole results in enhanced antineoplastic effects in breast tumors

Breast cancer is the most frequently diagnosed malignant neoplasia and the leading cause of cancer death among women worldwide [1]. One out of eight women will develop breast cancer during their lifetime. The standard medical treatment for breast cancer besides surgery and radiotherapy include cytotoxic chemotherapy, which targets rapidly dividing cells. However, this clinical approach is highly toxic, affects normal cells and causes a wide array of side effects. In the last decades, substantial changes in cancer therapy have been made. Among them, new anticancer drugs designed to recognize specific features in cancer cells are being produced. These drugs, created on the basis of their targeted mechanism of action, are expected to be more efficient with less toxicity. Approximately 80% of all breast cancers are susceptible for hormonal or antibody-based targeted therapy, based on the presence and/or abundance of the estrogen receptor alpha (ERα), progesterone receptor (PR) and/or the human epidermal growth factor receptor 2 (HER2). It is well known that absence of ERα, PR and HER2 precludes targeted therapies to these cell markers and often results in poorer outcomes [2]. Therefore, identification of new molecular targets expressed in breast tumors is needed. The ether à-go-go-1 potassium channel (EAG1) became an oncological target soon after the discovery of its involvement in cell proliferation and apoptosis [3‐6]. EAG1 promotes oncogenesis and tumor progression, and its pharmacological inhibition reduces tumor development [4, 6, 7]. Moreover, EAG1 is upregulated by cancer-associated factors such as estrogens and the human papilloma virus [8]. Interestingly, a substantial proportion of breast tumors including ERα-negative and triple-negative breast cancers express EAG1 [5, 9]. In this regard, the progression of breast cancer cells through the early G1 phase has been shown to be dependent on the activation of EAG1 channels [10‐12]. Previously, our laboratory showed that EAG1 expression and the rate of cell proliferation are inhibited in breast and cervical cancer cells by calcitriol, the active vitamin D metabolite [9, 13]. Calcitriol is an important endogenous as well as exogenous anticancer hormone. The antiproliferative effects of calcitriol have been extensively demonstrated in many cancerous cell types, most of them involving the ligand-activated vitamin D receptor (VDR) [14, 15]. Since the induction of cell cycle arrest and apoptosis by calcitriol depends on the expression of the VDR, this protein represents a good therapeutic target in treating cancer [16].

Previous in vitro studies by our group have shown that astemizole, a non-selective EAG1 blocker, synergized with calcitriol to inhibit breast cancer cell proliferation by modifying EAG1 gene expression and possibly its activity as well [17]. In addition, these studies also showed that astemizole upregulates VDR expression and downregulates the calcitriol-degrading enzyme CYP24A1; thus, increasing calcitriol bioactivity while decreasing its degradation. Taken together these observations and the fact that the VDR and EAG1 are expressed in 90% and 85%; respectively, of breast cancer tumors [18‐21], we hypothesized that a combined treatment targeting these two proteins in vivo could result in an improved therapeutic benefit for breast cancer management, including those tumors not treatable by hormonal therapy. In the present study we investigated the effects of calcitriol alone or in combination with astemizole on tumor growth in an in vivo preclinical model using athymic mice xenografted with two different human breast cancer cell lines: T-47D (ERα, VDR and EAG1 positive) and a ductal infiltrating carcinoma breast cancer-derived primary cell culture (MBCDF, ERα negative, VDR and EAG1 positive) [22]. These two cell lines were selected because they represent different types of breast tumors based on the expression of the ERα. In addition, both express the selected therapeutic targets and both were tumorigenic. Herein, we show for the first time that the concomitant in vivo administration of calcitriol and astemizole inhibited tumor growth more efficiently than each drug alone.

For a patient tailored anticancer therapy, the identification of potential targets in tumor tissue is paramount to predict therapeutic efficiency. Indeed, new antineoplastic drugs are expected to be less toxic to the patient and more tissue- and target-specific. Although some molecular subgroups of breast cancer are beneficed from a targeted therapy, the most aggressive tumors still lack molecular targets, representing a clinical challenge. Among the newly recognized therapeutic targets in oncology, EAG1 stands as a promising candidate, considering its involvement in oncogenesis and tumor growth [27]. Other important therapeutic target is the VDR; which is required to mediate calcitriol antineoplastic effects, such as the repression of EAG1 gene expression [9, 13, 28, 29]. Therefore, drugs that target the VDR and EAG1 represent novel approaches to fight against breast cancer. Interestingly, both EAG1 and the VDR are expressed in most breast tumors, independently of their general molecular signature [18‐21]. Considering the latter, we designed a combined targeted therapy directed to these biomarkers in vivo, based on the rationale of a dual blocking of EAG1 with the purpose to restrain its tumorigenic ability and consequently, tumor progression. For this, we used astemizole and calcitriol to inhibit EAG1 activity and gene expression; respectively, in order to completely obstruct EAG1 functionality. The design of the study was conceived to be tested in a murine model xenografted with two human breast cancer cell lines expressing both EAG1 and the VDR. Tumor growth reduction together with Ki-67 expression as proliferation marker, were used as biological endpoints representing therapeutic benefit. Taking into consideration these endpoints, the results showed that the combined regimen was significantly more efficient to produce antitumor effects than when either of the agents was tested alone. In addition, calcitriol significantly inhibited tumoral EAG1 mRNA and protein expression, an effect that was further increased by the co-administration with astemizole, showing for the first time the in vivo inhibition of this oncogenic potassium channel by these drugs in breast tumors.

Interestingly, the expression of EAG1 was also inhibited by astemizole, in a similar manner as observed previously in vitro [17]. This could probably be explained by astemizole blocking EAG1 channels located also in the inner nuclear membrane, which has been suggested to affect gene expression [30].

Besides the double inhibition of EAG1 by the combined treatment, additional individual antiproliferative effects of astemizole and calcitriol might also be taking place, such as long-term blocking of histamine H₁-receptors, induction of apoptosis or the modification of EAG1 activity and/or glycosylation patterns, which deserve to be further investigated.

Since calcitriol bioavailability and activity are potentiated by astemizole [17], its effects on calcium serum levels should be considered and avoided to prevent hypercalcemia, as an undesirable side effect. In this study, only the combined therapy was accompanied by a mild increase in serum calcium levels, which probably resulted from improved calcitriol bioactivity. In addition, no changes in the mean body weights between the experimental and control groups were observed, and the histopathological analysis of the lungs of treated animals by a specialized pathologist did not show any signs of toxicity, suggesting that the doses of calcitriol and astemizole, as used in this study, were well tolerated. These data suggested that the combined dosing regimen herein reported could potentially be tested in patients with breast cancer as an adjuvant therapy, with relative low adverse side effects. Alternatively, dietary vitamin D instead of calcitriol could be used since it is a safe, economical and easily available nutritional agent, that has proven to be equivalent to calcitriol in exerting anticancer effects in a preclinical model of breast cancer [31]. On the other hand, many different compounds may be used to target EAG1; however, for the purposes of this study we chose astemizole, given its well-known in vitro and in vivo antiproliferative effects on tumor cells through blocking ion currents [11, 17, 23, 32]. In addition, astemizole offers other advantages, such as the fact that it is a low-priced drug currently prescribed for treatment of simple allergic conditions or malaria in some countries and particularly because it does not cross the blood–brain barrier [33]. Regarding this, the observation that neither astemizole nor calcitriol modified the expression of EAG1 in the brains of the treated mice, as they did in breast cancer, was of particular importance since normal brain cells express EAG1. This observation may rule out alterations on the physiological role of EAG1 at the level of the central nervous system.

Overall, our results confirm previous in vitro findings [9, 17] and support earlier studies showing EAG1 as a promising target for the tailored treatment of human tumors [21]. As previously suggested, reevaluation of astemizole as an antineoplastic drug is needed [6].

The simultaneous administration of calcitriol and astemizole to mice xenografted with human breast cancer cells reduced tumor growth more efficiently than either drug alone. The mechanistic explanation for these results includes the inhibition of EAG1 expression, providing scientific bases to test the combined therapy in future clinical trials. The effect of the combined treatment was effective in both types of tumors (ER negative and ER positive), as seen in MBCDF and T-47D-xenografts, further supporting the potential use of this therapeutic approach in breast tumors that represent a clinical challenge, such as triple negative and those resistant to endocrine therapy. Some of the limitations of this study include the lack of serum calcitriol quantification and the analysis of tumoral VDR and CYP24A1 expression, which deserve to be further investigated.