Background
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that was first discovered as an intracellular protein that bound with high affinity to the environmental toxicant 2,3,7,8-tetrachlorodibenzo-
p-dioxin (TCDD) [
1]. Subsequent studies showed that AHR-mediated transcription was dependent on formation of a nuclear heterodimer composed of the AHR and AHR nuclear translocator (ARNT) proteins [
2] that bind AHR responsive elements (AhREs) on target gene promoters [
3]. Initial studies demonstrated that TCDD and structurally-related halogenated aromatic compounds induced a well-defined subset of genes and toxic responses [
4]. However, it is now apparent that this receptor plays a critical endogenous role in cellular homeostasis and multiple diseases and binds not only toxicants but also endogenous biochemicals, dietary flavonoids and several phytochemicals associated with health benefits, other synthetic/industrial chemicals, and many pharmaceuticals [
5‐
7]. The important role of the AHR and effects of AHR agonists or antagonists have been documented for various inflammatory conditions, stem cell stability and expansion, autoimmune diseases, and several different cancers and clearly demonstrate that this receptor is an important drug target [
8‐
15].
Research in this laboratory initially focused on the molecular mechanisms of inhibitory AHR-estrogen receptor (ER) crosstalk and development of selective AHR modulators (SAhRMs) for treatment of ER-positive breast cancer [
16,
17]. 6-Methyl-1,3,8-trichlordibenzofuran (6-MCDF) was initially developed as a relatively non-toxic AHR antagonist that inhibited TCDD-induced toxicity in rodent models [
18‐
22]. However, this compound also exhibited AHR agonist activity and activated inhibitory AHR-ERα crosstalk in breast cancer cells and decreased mammary tumor growth
in vivo[
17,
23,
24]. Subsequent studies showed that MCDF also blocked growth of ER-negative breast cancer cells [
25] and inhibited metastasis of triple negative MDA-MB-231 breast cancer cells to the lung by inducing the antimetastatic microRNA-335 (miR-335) [
26]. Recent studies showed that eight AHR-active pharmaceuticals including 4-hydroxtamoxifen, flutamide leflunomide, mexiletine, nimodipine, omeprazole, sulindac and tranilast exhibited structure- and cell context-dependent AHR agonist/antagonist activities in BT474 and MDA-MB-468 cells and several of these compounds also inhibited MDA-MB-468 cell migration [
27]. These results are typically observed for selective AhR modulators (SAhRMs) that exhibit tissue- and response-specific AhR agonist or antagonist activity due to differential expression of cofactors, different receptor/ligand conformations and epigenetic effects [
16]. Selective receptor modulators are also commonly observed for nuclear receptors such as the estrogen receptor (ER) and selective ER modulators have been extensively characterized for treatment of ER-positive breast cancer [
28].
In this study, we initially used the same set of AHR-active pharmaceuticals in triple-negative MDA-MB-231 cells with a primary objective of identifying a known pharmaceutical that may be effective for inhibiting breast cancer metastasis. Among the eight compounds, only omeprazole inhibited MDA-MB-231 breast cancer cell invasion and this response could be reversed, in part, by AHR antagonists or by knockdown of the AHR by RNA interference (RNAi). Omeprazole also inhibited lung metastasis of MDA-MB-231 cells (tail vein injection) in a mouse model and the antimetastatic pathway was linked to decreased expression of MMP-9 and AHR-dependent suppression of the pro-metastatic gene CXCR4. Decreased invasion and CXCR4 expression was also observed in MCF-7 and SKBR3 breast cancer cell lines treated with omeprazole. Thus, omeprazole may have potential clinical applications for inhibition of breast cancer metastasis due, in part, to its AHR agonist activity.
Discussion
The treatment and prognosis for breast cancer patients depends on multiple tumor characteristics including the size, stage, extent of tumor delocalization, and expression of various protein and mRNA prognostic factors [
35]. Patients that express ERα can be successfully treated with antiestrogens and aromatase inhibitors, and more aggressive tumors that overexpress the epidermal growth factor receptor 2 (
ErbB2) oncogene can be treated with the ErbB2 neutralizing antibody in combination therapies [
36]. Patients with ER-negative tumors are among the most difficult to treat and exhibit low survival rates due, in part, to metastasis from the breast to various distal sites.
Research in this laboratory and others [
33,
34] have demonstrated that the AHR is a potential drug target for treating ER-negative breast cancer. For example, the SAhRM MCDF acts as an AhR agonist to inhibit growth and/or metastasis of ER-negative breast tumors in animal models, and this was associated with induction of microRNA-335 and the subsequent suppression of the pro-metastatic
SOX4 gene [
26]. Several other studies with structurally diverse AHR ligands (agonists) demonstrated AHR-mediated inhibition of cell migration and/or invasion in ER-negative breast cancer cells and these responses were also accompanied by enhanced differentiation and downregulation of pro-metastatic genes such as
CXCR4 and
MMP-9[
31‐
34,
37‐
39].
A number of pharmaceutical agents approved for multiple uses are also AHR ligands, and some of these compounds including 4-hydroxytamoxifen and tranilast exhibit some anticancer activity in breast cancer cells [
37‐
39]. Our initial studies investigated the AHR agonist activities of eight AHR-active pharmaceuticals including tranilast and 4-hydroxytamoxifen in MDA-MB-468 and BT474 breast cancer cells and observed that their AHR activity was structure-, cell context- and response-specific [
27]. This variability in activity is illustrated by results for mexiletine which was an AHR antagonist in MDA-MB-468 cells and inhibited TCDD-induced
CYP1A1 gene expression but was a partial AHR agonist in BT474 cells [
27]. Moreover, in ER-negative MDA-MB-468 cells, several of the AHR pharmaceuticals including flutamide, leflunomide, nimodipine, omeprazole, sulindac and tranilast inhibited cell migration [
27]. In this study, we primarily focused on the effects of the AHR-active pharmaceuticals in the more aggressive MDA-MB-231 cell line which exhibits high basal rates of migration and also invasion in
in vitro assays. However, we also observed that omeprazole induced CYP1A1 in MCF-7 and MDA-MB-468 cells (Figure
4A) [
27]. The effects of the AHR-active pharmaceuticals on CYP1A1 and CYP1B1 expression in MDA-MB-231 cells were similar (Table
1); however, only omeprazole inhibited MDA-MB-231 cell invasion (Figures
1D and
2A) and we therefore selected this widely used proton pump inhibitor for further evaluation as an AHR-active antimetastatic agent in breast cancer. Previous studies suggest that omeprazole exhibits anti-inflammatory activity [
40] and anticancer activity, particularly in combination treatment studies [
41‐
43]. Since this investigation focused primarily on the effects of omeprazole alone, higher concentrations that were not cytotoxic were used in the cell culture experiments. Previous
in vivo studies used an omeprazole dose of 75 mg/kg for administration of drug combinations and this dose had no effect on tumor growth [
41]. A 100 mg/kg (daily) dose was used for investigating the
in vivo antimetastic activity of omeprazole (Figure
3C,D).
Omeprazole clearly inhibited MDA-MB-231 cell migration and invasion (Figures
2A and
3A) and this response was attenuated after knockdown of the AHR by RNAi or after cotreatment with AHR antagonists (Figure
2A-C). MDA-MB-468 cells did not exhibit invasion; however, omeprazole inhibited PMA-induced invasion of MCF-7 cells and this response was also attenuated after AHR knockdown (Figure
4D). Moreover, these
in vitro assays were complemented by inhibition of lung metastasis of MDA-MB-231 cells in mice administered the cells by tail vein injection and treated with omeprazole (Figure
3B). In contrast to the effects observed for MCDF [
26], omeprazole did not induce miR-335 expression in MDA-MB-231 (data not shown), but significantly decreased expression of the pro-metastatic genes
MMP-9 and
CXCR4 (Figure
5A,B) and similar results were observed in MCF-7 and MDA-MB-468 cells (Figure
4C). Previous studies show that one or both of these genes was decreased in ER-positive cells and tumors treated with structurally diverse AHR ligands [
31‐
34]. We also observed that
CXCR4 (and
PCNA) expression was also decreased in metastasized tumors (lung) (Figure
3D), suggesting that omeprazole not only decreased metastasis but directly targeted
CXCR4 and
PCNA in the metastasized tumors. We further investigated the role of the AHR in mediating the induction of
MMP-9 and
CXCR4 by omeprazole in MDA-MB-231 cells and our results show that although AHR silencing may decrease
MMP-9 activity (zymography), the loss of the receptor does not attenuate the effects of omeprazole on
MMP-9 (Figure
4B). In contrast, omeprazole-induced downregulation of
CXCR4 was significantly reversed by AHR silencing or cotreatment with AHR antagonists (Figure
5A,C), suggesting that downregulation of
MMP-9 and
CXCR4 by omeprazole in MDA-MB-231 cells is AHR-independent and -dependent, respectively.
Although the AHR and other nuclear receptors mediate induction and expression of genes, most mechanistic studies have focused on activation of genes and the ligand-dependent recruitment of the AHR and nuclear cofactors to DREs in Ah-responsive gene promoters [
44]. Treatment of MDA-MB-231 cells with omeprazole or TCDD resulted in recruitment of the AHR to the CYP1A1 gene promoter and this was accompanied recruitment of pol II (Figure
6C) and induction of
CYP1A1 gene expression (Additional file
1: Figure S1 and Additional file
1: Figure S3). The CXCR4 promoter contains two regions with
cis-elements consistent with DRE sequences (DRE-123 and DRE-4). Omeprazole decreased luciferase activity in MDA-MB-231 cells transfected with the pGL3-CXCR4 (-1121 to +95) construct which contains these DREs and this response was attenuated by AHR silencing or AHR antagonists (Figure
4C). Omeprazole and TCDD also induced AHR binding to the CXCR4 promoter (Figure
6B,C); however, in contrast to the recruitment of pol II to the CYP1A1 promoter, both TCDD and omeprazole decreased pol II interactions with the CXCR4 promoter (Figure
6C) and this was consistent with omeprazole-mediated repression of
CXCR4 gene expression. We did not observe ligand-dependent recruitment of the corepressor SMRT to the CXCR4 promoter and are currently investigating the role of other nuclear cofactors required for AHR-mediated suppression of
CXCR4 and other genes in cancer cells treated with omeprazole. Thus, the anticancer activity of omeprazole is due, in part, to the AhR but this does not exclude a role for other AhR-independent pathways.
Conclusions
In summary, results of this study demonstrate that among several AHR-active pharmaceuticals, omeprazole exhibits antimetastatic activity for triple-negative MDA-MB-231 breast cancer cells, and
CXCR4 is one of the key target genes not only for omeprazole but also for other AHR agonists [
31‐
34]. AHR-dependent downregulation of
CXCR4 by omeprazole significantly contributed to the antimetastatic activity of this compound since silencing of
CXCR4 by RNAi in MDA-MB-231 cells also inhibited invasion of these cells in a Boyden chamber assay (Figure
5D). Since
CXCR4 has both functional and prognostic significance for metastasis in breast tumors and cells [
45], the antimetastatic activity of omeprazole and other AHR-active pharmaceuticals including other benzimidazole protein pump inhibitors are currently being investigated. The anticancer activity of drugs used for treatment of gastroesophageal reflux disease is not well established [
46] and it is possible that chemotherapeutic effects of omeprazole for inhibition of breast cancer metastasis may require higher doses than are typically used for treating acid reflux. However, lower doses of omeprazole may be effective for drug combination therapies [
41] and these are currently being investigated.
Competing interest
The authors declare that have no competing interest.
Authors’ contributions
UHJ carried out most of the in vivo and in vitro studies. SOL assisted UHJ in the in vivo studies and the immunostaining experiments. CP carried out the pathology studies. SS supervised the project and wrote the paper. All authors read and approved the final manuscript.