The role of the aryl hydrocarbon receptor in the development of cells with the molecular and functional characteristics of cancer stem-like cells
- Elizabeth A. Stanford1, 2,
- Zhongyan Wang1,
- Olga Novikov1, 2,
- Francesca Mulas3,
- Esther Landesman-Bollag4,
- Stefano Monti3,
- Brenden W. Smith2, 4, 5,
- David C. Seldin^4,
- George J. Murphy4, 5 and
- David H. Sherr1Email author
© Stanford et al. 2016
Received: 18 October 2015
Accepted: 22 February 2016
Published: 16 March 2016
Self-renewing, chemoresistant breast cancer stem cells are believed to contribute significantly to cancer invasion, migration and patient relapse. Therefore, the identification of signaling pathways that regulate the acquisition of stem-like qualities is an important step towards understanding why patients relapse and towards development of novel therapeutics that specifically target cancer stem cell vulnerabilities. Recent studies identified a role for the aryl hydrocarbon receptor (AHR), an environmental carcinogen receptor implicated in cancer initiation, in normal tissue-specific stem cell self-renewal. These studies inspired the hypothesis that the AHR plays a role in the acquisition of cancer stem cell-like qualities.
To test this hypothesis, AHR activity in Hs578T triple negative and SUM149 inflammatory breast cancer cells were modulated with AHR ligands, shRNA or AHR-specific inhibitors, and phenotypic, genomic and functional stem cell-associated characteristics were evaluated. The data demonstrate that (1) ALDHhigh cells express elevated levels of Ahr and Cyp1b1 and Cyp1a1, AHR-driven genes, (2) AHR knockdown reduces ALDH activity by 80 %, (3) AHR hyper-activation with several ligands, including environmental ligands, significantly increases ALDH1 activity, expression of stem cell- and invasion/migration-associated genes, and accelerates cell migration, (4) a significant correlation between Ahr or Cyp1b1 expression (as a surrogate marker for AHR activity) and expression of stem cell- and invasion/migration-associated gene sets is seen with genomic data obtained from 79 human breast cancer cell lines and over 1,850 primary human breast cancers, (5) the AHR interacts directly with Sox2, a master regulator of self-renewal; AHR ligands increase this interaction and nuclear SOX2 translocation, (6) AHR knockdown inhibits tumorsphere formation in low adherence conditions, (7) AHR inhibition blocks the rapid migration of ALDHhigh cells and reduces ALDHhigh cell chemoresistance, (8) ALDHhigh cells are highly efficient at initiating tumors in orthotopic xenografts, and (9) AHR knockdown inhibits tumor initiation and reduces tumor Aldh1a1, Sox2, and Cyp1b1 expression in vivo.
These data suggest that the AHR plays an important role in development of cells with cancer stem cell-like qualities and that environmental AHR ligands may exacerbate breast cancer by enhancing expression of these properties.
Given the emerging evidence that common environmental carcinogens play a significant role in cancer , increased attention has been paid to molecular mechanisms through which pollutants affect tumor formation, invasion and/or progression [2–4]. Historically, most studies on environmental chemical carcinogenesis centered on the ability of genotoxic chemicals to damage DNA, induce mutations, and initiate cancers [5–7]. However, recent data suggest alternative, non-genotoxic pathways involving cellular receptors that can be activated by environmental ligands. One such receptor is the aryl hydrocarbon receptor (AHR). The AHR is the only ligand-activated member of the Per-ARNT-Sim (bHLH/PAS) family of transcription factors, all of which play important roles as environmental- and physiological stress-sensing proteins . The AHR has been best studied for its ability to be activated by dioxins, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons , all of which are high priority chemicals on the U.S. Agency for Toxic Substances and Disease Registry list of pollutants of greatest concern to human health (http://www.atsdr.cdc.gov/SPL/resources).
Ligand-bound AHR induces P450 enzymes such as CYP1B1 and CYP1A1, which are capable of generating mutagenic intermediates. However, more recent work suggests that the AHR, which is expressed at aberrantly high levels and is chronically active in several cancers, plays an ongoing role in tumor progression by enhancing tumor invasion and migration [10–15]. The contribution of the AHR to the later stages of cancer may be mediated by non-genotoxic endogenous ligands, which chronically drive AHR transcriptional activity [16, 17]. Here, it is postulated that environmental ligands mimic this effect and drive cancer progression, at least in part, by increasing the development and/or function of cells exhibiting cancer stem-like cell (CSLC) properties.
Recent evidence suggests that invasion and eventual metastasis leading to patient death is mediated, to a disproportionate extent, by chemoresistant, long-lived cancer stem cells, sometimes referred to as tumor-initiating cells [18–25]. Breast cancer stem cells can be defined by (1) expression of genes associated with ‘normal’ tissue stem cells (e.g. Notch1,2, Sox2, Pou5F1/Oct4) and with invasion and migration (e.g. Twist1,2, Vim, Snai1, Snai2) [26–30]; (2) formation of spheroid colonies in ultra-low adherence cultures ; (3) elevated levels of aldehyde dehydrogenases (ALDH), enzymes associated with chemoresistance, high histological tumor grade, and poor prognoses [19, 21, 32]; (4) the propensity to self-renew while spawning progenitor cells [31, 33]; and (5) an increased tumor initiation capacity in xenografts [31, 33]. Here, we operationally define ‘breast cancer stem-like cells’ (BCSLC) as tumor cells robustly expressing the five aforementioned characteristics in a continuum of ‘stem-ness’ in which some cells are more stem-like than others at any given time. Clearly, identifying factors responsible for the development of cells with cancer stem cell qualities is an important step towards understanding why many patients relapse, even several years after remission.
The AHR plays an important role in tissue-specific embryonic development, hematopoietic stem cell self-renewal, pluripotent stem cell and neural stem cell differentiation, and megakaryocyte/erythroid stem cell growth [34–40]. Here, complementing parameters of ‘stem-ness’, including ALDH enzyme activity, stem cell-, invasion- and migration-associated gene expression, tumorsphere formation, migration rate, chemoresistance, and tumor formation at limiting concentrations in xenografts were assessed to test the hypothesis that the AHR similarly influences development and function of BCSLCs. The potential for the AHR to directly interact with the Sox2 gene, a master regulator of normal tissue-specific stem cell self-renewal and differentiation, was of particular interest.
These studies were performed primarily with ER−/PR−/Her2− triple negative breast cancer (TNBC) cell lines: Hs578T, derived from a carcinomosarcoma, and SUM149, derived from an inflammatory breast cancer (IBC). TNBC lines were selected for these studies primarily because no effective targeted therapeutic is yet available for this class of breast cancers and because we wanted to evaluate AHR signaling in the absence of its well-established interactions with the estrogen receptor . Results in those lines were compared with genomic outcomes in 79 breast cancer cell lines and more than 1,850 primary cancers. Our results show that the AHR is involved in the control of phenotypic, genomic, and functional cancer stem cell markers in ER−/PR−/Her2− cells, strongly implicating an important role for the AHR in acquisition of stem cell-like qualities, encouraging development of AHR-targeted therapeutics, and raising the possibility that environmental AHR ligands may drive BCSLC development or activity.
AHR expression is elevated in ALDH1high TNBCs
We have previously published data demonstrating elevated expression of transcriptionally (‘constitutively’) active AHR in human breast cancer cell lines [10, 15, 42, 43]. The expression of nuclear AHR in ER−/PR−/Her2− human breast cancer-derived Hs578T cells and in inflammatory ER−/PR−/Her2− breast cancer-derived SUM149 cells (Additional file 1: Figure S1A) was consistent with these reports. Furthermore, a predominance of nuclear AHR in primary human breast cancers (Additional file 1: Figure S1B, middle and bottom panels), but not in normal breast tissue (Additional file 1: Figure S1B, top panel), supports the conclusion that the AHR is constitutively active in primary cancers as well. Importantly, non-epithelial cells did not express AHR, normal epithelial cells in ducts had a low level of AHR staining, similar to our previous findings in rats , and all AHR staining seen in normal epithelial cells was cytoplasmic, indicating inactive AHR. Note that the stains presented here are representative of similar staining observed in 50 human breast cancer samples fixed on a tissue microarray.
Work from several laboratories indicates a role for the AHR in tissue-specific stem cell development [34–38], suggesting a general role for the AHR in stem cell biology. We and others have demonstrated that the AHR is highly expressed and constitutively active in breast cancers and that its activity correlates with tumor aggressiveness [10, 44–47]. Since cancer stem cells contribute to tumor progression, we postulated that the AHR plays a role in the development of breast cancer cells with stem cell-like characteristics (BCSLC).
Several investigators have shown that CD44+/CD24− cell staining is not an entirely consistent indicator of tumor initiating ability in ER−/PR−/Her2− breast cancer cells due to over-staining of TNBCs [23, 48–51]. Over-expression or non-specific staining for these prototypic cancer stem cell markers also precluded their use in our studies (data not shown). Therefore, ALDH activity, which appears to be a more selective functional marker for TNBC stem-like cells [19, 23, 52, 53], was used here for marking of and enriching for cancer stem-like cells.
To determine if elevated Ahr and Cyp1b1 expression in ALDH1high cells reflects a role for the AHR in maintaining stem cell properties and if environmental AHR ligands have the potential to increase these properties in TNBCs, AHR expression or activity was modulated with a doxycycline (dox)-inducible Ahr-specific shRNA (shAhr), AHR inhibitors (CH223191 and CB7993113) [42, 54, 55], or four AHR agonists: (1) 6-formylindolo[3,2-b]carbazole (FICZ), a high affinity AHR ligand, tryptophan photo-metabolite, and potential endogenous ligand ; (2) β-naphthoflavone (β-NF), a flavone with moderate affinity for the AHR; (3) 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD), a high affinity, persistent environmental AHR ligand and ‘gold standard’ AHR ligand; or (4) 7,12 dimethylbenzanthracene (DMBA), a readily metabolizable polycyclic aromatic hydrocarbon.
Increasing AHR activity increases expression of BCSLC-related genes
To determine if several stem cell-associated genes are regulated by the AHR, Hs578T cells were treated for 48 hours with vehicle or FICZ, stained with ALDEFLUORTM, and sorted for ALDHhigh and ALDHlow cells. Consistent with previous studies demonstrating BCSLC plasticity , pre-sorting Hs578T ALDHhigh and ALDHlow cells prior to treatment and culture for 48 hours was precluded by the tendency for sorted Hs578T subpopulations to revert to the original distribution of ALDHhigh (~5 %) and ALDHlow (~95 %) cells within 24 hours (data not shown).
Consensus aryl hydrocarbon receptor response elements (AHREs) in human stem cell- and migration/invasion-associated gene promoters
# of Consensus AHREs and location relative to TSS
8 (−206, −267, −840, −859, −944, −1028, −1678, −2392)
Stem cell markers
7 (−617, −749, −1284, −1430, −1678, −2577, +59)
9 (−83, −140, −169, −430, −683, −1076, −2019, −2103, +146)
3 (−1403, −2203, −2289)
5 (−64, 328, −369, −2275, −2428)
13 (−75, −284, −367, −655, −743, −982,-1270, −1838, −1844, −2114, −2140, +52, +196)
7 (−270, −559, −904, −1821, −2114, −2686, +190)
Invasion and migration markers
5 (−82, −148, −1836, −1944, +216)
6 (−360, 384, −927, −1003, +224, +294)
2 (−314, −2114)
5 (−836, −1936, −2037, −2190, −2734)
5 (−576, −2016, −2821, −2823, +50)
Increasing AHR activity increases expression of migration- and invasion-associated genes
As a functional readout of migration, the effects of AHR modulation on the ability of SUM149 cells to migrate in a 48 hour scratch-wound assay were determined. SUM149 cells were chosen for this experiment since, unlike Hs578T cells, ALDHhigh SUM149 cells remained ALDHhigh in vitro for at least 96 hours, unless the AHR inhibitor, CH223191 was added (Additional file 3: Figure S3A). ALDHlow, SUM149 cells tended to revert to ALDHhigh phenotype but this reversion was inhibited by CH223191 treatment (Additional file 3: Figure S3B). Similar results describing the plasticity of stem-like cells have been previously reported .
Generalization of the correlation between Ahr or Cyp1b1 and BCSLC- and invasion/migration-associated genes
The experiments described above confirm that AHR hyper-activation with FICZ induces both BCSLC- and migration/invasion-associated genes in an AHR-dependent fashion in Hs578T cells. If these associations are generalizable to other breast cancer cell lines, then it would be predicted that Ahr expression and expression of Cyp1b1, as a marker for AHR activity, would correlate, in multiple breast cancer cell lines, with expression of the BCSLC- and migration/invasion-associated gene sets identified in Hs578T cells. For such an analysis, we used microarray/RNA-seq data compiled by the Broad Institute on 79 primary human breast cancer cell lines, i.e. the Cancer Cell Line Encyclopedia (CCLE) . Use of Cyp1b1 as a marker for AHR activity in this context is supported by (1) our findings , and those of others , demonstrating that baseline Cyp1b1 mRNA levels are maintained in part by ‘constitutively active’ AHR in human breast cancer cell lines, and (2) the observation that, of all breast cancer cell lines in the CCLE, the nearest neighbor to Ahr of >20,000 gene probes is Cyp1b1 (P = 0.0019; this is not to say that there are no other factors regulating Cyp1b1 expression ). Gene set enrichment analyses (GSEA) were performed with the aim of testing whether the gene set listed in Table 1 is significantly and coordinately correlated with Ahr or Cyp1b1 expression. Indeed, Ahr expression was significantly correlated (false discovery rate = 0.025) with the putative AHR target gene set shown in Table 1 (Additional file 5: Figure S5A). Similarly, there was a significant correlation between Cyp1b1 and the expression of the putative AHR target gene set (FDR = 0.021; Additional file 5: Figure S5B). Interestingly, the ‘outlier’ with a negative correlation score for both the Ahr and Cyp1b1 analyses, was Msi1 (Additional file 5: Figure S5A and S5B, red arrow), the one stem cell-associated gene we tested that did not increase following AHR hyper-activation (Fig. 3).
To generalize results to primary human cancers, a similar GSEA analysis was performed using transcriptomic data from 977 primary human breast cancers catalogued in the Cancer Genome Atlas (TCGA) database  and 995 primary human breast cancers in the Curtis database . As shown for cell lines in the CCLE, there was a significant association (FDR = 0.047) between Ahr expression and the gene set listed in Table 1 (Additional file 6: Figure S6A). A stronger association (FDR = 0.0001) was seen between Cyp1b1 expression and expression of the putative AHR target gene set (Additional file 6: Figure S6B). As with the CCLE database, Msi1 was not correlated with either Ahr or Cyp1b1 in the TCGA database (Red arrows, Additional file 6: Figure S6A, S6B). Similar data were obtained using the Curtis dataset (not shown). Collectively, data mined from three large breast cancer databases (CCLE, TCGA, and Curtis) show a significant and generalizable association between Ahr or AHR activity (Cyp1b1 expression) and cancer stem cell- and migration/invasion-associated gene sets, an outcome consistent with regulation of these genes by a constitutively active (i.e. endogenous AHR ligand-activated) AHR.
Decreasing AHR activity decreases tumorsphere formation
AHR controls expression of cancer stem cell-associated properties in an inflammatory breast cancer cell line
Decreasing AHR activity decreases chemoresistance, a hallmark of BCSLCs
Half maximal effective concentrations (EC50) of two chemotherapeutics in the presence or absence of an AHR inhibitor
Chemotherapeutic EC50 (μM)
Paclitaxel + CH
Paclitaxel + CH
Adriamycin + CH
Adriamycin + CH
shAhr-mediated AHR knockdown decreases expansion of tumors initiated with ALDHhigh and ALDHlow SUM149 cells
Accumulating data suggest that the AHR plays an important role in breast cancer, in general, and in progression to end-stage invasion and migration in particular. For example, the AHR is hyper-expressed and transcriptionally active in most TNBC and IBC cell lines, and its expression is associated with tumor invasion [10, 13, 15, 79]. The data presented here strongly suggest that the AHR drives tumorigenesis in part through induction or maintenance of cells with cancer stem cell-like properties.
The involvement of the AHR in BCSLC biology is suggested by its emerging role in normal tissue-specific stem cell development. For example, the AHR, presumably activated by endogenous ligand(s), helps maintain hematopoietic stem cell self-renewal and block differentiation [36, 37, 80], and drives bipotential blood stem cell differentiation . AHR repression in embryonic stem cells likely maintains pluripotency, and the AHR controls embryonic stem cell differentiation into cardiomyocytes . Data presented here extend these studies by demonstrating that the AHR is involved in the phenotype (ALDH1 activity and Aldh1a1 expression), genomics (up-regulation of stem cell- and migration/invasion-associated genes), and function (migration, chemoresistance, tumorigenicity) of BCSLCs.
Elevated ALDH expression identifies BCSLCs and is associated with increased expression of chemoresistance proteins, increased tumor cell invasion, higher tumor grade, and poor survival in breast cancer patients [19, 21, 28, 32, 53]. Indeed, in our hands, ALDHhigh cells exhibited increased chemoresistance (Fig. 9), elevated expression of stem cell- and migration/invasion-associated genes (Figs. 3 and 5), faster migration (Fig. 6), higher tumor-initiating capacity and increased tumor growth rates in vivo (Figs. 10, 12 and 13). AHR control of chemoresistance is particularly interesting given important recent studies demonstrating that TCDD decreases and AHR inhibition increases apoptosis induced by UV light or chemotherapeutics in six breast cancer cell lines . AHR-mediated chemoresistance takes on even greater significance given recent studies showing that chemoresistance may be a more meaningful marker of metastatic behavior than markers of epithelial-to-mesenchymal transition . These data support the hypothesis that AHR inhibitors may represent effective, targeted therapeutics when used in combination with conventional chemotherapeutics. Collectively, these data strongly support the conclusion that ALDHhigh cells are at least breast cancer stem-like cells if not bona fide breast cancer stem cells.
We previously demonstrated that constitutively active AHR in breast cancer lines preferentially drives Cyp1b1 expression while an exogenous ligand, e.g. DMBA, tends to induce greater fold-increases in Cyp1a1 than Cyp1b1 . Interestingly, higher relative levels of Cyp1b1 were noted in ALDHhigh cells, as compared with ALDHlow cells (Fig. 3a), suggesting the possibility that a higher level of AHR activity, as represented by baseline Cyp1b1 levels, characterizes ALDHhigh-stem like cells.
AHR hyper-activation with FICZ increased expression of Aldh1a1 and stem cell-associated genes demonstrating a causal relationship between AHR activity and expression of these genes (Fig. 3b,c). This gene set has been implicated in generating both normal tissue stem cells and BCSLCs. Notch1 and Notch2 are critical to symmetric and asymmetric cell division, stem cell differentiation in embryonic and adult stem cells [26, 81], and are potential therapeutic targets [76, 82]. Bmi1 is required for the maintenance of somatic stem cells through repression of cellular senescence and cell death , and is involved in the control of BCSLC growth, chemoresistance, and tumorsphere formation . Stella is involved in maintenance of gene-specific DNA methylation in the early embryo, and is a marker for some BCSLC types . Sox2, Oct4 and Nanog are traditional embryonic stem cell markers used to reprogram cells to a pluripotent state, and are expressed at elevated levels in BCSLCs [27, 30, 85, 86]. Sox2 is up-regulated in TNBCs and has been implicated in tumorsphere formation and control of tumor initiation [27, 62, 63]. Therefore, the finding that the AHR directly interacts with the Sox2 promoter (Fig. 4a) strongly suggests that the AHR is at the apex of an important signaling pathway that controls cancer progression by increasing phenotypic and functional expression of cancer stem cell-associated markers within the tumor cell population.
Our data also suggest that the AHR plays a key role in regulating BCSLC migration. Snail, Slug, Twist1, Twist2, Tgfb1, and Fibronectin (Fn1), which contribute to cell invasion and cell migration [28, 87, 88], are all up-regulated in FICZ-treated ALDHhigh cells (Fig. 5c). As would be predicted from these results, AHR inhibition slows (Fig. 6) and AHR hyper-activation accelerates (Additional file 4: Figure S4) cell migration in the scratch-wound assay.
Importantly, these findings on AHR-regulated genes appear generalizable since strong correlations were seen between Ahr or Cyp1b1 and the stem cell- and migration/invasion-associated gene sets in databases of 79 human breast cancer cell lines characterized in the CCLE and over 1850 primary human breast cancers catalogued in the TCGA and Curtis databases (Additional files 5 and 6: Figure S5 and S6). Furthermore, these results suggest the possibility that the AHR contributes to cell invasion and migration through up-regulation of stem cell- and invasion/migration-associated genes.
Consistent up-regulation of CYP1B1 in breast cancers  suggests that this enzyme plays an important role in cancer, potentially by influencing cell migration . It is, therefore, formally possible that at least some of the effects observed here reflect AHR ligand binding to CYP1B1. While this possibility cannot be ruled out, particularly for ligands such as FICZ, which are metabolized by CYP1B1 , it seems unlikely as a general rule since TCDD, which is not metabolized by CYP1B1 and does not bind CYP1B1 (data not shown), generates the same outcomes (increase in stem cell-associated genes, cell migration) as the other ligands.
AHR inhibition or knockdown in either Hs578T or SUM149 cells significantly reduced the number and size of tumorspheres formed in low adherence conditions over several generations (Figs. 7 and 8e,f). The formation of these colonies is generally considered to be a function of asymmetric BCSLC division and production of progenitor cells which constitute the majority of the cells in the spheres [31, 70, 72]. Therefore, it is possible that the AHR controls the asymmetric differentiation of BCSLC and/or the growth of their progenitors.
Furthermore, AHR knockdown with either of two shAhr constructs significantly slowed the initiation and outgrowth of both ALDHlow and ALDHhigh cell-derived tumors (Figs. 10b, c, 12b, c, 13). This decrease in tumor outgrowth was accompanied by a decrease in Cyp1b1, Aldh1a1, and Sox2 expression (Fig. 11), further linking AHR activity to expression of these genes.
Results presented here are reminiscent of several studies demonstrating that baseline (endogenous ligand-induced) AHR activity in immortalized cells favors tumor growth or aggressive behavior [12–14, 92–97]. Paradoxically, several studies indicate that exogenous AHR ligands can reduce tumor growth or invasion [41, 92, 96, 97]. As elegantly described in a recent review , these seemingly contradictory results may, in part, reflect context- or tumor stage-specific differences. For example, AHR agonists may inhibit growth in ER+ breast cancers in part through AHR-mediated down-regulation of ER expression or activity . However, in similar cell types [47, 99, 100], similar AHR agonist- and antagonist-mediated outcomes could be due to more subtle effects on AHR activation or signaling. For instance, it has been postulated that, while endogenous AHR ligands drive signaling towards, for example, increased invasion, exogenous AHR ligands ‘divert’  or ‘disrupt’  the response towards signaling pathways which oppose tumor invasion, e.g. differentiation . Furthermore, exogenous ligands, e.g. Tranilast, that decrease invasion [99, 100], may act as partial agonists that compete with endogenous ligands for AHR binding but which are weaker activators of AHR transcriptional activity, thereby reducing baseline AHR signaling . Finally, outcomes may be ligand-, cell subset-, or dose-specific. Thus, high affinity AHR ligands, such as TCDD, induce stem cell characteristics including ALDH expression and accelerated migration, particularly at low doses (e.g. 0.2–1 nM; Additional file 4: Figure S4, and data not shown), while higher doses (10 nM) may reduce invasiveness of the majority non-BCSLC population .
Finally, a limited number of previous studies have addressed the role of the AHR in breast cancer stem cell generation [96, 97, 99, 102]. While these studies all point towards a role for the AHR in cancer stem-like cell generation, there is as yet no clear consensus on how this occurs or even on whether the AHR favors or inhibits BCSLC production/function. For example, Zhao et al.  showed that AHR activation with β-NF or 3-MC or over-expression of a PasB mutant AHR decreased tumorsphere formation; in our hands, only 3-MC reduced secondary tumorsphere formation in SUM149 and MCF-7 cells (data not shown). In what may seem like a contradiction, Zhao et al.  later published that MCF-7 mammosphere formation was suppressed by AHR inhibition with CH223191 as well as by siRNA-mediated AHR knockdown in MDA-MB-453 cells. In Dubrovska et al. , AHR inhibitors reduced the percentage of ALDHhigh MCF-7 cells in tamoxifen-resistant MCF-7 (as shown in our studies with triple negative Hs578T and SUM149 cells) but produced the opposite effect in wildtype MCF-7 cells. At least some of these differences can be attributed to the different subtypes of breast cancer cells (i.e. ER+, Luminal A-type or Her-2 over-expressing MCF-7 cells versus ER−, basal-like, triple negative Hs578T and SUM149 cells). In any case, further experimentation is required to determine how the AHR influences ‘stem-ness’ in breast cancer cells.
Studies presented here indicate that the AHR influences, in TNBC and IBC cells, critical markers associated with ‘stem-ness’. The ability of several exogenous AHR ligands, including TCDD and DMBA to up-regulate phenotypic, genomic, and/or functional markers of BCSLCs strongly suggests the potential for ubiquitous environmental AHR ligands to accelerate progression to lethal, invasive cancers. Furthermore, the demonstration that AHR inhibition significantly reduces expression of these phenotypic and functional cancer stem cell markers encourages the testing of AHR inhibitors, for example, to significantly increase the sensitivity of BCSLCs to conventional chemotherapeutics. In general, these results suggest that non-toxic AHR modulators may represent important therapeutics for otherwise refractory TNBC and IBC, and potentially for brain and other cancers in which the AHR appears to play a role.
DMSO, β-NF, DMBA, TCDD, paclitaxel, doxorubicin, and doxycycline were obtained from Sigma-Aldrich (St. Louis, MO). FICZ, CH223191, and CB7993113 were provided by Dr. M. Pollastri (Northeastern University).
Cell line acquisition, cell culture, and media
Hs578T and MCF-10F cells were purchased from ATCC and cultured according to ATCC recommendations (ATCC, Manassas, VA). SUM149 cells were a generous gift from Dr. Stephen Ethier (Wayne State University, Detroit, MI). SUM149 cells were maintained in F-12 K Medium (Mediatech, Herndon, VA) containing 5 % FBS (Sigma-Aldrich), 0.5 μg/mL hydrocortisone (Sigma-Aldrich), 2 mM L-glutamine (Mediatech), 100 IU penicillin/100 μg/mL streptomycin (Mediatech), 10 μg/mL insulin (Sigma-Aldrich), and 5 μg/mL Plasmocin (Invivogen, San Diego, CA).
Inducible, stable Ahr-specific shRNA cells
Doxycycline (dox)-inducible TurboRFP-shAhr TRIPZ lentiviral vectors (Open Biosystems, Huntsville, AL) were used to make viral transduction particles. Hs578T and SUM149 cells were transduced at optimal MOIs of 25 and 50, respectively, in medium containing hexadimethyrine bromide (8 μM g/mL polybrene; Sigma-Aldrich). Transduced cells were maintained in 1.5 μg/mL puromycin (Invitrogen, Grand Island, NY). RFP expression was maximal 48 hours after dox treatment (1.5 μg/mL) of transduced cells. For in vivo experiments, two different inducible shAHR plasmids were constructed and used to generate two independent doxycycline-inducible shAHR-expressing SUM149 lines.
Cells were dosed with 0.5 μM FICZ, 1 μM β-NF, 10 μM CH223191, 10 μM CB7993113, 1 nM TCDD, 1 μM DMBA, 1.5 μg/mL dox, vehicle (0.1 % DMSO), and/or left untreated every 24 hours. After 48 hours, ALDEFLUOR™ assays were performed according to the manufacturer’s instructions (Stem Cell Technologies, Vancouver, Canada). Briefly, cells (106 cells/mL) were treated with 5 μL/mL ALDEFLUORTM substrate in 1 mL of ALDEFLUORTM buffer. Negative controls were treated with both ALDEFLUORTM substrate and 50 mmol/L diethylaminobenzaldehyde (DEAB), an ALDH-specific inhibitor. Samples were incubated for 35 minutes at 37 °C in the dark. After 35 minutes, cells were centrifuged, the supernatant was removed and the remaining pellet was suspended in ice-cold ALDEFLUORTM buffer and kept on ice. Before samples were read on the flow cytometer, propidium iodine was added (1.5 μg/mL) to quantify viability (propidium iodine was not used on TurboRFP-shAhr transduced cells due to overlapping emissions). Cells were immediately assayed with an LSRII flow cytometer (Becton Dickinson Biosciences, San Jose, CA) using DEAB controls as baselines to gate ALDHhigh and ALDHlow cell populations. ALDHhigh and ALDHlow cells were sorted on a MoFlo Legacy (Beckman Coulter, Indianapolis, IN). All flow cytometry data was analyzed using Flowjo software (Ashland, OR) according to Stem Cell Technologies’ manufacturer instructions. Briefly, DEAB-treated control samples were used to make ALDHhigh and ALDHlow gates as pictured in Fig. 1. The percent of cells that fell into each gate was then quantified as ALDHhigh or ALDHlow subsets.
Cells were lysed and protein extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific, Grand Island, NY), according to manufacturer instructions. Protein concentration was then quantified via a Bradford protein assay. Equal amounts of protein (40 μg) were subjected to 10 % SDS-PAGE and then transferred to a nitrocellulose membrane. Non-specific binding sites were blocked with blocking buffer containing Tris-buffered saline and 0.1 % Tween-20 with 5 % nonfat milk powder for 1 hour at room temperature, and the blot was incubated with specific antibody in blocking buffer (SOX2, Lamin A/C and α-Tubulin antibody in 1:1000 dilution, respectively) at 4 °C overnight. After washing, the blot was incubated with an appropriate secondary antibody conjugated with horseradish peroxidase for 1 hour at room temperature. After washing, the detection was performed using the enhanced chemiluminescence system. The antibody of SOX2 was purchased from Cell Signaling Technology (Danvers, MA; Cat #: 2748), Lamin A/C from Cell Signaling Technology (Cat #: 2032), and α-Tubulin from EMD Millipore (Billerica, MA; Cat #: CP06). Image J (National Institutes of Health, Bethesda, MD) was used to perform densitometry analysis. Fold-change from naïve is presented following normalization to loading control (Lamin A/C for nuclear extract and α-tubulin for cytoplasmic extract).
Cells were treated as above. After 48 hours, cells were harvested, dosed, and 3 × 103 cells plated in complete MammoCult Medium (STEMCELL Technologies) containing 0.5 μM hydrocortisone, 2 mM L-glutamine, 100 IU penicillin/100 μM g/mL streptomycin, and 1 % methylcellulose (Sigma Aldrich) in ultra-low adherent 24-well plates (Corning Inc.). Colonies were quantified with a Celigo S Imaging Cytometer (Brooks Automation, Chelmsford, MA) after 8 days. For secondary sphere formation, tumorspheres were mechanically and enzymatically dissociated into a single cell suspension, re-dosed, re-plated, and imaged as above.
mRNA was extracted using RNeasy® Plus Mini Kit (Qiagen, Valencia, CA) and cDNA prepared using the GoScript™ Reverse Transcription System (Promega, Madison, WI) with a 1:1 mixture of random and Oligo (dT)15 primers according to manufacturer’s instructions. All RT-qPCR reactions were performed using the GoTaq® RT-qPCR Master Mix System (Promega). Validated primers were purchased from Qiagen Inc. (Valencia, CA): human Cyp1b1 – QT00209496, Cyp1a1 – QT00012341, Twist1 – QT00011956, Snai1 – QT00010010, Snai2 – QT00044128, VIM – QT00095795, Twist2 – QT02454004, FN1 – QT00038024, Notch1 – QT01005109, Notch2 – QT00072212, Aldh1a1 – QT00013286, Aldh1a3 – QT00077588, Pou5f1 – QT00210840, Sox – QT00237601, Nanog – QT01844808, Dppa3 – QT01667197, Msi1 – QT00025389, Human Bmi1 – QT00052654, Tgfb1 – QT00000728, Ahr – QT02422938, and Gapdh – QT01192646. RT-qPCR reactions were performed using a 7900HT Fast Real-Time PCR instrument (Applied Biosystems, Carlsbad, CA), with hot-start activation at 95 °C for 2 min, 40 cycles of denaturation (95 °C for 15 sec), and annealing/extension (55 °C for 60 sec). Relative gene expression was determined using the Pfaffl method  and the threshold value for Gapdh mRNA was used for normalization.
Chromatin immunoprecipitation (ChIP) assay
ChIP studies were performed using an AHR-specific antibody (ab2769; Abcam, Cambridge, MA) and the ChIP kit (ab500; Abcam) according to the manufacturer’s protocol. Cells were fixed and sonicated to produce fragments averaging 500 bp. Following immunoprecipitation with AHR-specific antibody or normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX), DNA was purified and amplified using the following primers: Cyp1b1 primer: 5’-GTTTGGCGCTGGGTTAC-3’ and 5’-AGGTCGGAGCTGACTCTCT-3’ , Sox2 primer: 5’-CTGTGAGAAGGGCGTGAGAG-3’ and 5’- AAACAGCCAGTGCAGGAGTT-3’. The relative DNA amount was calculated using the ΔΔCt method. AHR and IgG control pull-down signal were normalized to input signal.
Hs578T or SUM149 cells were co-transfected with the pGudluc reporter plasmid (0.5 μg) (generously provided by Dr. M. Denison, UC, Davis), and CMV-green (0.1 μg; for normalization) using TransIT-2020 transfection reagent (Mirus, Madison, WI). The transfection medium was replaced after 24 hours. The cells were left untreated or dosed with vehicle (DMSO, 0.1 % final concentration), 0.5 μM FICZ or CH223191 (10 μM), and harvested after 24 hours in Glo Lysis Buffer (Promega, San Luis Obispo, CA). Luciferase activity was determined with the Bright-Glo Luciferase System according to the manufacturer’s instructions (Promega). Luminescence and fluorescence were determined using a Synergy2 multifunction plate reader (Bio-Tek, Winooski, VT).
ALDHhigh and ALDHlow cells were sorted and grown to confluence in 12-well plates. A p200 pipet tip was used to make an ‘X’ in each well and non-adherent cells were removed with PBS washes. Media was added and cells treated with vehicle, 10 μM CH223191, 1 nM TCDD, or 0.5 μM FICZ. Media was changed and cells were re-dosed daily. TScratch software (Tobias Gebäck and Martin Schulz, ETH Zürich) was used to quantify the closure of the scratch over time.
Eight-week old, female non-obese diabetic-severe combined immunodeficiency (NOD/SCID) mice were purchased from Jackson Laboratory (Bar Harbor, ME). To determine if the AHR influences this parameter of BCSLCs, two separate in vivo experiments were performed. For both experiments, SUM149 cells that were stably transduced with either of two dox-inducible shAHR were sorted into ALDHhigh and ALDHlow cell populations. For the first in vivo experiment, 3000 ALDHhigh and ALDHlow cells in 100 μL of 50:50 Matrigel/DMEM were injected into the right and left mammary fat pads, respectively, of female NOD/SCID mice. For the second in vivo experiment, titered numbers (2,500, 5,000, or 10,000) of ALDHhigh and ALDHlow cells in 100 μL of 50:50 Matrigel/DMEM were injected into the right and left mammary fat pads, respectively, of female NOD/SCID mice. For both experiments, control mice drank water with 5 % sucrose, while the treated mice were provided with water containing 5 % sucrose and 2 mg/mL doxycycline to induce the shAHR. Tumor growth was quantified using Vernier calipers and animals were sacrificed when the total tumor burden reached 15 mm. No metastases were noted at this time. Necropsies were performed to resect the tumors from both sides. RNA was isolated from each of the primary tumors for gene expression analyses for the first in vivo experiment. Animals were housed at the Association for Assessment and Accreditation of Laboratory Animal Care certified Boston University Medical Laboratory Animal Science Center and used in accordance with the NIH Guide for the Care and Use of Laboratory Animals. A Boston University Medical Campus Institutional Animal Care and Use Committee approved protocol and National Institutes of Health Guide for the Care and Use of laboratory Animals were followed.
Hs578T and SUM149 cells were grown overnight on glass cover slips. Upon harvest, cells were washed with cold PBS, fixed with 4 % fresh paraformaldehyde for 10 minutes, permeabilized in 0.5 % Triton X-100 for 10 minutes, and blocked with 2 % BSA overnight. Cells were incubated with anti-AHR antibody H-211 (Santa Cruz Biotechnology, Dallas, TX) for 2 hours, washed in PBS and incubated with Alexa Fluor® 594 conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR) for 60 minutes. Cover slips were washed and mounted on slides with ProLong® Gold Antifade Reagent (Life Technologies, Carlsbad, CA). Photomicrography was performed with a Nikon Deconvolution Wide-Field Epifluorescence Microscope using NIS Elements software. No background fluorescence was detectable in samples treated with the secondary antibody alone.
Immunohistochemistry was performed on slides of paraffin-embedded, 5 μm-thick sections of breast invasive ductal carcinoma in a tissue microarray (US Biomax, Inc., Rockvilla, MD) by standard protocol on an intelliPATH Automated Slide Staining System from Biocare Medical (Concord, CA). Briefly, the slides were heated for 15 minutes at 60 °C followed by deparaffinization starting with xylene and rehydrated through graded alcohols to distilled water. Antigen-retrieval was then performed using Diva Decloaker (Biocare Medical) reagent at 100 °C for 35 minutes, and then at 85 °C for 10 minutes. Slides were incubated with Biocare Medical Peroxidase 1 solution for 10 minutes at room temperature, washed with TBST, blocked with Biocare Medical Background Sniper for 30 minutes and washed. Primary AHR-specific antibody (clone H-211, 1:50 dilution, Santa Cruz Biotechnology) was diluted in Biocare Medical Da Vinci Green Diluent and incubated for 2 hours at room temperature followed by washing in TBST. Incubation in Biocare Medical Mach 4 Universal HRP Polymer was then performed for 30 minutes followed by washing in TBST. DAB was diluted in DAB substrate buffer and applied to slides for 5 minutes followed by washing in deionized-H2O. A light hematoxylin stain was applied, the slides were dehydrated, air dried, and mounted, using EcoMount and a coverslip. Microphotography was performed with an Olympus Upright Microscope using QCapture software. No background stain was detectable in the absence of AHR-specific antibody.
Statistical analyses were performed with Prism (GraphPad Software, La Jolla, CA) or StatPlus (Alexandria, VA) unless otherwise noted. Data are presented as mean ± standard error where applicable. One-way analysis of variants (ANOVAs; simple) were used to determine significance. For experiments measuring relative fold-changes in gene expression (determined using the Pfaffl method  with Gapdh mRNA used for normalization), statistical analyses were performed using SAS v9.3. For comparisons of fold-change in vehicle-treated ALDHhigh versus vehicle-treated ALDHlow, and FICZ-treated ALDHlow versus vehicle-treated ALDHlow cells, Gapdh-normalized expression levels were normalized to expression levels in vehicle-treated ALDHlow cells. For comparisons of fold-change with FICZ-treated ALDHhigh versus vehicle-treated ALDHhigh cells, Gapdh-normalized expression levels were normalized to expression levels in vehicle-treated ALDHhigh cells. Statistical significance was determined with the Wilcoxon rank sum test. Statistical analyses of the mouse model compared the average rate of change over time between groups using a random effects model with a random intercept for each mouse. Day 22 was used as the starting (baseline) value to calculate the rate of change. All mouse analyses were performed using SAS v9.3 using a 0.05 level of significance.
Availability of supporting data
The data sets supporting the results of this article are included within the article and its additional files (Additional file 7, excel document). For further information, please contact the corresponding author.
Supported by P42ES007381, PO1 ES011624, The Art beCAUSE Breast Cancer Foundation, The Mary Kay Foundation, and the Avon Foundation. This publication was developed under STAR Fellowship Assistance Agreement no. FP-917648-01-0 awarded by the U.S. Environmental Protection Agency (EPA). It has not been formally reviewed by EPA. The views expressed in this publication are solely those of Elizabeth Stanford, and EPA does not endorse any products or commercial services mentioned in this publication. The authors would like to acknowledge Ms. B. Campbell for her technical assistance, Dr. J. Weinberg for statistical consultations, Dr. M. Pollastri for synthesizing FICZ and CH223191, Dr. M. Denison for his gift of the pGudLuc vector, Dr. S. Ethier for the Sum149 cells, the Boston University Flow Cytometry Core Facility for their support and assistance, Dr. Michael Kirber and the BUMC Cellular Imaging, and Ms. Nathalie Bitar in the Boston University Immunohistochemistry Core Facility. Finally, we would like to thank Dr. Ana De La Cueva Herrera for her assistance with mammary fat pad injections.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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