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 [44], 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 (BCS L C).

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.

A fluorescence-based ALDH1 enzyme activity assay [19, 20, 23, 52, 53] was used to quantify ALDH1 activity in TNBC Hs578T cells, which express relatively high levels of transcriptionally active AHR [15]. Cells were sorted by flow cytometry into ALDH1high and ALDH1low subsets. Approximately 5 % of Hs578T cells expressed high levels of ALDH1 activity (ALDHhigh; Fig. 1a, right panel), a result consistent with previous studies of BCS L Cs [20]. To determine if the Ahr and an AHR target gene, Cyp1b1, are more highly represented in ALDHhigh cells, Ahr and Cyp1b1 mRNAs were quantified by RT-qPCR. Ahr and Cyp1b1 mRNAs were significantly higher in ALDHhigh cells than ALDHlow cells (P <0.05–0.005; Fig. 1b).

Fig. 1 Ahr and Cyp1b1 expression is increased in ALDH1high Hs578T cells. (a) ER−/PR−/HER2− Hs578T cells were stained with ALDEFLUOR™ in the presence or absence of diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, and ALDH activity (production of fluorescent substrate) was quantified by flow cytometry. Regions were set using dot plots from DEAB-treated cells. Data are representative of 24 experiments. (b) Ahr and Cyp1b1 mRNA expression in sorted ALDHhigh and ALDHlow cells was quantified by RT-qPCR. Data from three independent experiments were analyzed using the Pfaffl method [103], normalized to the Gapdh signal, and presented as mean fold-change from ALDHlow ± standard error. Asterisks indicate a significant increase in the mRNA fold-change, *P <0.05, **P <0.005 Full size image

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 [56]; (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.

Dox-induced shAhr or the AHR-specific inhibitor CH223191 significantly reduced (P <0.01–0.0001) Ahr expression and AHR-dependent (pGudLuc) reporter activity, respectively (Fig. 2a), significantly decreased (P <0.05–0.0005) the percentage of ALDHhigh cells by over 80 % (Fig. 2b, c), and reduced overall ALDH1 activity in the entire Hs578T population (Fig. 2d), suggesting that ‘constitutively active’ (endogenous ligand-activated) AHR maintains baseline ALDH1 levels. Similar data were obtained with our recently described AHR inhibitor, CB7993113 [42] (not shown). Conversely, FICZ, β-NF, TCDD, or DMBA significantly increased the percentage of ALDH1high cells and ALDH1 activity in the entire Hs578T population (P <0.05–0.005; Fig. 2b,c, d). In all cases, AHR agonist-induced increases were significantly inhibited by CH223191 (P <0.05–0.001). Similar results were obtained with immortalized but non-malignant triple negative MCF-10F cells (Additional file 2: Figure S2) and with ER+ luminal-type MCF7 cells [57] (data not shown). These results suggest that AHR ligand-induced ALDH up-regulation is likely generalizable to different breast cancer subtypes.

Fig. 2 AHR modulation alters ALDH1 activity in Hs578T cells. (a) Wildtype or doxycycline (dox)-inducible shAhr-transduced Hs578T cells were transfected with CMV-green control plasmid and AHR-driven pGudLuc reporter and treated for 48 hours with 10 μM AHR inhibitor CH223191 or with doxycycline (1.5 μg/mL) to induce the shAhr. Ahr mRNA was quantified by RT-qPCR and normalized to Gapdh mRNA expression. pGudLuc activity was assayed by luminescence and normalized to CMV-green expression. Values were normalized to Ahr or pGudLuc levels in untreated Hs578T cells. Data are presented as mean ± standard error. Left panel n = 6, middle panel n = 3, right panel n = 6. Asterisks indicate a significant decrease in the mRNA fold-change or reporter activity, *P <0.01, **P <0.001, ***P <0.0001. (b) Representative flow cytometry plots of ALDEFLUOR™ staining of wildtype Hs578T cells or dox-inducible shAhr-transduced Hs578T cells treated for 48 hours are presented. Dox-inducible shAhr transduced Hs578T cells were treated for 48 hours with dox. Hs578T wildtype cells were treated for 48 hours with vehicle, 10 μM CH223191, 1 μM β-NF, 0.5 μM FICZ, 1 nM TCDD, or 1 μM DMBA. Regions representing ALDHhigh cells were drawn based on the signal generated in the presence of DEAB. (c) Hs578T cells were treated as in (b) and assayed for the percentage of ALDHhigh cells. Data were normalized to results obtained with naïve cells (mean baseline = 4.7 % ALDHhigh cells) and presented as mean fold-change from naive ± standard error. Number of experiments by condition: shAHR-dox n = 10, shAHR + dox n = 10, DMSO n = 24, CH223191 n = 9, FICZ n = 16, FICZ + CH223191 n = 5, β-NF n = 5, β-NF + CH223191 n = 5, TCDD n = 10, TCDD + CH223191 n = 3, DMBA n = 10, and DMBA + CH223191 n = 4. Asterisks indicate a significant decrease in the percentage of ALDHhigh cells, *P <0.05, **P <0.001, ***P <0.0005. A cross indicates a significant increase in ALDHhigh cells, + P <0.05, ++ P <0.01, +++ P <0.005. (d) Depicted are flow cytometry dotplots of dox-inducible shAhr-transduced Hs578T cells without dox (red dots) versus with dox (black dots), wildtype Hs578T cells treated with vehicle (red dots) versus CH223191 (black dots), or wildtype Hs578T cells treated with vehicle (red dots) versus FICZ (black dots). Data are representative plots of independent experiments of the data presented in (c) Full size image

Increasing AHR activity increases expression of BCS L C-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 BCS L C plasticity [58], 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).

As expected, vehicle-treated ALDHhigh cells produced higher levels of Aldh1a1, Ahr, Cyp1b1, and Cyp1a1 than vehicle-treated ALDHlow cells (P <0.005; Fig. 3a). Aldh3a1 mRNA, previously associated with AHR activity [59], was not detected (>33 cycles) in either vehicle or AHR agonist (FICZ or β-naphthoflavone)-treated Hs578T cells (data not shown). ALDHhigh cells also expressed significantly higher levels of seven of the eight stem cell-associated genes studied (P <0.05–0.005) with Msi1 being the exception (Fig. 3a). As expected from the ALDH enzyme activity assay (Fig. 2), FICZ treatment increased Aldh1a1, Cyp1b1, and Cyp1a1 expression in both ALDHlow and ALDHhigh cells (P <0.05–0.01; Fig. 3b, c). Consistent with previous studies, AHR ligand induced significantly higher levels of Cyp1a1 than Cyp1b1 (P <0.05), while baseline Cyp1b1 levels tended to be higher than Cyp1a1 levels [15]. FICZ also increased expression of seven of eight stem cell-associated genes in both cell subsets (P <0.05–0.005), again with Msi1 being the outlier (Fig. 3b, c). These results support the hypothesis that constitutively active and/or exogenous agonist-induced AHR up-regulates multiple stem cell-associated genes. Several of these genes express multiple consensus AHR response elements (Table 1), suggesting that they may be directly regulated by the AHR.

Fig. 3 AHR hyper-activation increases expression of stem cell-associated genes. Hs578T cells were treated with vehicle or 0.5 μM FICZ for 48 hours, sorted into ALDHhigh and ALDHlow cell populations, and then assayed by RT-qPCR for the relative levels of the eight stem cell-associated genes indicated. Gene expression was then normalized to Gapdh levels and fold-change from vehicle-treated ALDHlow or ALDHhigh cells was calculated. Data from nine independent experiments are presented as the mean fold-change ± standard error for all genes except for Cyp1a1, in which six independent experiments are presented. In all cases, statistical significance was determined with the Wilcoxon rank sum test to determine if the distributions of results, relative to 1 as the standard (represented by the dotted line on each graph), are different between the comparison groups. Asterisks indicate a significant increase in the mRNA fold-change, *P <0.05, **P <0.01, ***P <0.005. (a) Expression levels of stem cell-associated genes were normalized to expression levels in vehicle-treated ALDHlow cells and the distribution of outcomes from vehicle-treated ALDHhigh versus vehicle-treated ALDHlow cells compared. (b) Stem cell-associated gene expression levels were normalized to expression levels in vehicle-treated ALDHlow cells and the distribution of outcomes from vehicle-treated ALDHlow versus FICZ-treated ALDHlow cells was compared. (c) Stem cell-associated gene expression levels were normalized to expression levels in vehicle-treated ALDHhigh cells and the distribution of outcomes from vehicle-treated ALDHhigh versus FICZ-treated ALDHhigh cells was compared Full size image

Table 1 Consensus aryl hydrocarbon receptor response elements (AHREs) in human stem cell- and migration/invasion-associated gene promoters Full size table

Given the pivotal role for Sox2 in stem cell self-renewal, BCS L C development and breast cancer outcomes [27, 60–63], AHR/Sox2-specific ChIP assays were performed to determine if the AHR directly interacts with the Sox2 promoter. ChIP assays measuring AHR-Cyp1b1 promoter binding served as positive controls [15]. Indeed, there was a significant basal level of AHR binding to both Cyp1b1 and Sox2 promoter fragments (P <0.005; Fig. 4a), each of which contains several AHR response elements within 500 bp of the PCR primer binding sites (Table 1). AHR inhibition with CH223191 significantly decreased AHR-Sox2 and AHR-Cyp1b1 binding (P <0.05). AHR hyper-activation with FICZ significantly increased AHR-Cyp1b1 and AHR-Sox2 binding by approximately 3-fold and 2-fold, respectively (P <0.05). The AHR-Sox2 increase was blocked with CH223191 treatment (P <0.05; Fig. 4a). Furthermore, treatment of SUM149 cells or MCF-7 cells, which are known to express relatively high SOX2 levels [64], with FICZ, TCDD, or β-NF consistently increased nuclear SOX2 (Fig. 4b, c), a result consistent with increased levels of transcriptionally active SOX2 following AHR hyper-activation. Finally, ectopic Sox2 expression significantly increased ALDH1 activity (P <0.005; Fig. 4d). These data strongly suggest that the AHR directly interacts with Sox2, a critical BCS L C-associated gene, which in turn regulates ALDH1 expression, an enzyme associated with chemoresistance [53].

Fig. 4 Modulation of AHR activity affects AHR binding to Cyp1b1 and Sox2 promoters and SOX2 protein production. (a) Hs578T cells were treated with vehicle, 10 μM AHR inhibitor CH223191, 0.5 μM FICZ, or 0.5 μM FICZ + 10 μM AHR inhibitor CH223191 for 48 hours and ChIP assays were performed with human AHR-specific antibody and Cyp1b1- or Sox2-specific promoters as described in the Materials and Methods. Data are presented as mean fold-change ± standard error, IgG control n = 4, vehicle n = 5, CH223191 n = 4, FICZ n = 5, FICZ + CH223191 = 4. An asterisk indicates a significant increase relative to vehicle controls, *P <0.05. A pound sign indicates a significant decrease relative to vehicle controls, # P <0.05. A cross indicates a significant increase relative to IgG controls, + P <0.005. A caret sign indicates a significant decrease relative to FICZ treatment, ^P <0.05. Relative positions of putative AHR response elements and amplified fragments are represented in the embedded map. (b) SUM149 or (c) MCF-7 cell cytoplasmic and nuclear protein extracts were probed for SOX2 protein expression following treatment with vehicle or AHR agonists: 0.5 μM FICZ, 10 μM B(a)P or 1 nM TCDD. C = cytoplasmic extract, N = nuclear extract. The number above each band indicates fold-change from naïve after normalization to loading control, based on ImageJ densitometry analysis. (d) Hs578T cells were transfected with a CMV promoter-driven Sox2 plasmid and ALDH activity assayed 48 hours later. Data from four independent experiments are presented as percent ALDHhigh ± standard error. Asterisk indicates a significant increase in the %ALDHhigh cells, *P <0.005 Full size image

Increasing AHR activity increases expression of migration- and invasion-associated genes

BCS L Cs are more invasive than the bulk tumor population and have increased expression of migration- and invasion-associated markers [21, 22, 28, 29, 53, 58, 65]. To determine if the increase in stem cell markers described above correlates with markers of migration and invasion, Hs578T cells were treated with vehicle or FICZ for 48 hours, sorted for ALDHhigh and ALDHlow cells, and evaluated for expression of seven genes associated with increased tumor migration and/or invasion. As seen for stem cell markers (Fig. 3), ALDHhigh cells expressed significantly higher levels (P <0.005–0.0005) of Snai1, Twist 1, Twist2, Tgfb1, and Vim than ALDHlow cells, with Twist2 showing the greatest fold-change (Fig. 5a). Although Snai2 and Fn1 tended to be higher in ALDHhigh cells, neither was statistically significant in nine independent experiments. These data are consistent with the BCS L C properties of ALDHhigh cells. AHR hyper-activation with FICZ significantly (P <0.05–0.0005) increased Snai1, Twist1, Twist2, and Vim in both ALDHhigh and ALDHlow cells (Fig. 5b,c) and Tgfb1 was marginally increased in FICZ-treated ALDHlow cells (Fig. 5c).

Fig. 5 AHR hyper-activation increases expression of migration and invasion-associated genes in Hs578T cells. Hs578T cells were treated with vehicle or 0.5 μM FICZ for 48 hours, sorted into ALDHhigh and ALDHlow cells, and then assayed by RT-qPCR for the relative levels of the seven migration and invasion-associated genes indicated. Gene expression was then normalized to Gapdh mRNA levels and fold-change from vehicle-treated ALDHlow or ALDHhigh cells was calculated. Data from nine independent experiments are presented as mean fold-change ± standard error. In all cases, statistical significance was determined with the Wilcoxon rank sum test to determine if the distributions of results, relative to 1 as the standard (represented by the dotted line on each graph), are different between the comparison groups. Asterisks indicate a significant increase in the mRNA fold-change, *P <0.05, **P <0.005, ***P <0.0005. (a) Migration- and invasion-associated gene expression levels were normalized to expression levels in vehicle-treated ALDHlow cells and the distribution of outcomes from vehicle-treated ALDHhigh versus vehicle-treated ALDHlow cells was compared. (b) Gene expression levels were normalized to expression levels in vehicle-treated ALDHlow cells and the distribution of outcomes from vehicle-treated ALDHlow versus FICZ-treated ALDHlow cells was compared. (c) Gene expression levels were normalized to expression levels in vehicle-treated ALDHhigh cells and the distribution of outcomes from vehicle-treated ALDHhigh versus FICZ-treated ALDHhigh cells was compared Full size image

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 [58].

As expected, vehicle-treated ALDHhigh cells ‘repaired’ the wound significantly faster than vehicle-treated ALDHlow cells, as quantified by a decrease in exposed surface area (Fig. 6; P <0.05 at 48 hours). Furthermore, wound repair with both subpopulations was significantly inhibited by CH223191 treatment (Fig. 6; P <0.05–0.0005). Similar data were obtained with unsorted Hs578T and SUM149 cells and with another AHR inhibitor, CB7993113 (not shown). No cell divisions were observed over this 48-hour period as assessed by CFSE staining and analysis by flow cytometry (not shown). In addition, both 0.5 μM FICZ and 1 nM TCDD significantly accelerated migration of unsorted, ALDHlow (not shown), and ALDHhigh SUM149 subsets (Additional file 4: Figure S4). A significant increase in migration rate also was seen for ALDHhigh cells following a 48-hour treatment with a lower TCDD dose (0.2 nM, not shown).

Fig. 6 AHR down-regulation decreases migration of SUM149 cells. (a) Presented are representative images of SUM149 cell migration at 24 and 48 hours after cells were sorted into ALDHhigh and ALDHlow populations, cultured to confluence, scratched, and treated with vehicle or 10 μM CH223191. Data are representative of three independent experiments. Black lines indicate the borders of the original scratch. (b) SUM149 cells were treated as in (a) and percent exposed area was quantified. Data from three experiments were normalized to results obtained with naïve cells and presented as mean percent exposed area ± standard error. Asterisks indicate a significant increase in exposed area, *P <0.05, **P <0.0005. A cross indicates a significant decrease in exposed area, + P <0.05 Full size image

Generalization of the correlation between Ahr or Cyp1b1 and BCS L C- and invasion/migration-associated genes

The experiments described above confirm that AHR hyper-activation with FICZ induces both BCS L C- 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 BCS L C- 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) [66]. Use of Cyp1b1 as a marker for AHR activity in this context is supported by (1) our findings [15], and those of others [67], 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 [67]). 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 [68] and 995 primary human breast cancers in the Curtis database [69]. 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

BCS L C can form tumorspheres and produce progenitor cells in ultra-low adherence conditions over several passages [20, 31, 70–73]. To determine if the AHR contributes to this functional readout of BCS L Cs, Hs578T cells were cultured in Mammocult media under ultra-low adherence conditions and AHR activity and expression were modulated with CH223191 or with a dox-inducible shAhr. Both the size and total number of tumorspheres were significantly reduced (P <0.05–0.005) by CH223191 or a dox-induced shAhr in primary, secondary (Fig. 7a,b), tertiary, and quaternary (not shown) cultures. No effect on cell viability (trypan blue exclusion) was seen (the percent viability is indicated in the upper right corner of each image in Fig. 7a). Similar results were obtained with CB7993113 (not shown). These results suggest that the AHR regulates tumorsphere formation and the ability of BCS L Cs to (asymmetrically) divide and/or differentiate into progenitor cells in low-adherence, selective conditions, and/or controls the ability of progenitor cells to divide.

Fig. 7 AHR down-regulation decreases Hs578T tumorsphere formation. (a) Dox-inducible shAhr-transduced Hs578T (‘shAhr’) or wildtype Hs578T cells were left untreated or treated for 48 hours with vehicle, doxycycline, or 10 μM CH223191 as indicated and cultured in Mammocult media under ultra-low adherence conditions. Representative images of primary (day 8) and secondary (day 16, following passage at day 8) tumorspheres are presented. The percentage of viable cells is included on each image. Vehicle and CH223191 treatment groups are representative of six independent experiments. shAHR, no dox and shAHR + dox treatment groups are representative of five independent experiments. (b) Hs578T cells were treated as in (a) and tumorsphere formation was quantified. Data from six experiments were normalized to results obtained with naïve cells and presented as mean fold-changes from naive ± standard error. Asterisks indicate a significant decrease in the percentage of tumorspheres, *P <0.05, **P <0.005 Full size image

AHR controls expression of cancer stem cell-associated properties in an inflammatory breast cancer cell line

IBC is a particularly aggressive form of cancer characterized by a ~50 % survival rate at 2 years [74]. To determine if AHR control of stem cell characteristics is generalizable to this cancer subtype, SUM149 cells, derived from an IBC, were studied for expression of Ahr and Cyp1b1 in ALDHhigh and ALDHlow subpopulations, for the contribution of the AHR to ALDH1 activity, and for the ability to form tumorspheres. As shown for TNBC Hs578T cells, ALDHhigh SUM149 cells expressed significantly higher Ahr and Cyp1b1 levels than ALDHlow cells (P <0.05–0.005; Fig. 8a). CH223191 or Ahr-specific shRNA significantly decreased AHR activity or expression greater than 60 % (P <0.01–0.0001; Fig. 8b), the percentage of ALDHhigh cells by over 80 % (P <0.05–0.0005; Fig. 8c,d), and overall ALDH1 activity in the entire population (not shown). Conversely, FICZ, β-NF, TCDD, and DMBA significantly increased the percentage of ALDHhigh cells (P <0.01–0.005; Fig. 8c,d) and CH223191 treatment in tandem significantly reduced this increase (P <0.05–0.01; Fig. 8c,d). Finally, fewer and smaller tumorspheres were formed following CH223191 treatment (P <0.05–0.01; Fig. 8e,f). No changes in cell viability were detected (the percent viability is indicated in the upper right corner of each image in Fig. 8e). These data parallel those found with Hs578T cells (Fig. 7) and suggest that AHR control of these stem cell properties is generalizable to other ER− breast cancer subtypes.

Fig. 8 AHR modulation affects markers associated with BCS L C in SUM149 cells. (a) Ahr, Cyp1b1 and Aldh1a1 mRNA expression in sorted ALDHhigh and ALDHlow SUM149 cells was quantified by RT-qPCR. Data from three independent experiments were analyzed using the Pfaffl method, normalized to the Gapdh signal, and presented as mean fold-change from ALDHlow ± standard error. Asterisks indicate a significant increase in the mRNA fold-change, *P <0.05, **P <0.01, ***P <0.005. (b) Wildtype or dox-inducible shAhr-transduced SUM149 cells were transfected, treated and quantified as indicated in Fig. 2a. Data are presented as mean ± standard error. Left panel n = 5, center panel n = 4, right panel n = 6. Asterisks indicate a significant decrease in the mRNA fold-change or reporter activity, *P <0.01, **P <0.001, ***P <0.0001. (c) Representative flow cytometry plots of ALDEFLUOR™ staining of dox-inducible shAhr-transduced SUM149 cells cultured, treated, and depicted as described in Fig. 2b. (d) Dox-inducible shAhr-transduced or wildtype SUM149 cells were treated, stained, and quantified as in Fig. 2c. Data were normalized to results obtained with naïve cells (mean baseline =10.4 % ALDHhigh cells) and presented as mean fold-change from naive ± standard error. shAHR-dox n = 4, shAHR + dox n = 4, DMSO n = 12, CH223191 n = 5, FICZ n = 6, FICZ + CH223191 n = 5, β-NF n = 4, β-NF + CH223191 n = 4, TCDD n = 8, TCDD + CH223191 n = 3, DMBA n = 5, and DMBA + CH223191 n = 3. Asterisks indicate a significant decrease in the percentage of ALDHhigh cells, *P <0.05, **P <0.01, ***P <0.0005. A cross indicates a significant increase in ALDHhigh cells, + P <0.01, ++ P <0.005. (e) Representative images of primary (day 8) and secondary (day 16) tumorspheres after SUM149 cells were treated for 48 hours with vehicle or 10 μM CH223191. The percent viable cells are included on each image. Data are representative of four independent experiments. (f) SUM149 cells were treated as in (e) and tumorsphere formation was quantified. Data from four experiments were normalized to results obtained with naïve cells and presented as mean fold-change from naive ± standard error. Asterisks indicate a significant decrease in the percentage of tumorspheres, *P <0.05, **P <0.01 Full size image

Decreasing AHR activity decreases chemoresistance, a hallmark of BCS L Cs

Chemoresistance is another widely studied functional BCS L C marker [21, 22, 53, 72, 73, 75, 76]. To determine if the AHR influences chemoresistance, ALDHhigh and ALDHlow Hs578T cells were treated with titrated doses of adriamycin or paclitaxel, chemotherapeutics with distinct mechanisms of action, with or without CH223191. Cell viability was assayed 24 hours later. As expected of BCS L Cs, ALDHhigh cells were more resistant to the chemotherapeutics than ALDHlow cells (see half maximal effective concentrations (EC 50 ) in Table 2). CH223191 had no effect on viability (not shown). However, CH223191 significantly (P <0.05–0.005) increased sensitivity to both adriamycin and paclitaxel in both ALDHlow and ALDHhigh cells (Fig. 9). The EC 50 of adriamycin-treated ALDHhigh cells (EC 50 = 1.99 μM) was three times greater than that of adriamycin + CH223191-treated cells (EC 50 = 0.60 μM; Fig. 9b; Table 2). These results are consistent with previous reports demonstrating AHR control of chemotherapeutic-induced breast cancer cell apoptosis [77]. Furthermore, they indicate that migration/invasion-associated genes, and functional markers of BCS L Cs (tumorsphere formation, rapid migration, chemoresistance) are influenced by the AHR.

Table 2 Half maximal effective concentrations (EC 50 ) of two chemotherapeutics in the presence or absence of an AHR inhibitor Full size table

Fig. 9 AHR down-regulation decreases chemotherapeutic resistance of both ALDHhigh and ALDHlow Hs578T cells. MTT assays were used to measure cell viability after Hs578T cells were sorted into ALDHlow (a and c) and ALDHhigh (b and d) populations and treated with adriamycin (a and b) or paclitaxel (c and d) with and without 10 μM CH223191 for 24 hours. CH223191 treatment alone did not affect cell viability (≥95 % viability in the presence or absence of CH223191 only). Data from six independent experiments were normalized to vehicle-treated cells and presented as mean percent viable cells ± standard error. Asterisks indicate a significant increase in cell death, *P <0.05, **P <0.005, ***P <0.005 Full size image

shAhr-mediated AHR knockdown decreases expansion of tumors initiated with ALDHhigh and ALDHlow SUM149 cells

Cancer stem cells tend to generate tumors more efficiently in vivo than non-cancer stem cells [18–20, 31, 72]. To determine if AHR, which increases expression of stem cell-associated properties in vitro, influences tumor cell fate in vivo, SUM149 cells, stably transduced with a dox-inducible shAhr (Fig. 8), were sorted and 3,000 ALDHhigh and ALDHlow cells were injected into the right and left mammary fat pads, respectively, of female NOD/SCID mice. Half of the mice were given doxycycline-containing water to induce the shAhr. ALDHhigh cells generated palpable tumors more rapidly and these tumors grew faster than ALDHlow cells (growth rates of 0.26 vs. 0.19 mm/day, P <0.0001; Fig. 10a). Furthermore, dox-induced shAhr significantly reduced growth rates from 0.26 to 0.18 and from 0.19 to 0.11 mm/day in ALDHhigh and ALDHlow cells, respectively (P <0.0005; Fig. 10b,c). Consistent with in vitro experiments, Ahr, Cyp1b1, Aldh1a1, and Sox2 mRNA levels were reduced in tumors from doxycycline-treated mice (Fig. 11).

Fig. 10 AHR down-regulation decreases tumor formation in xenograft mice. (a) Doxycycline-inducible shAhr-expressing SUM149 cells were sorted for ALDHhigh or ALDHlow activity and 3,000 cells were grafted into the mammary fat pads of NOD/SCID mice (10 mice/group). Tumor volumes were measured over the next 56 days. Data are presented as mean tumor volume ± standard error; P <0.0001. The average rate of tumor growth of tumors initiated with ALDHhigh cells (0.26 mm/day) was significantly different than the rate of growth of tumors initiated with ALDHlow cells (0.19 mm/day), P <0.0005. A cross indicates a significant increase in average tumor size beginning at day 25, + P <0.01. (b) Doxycycline-inducible shAhr-expressing SUM149 cells were sorted for low ALDH expression and 3,000 cells were grafted into the mammary fat pads of NOD/SCID mice. Mice then were given water + 5 % sucrose or water with doxycycline + 5 % sucrose and tumor volumes were quantified over the next 56 day period. The average rate of tumor growth of ALDHlow cells in control mice (0.19 mm/day) was significantly different than that of ALDHlow cells in dox-treated mice (0.11 mm/day), P <0.0005. (c) Doxycycline-inducible shAhr-expressing SUM149 cells were sorted for high ALDH expression and 3,000 cells were grafted into the mammary fat pads of NOD/SCID mice. Mice were then given water + 5 % sucrose or water with doxycycline + 5 % sucrose. Tumor volumes were measured over the next 56 day period. The average rate of tumor growth in water-treated control mice (0.26 mm/day) was significantly different than the rate of tumor growth in dox-treated mice (0.18 mm/day), P <0.0005. A cross indicates a significant increase in tumor size beginning at day 25, + P <0.01 Full size image

Fig. 11 AHR down-regulation decreases expression of Ahr, Aldh1a1, Cyp1b1, and Sox2 in xenografted mouse tumors. Twenty NOD/SCID mice were injected with 3,000 dox-inducible shAhr-transduced ALDHhigh or ALDHlow SUM149 cells as described in Fig. 10. Half of the mice were provided with water containing 2 mg/mL doxycycline to induce the shAhr and tumors were harvested after 42–72 days when they reached 15 mm in total size (control mice n = 20, mice with doxycycline treatment n = 19). (a) Ahr, (b) Cyp1b1, (c) Aldh1a1, and (d) Sox2 mRNA expression levels were assayed by RT-qPCR. Since the AHR controls expression of Cyp1b1, Aldh1a1, and Sox2 in both ALDHhigh and ALDHlow cells (e.g. Fig. 3), data generated with tumors from ALDHhigh and ALDHlow tumors were pooled for statistical purposes. Data are presented as fold-change relative to the average CT value from control mice, i.e. no dox, grafted with ALDHlow cells ± standard error. Asterisks indicate a significant decrease in the mRNA fold-change, *P <0.05, **P <0.01, ***P <0.005 Full size image

Although ALDH1 activity and Aldh1 expression have been identified as a valid marker for BCS L Cs [19, 23, 50, 53, 78] and despite ALDHhigh cells having formed tumors sooner than ALDHlow cells in the experiment described above, we wanted to confirm that ALDHhigh cells, in our hands, exhibit a hallmark property of cancer stem cells in vivo, i.e. efficient formation of tumors following xenografting. Therefore, SUM149 cells, stably transduced with a different doxycycline-inducible shAHR, were sorted into ALDHhigh and ALDHlow subpopulations and xenografted at titered numbers (10,000, 5,000, 2,500 cells) into the mammary fat pads of female NOD/SCID recipients (six mice/group). Tumor volume was then tracked over a 69-day period. As predicted of tumors derived from cells with cancer stem-like properties, tumors generated from ALDHhigh cells were detected sooner than tumors generated from ALDHlow cells at each respective cell number, thereby demonstrating a consistently higher efficiency of tumor initiation (Figs. 12 and 13). Tumors grew faster after xenograft of 10,000 ALDHhigh as compared with ALDHlow cells. Furthermore, induction of shAhr with doxycycline significantly delayed tumor formation and subsequent growth of tumors generated from both ALDHhigh cells and ALDHlow cells regardless of cell number transferred to recipients. These data strongly support the use of ALDH activity as a marker for breast cancer cells with cancer stem cell-like properties and the conclusion that AHR influences the efficiency with which all cells along a continuum of low to high ALDH expression can initiate tumors.

Fig. 12 AHR down-regulation decreases the efficiency of tumor formation in vivo. Titered numbers (10,000, 5,000, or 2,500) of sorted ALDHhigh or ALDHlow dox-inducible shAhr-transduced SUM149 cells were grafted into the mammary fat pads of NOD/SCID mice (six mice/group). Mice were given water + 5 % sucrose or water with doxycycline + 5 % sucrose to induce the shAhr. Data are presented as mean tumor volume ± standard error. Asterisks indicate a significant difference in tumor volume, P <0.05. Statistical analyses were not performed when none of the mice in a given control group developed tumors at that time point Full size image