Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m⁶A-demethylation of NANOG mRNA

HIF-Dependent ALKBH5 Expression Promotes m⁶A RNA Demethylation in Hypoxic Breast Cancer Cells.

Breast cancers are divided into three groups based on expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). We first investigated whether ALKBH5 expression is induced by hypoxia in nine human breast cancer cells that are ER⁺ (ZR75.1), ER⁺PR⁺ (MCF-7 and T47D), ER⁺PR⁺HER2⁺ (BT-474), HER2⁺ (HCC-1954), or triple-negative (MDA-MB-231, MDA-MB-435, SUM-149, and SUM-159) (37). These nine breast cancer cell lines were exposed to 20% or 1% O₂ for 24 h, and RNA was isolated for reverse transcription and quantitative real-time PCR (RT-qPCR). ALKBH5 mRNA levels were significantly increased under hypoxic conditions by greater than 1.6-fold in SUM-159 and MDA-MB-435 cells and greater than twofold in MDA-MB-231 and MCF-7 cells (Fig. 1A).

To determine whether hypoxia-induced ALKBH5 expression was dependent on HIF-1α, HIF-2α, or both, we analyzed MDA-MB-231 and MCF-7 subclones that were stably transfected with lentiviral vectors encoding shRNA to inhibit the expression of HIF-1α (sh1α), HIF-2α (sh2α), or both HIF-1α and HIF-2α [double knockdown (DKD)], as well as a subclone expressing a nontargeting control shRNA (NTC) (21, 38). In contrast to the NTC subclone, hypoxia-induced ALKBH5 mRNA expression was abrogated in sh1α, sh2α, and DKD subclones of MDA-MB-231 (Fig. 1B) and MCF-7 (Fig. 1C). Immunoblot assays revealed that ALKBH5 protein levels were increased by hypoxia in NTC subclones of MDA-MB-231 (Fig. 1D) and MCF-7 (Fig. 1E), but not in HIF-knockdown subclones. Thus, hypoxia induces ALKBH5 expression in a subset of ER⁺ and ER⁻ breast cancer cell lines in an HIF-dependent manner.

Next, we investigated whether increased ALKBH5 expression in hypoxic cells leads to changes in levels of m⁶A RNA modification in total cellular RNA pools. We hypothesized that m⁶A levels in RNA represent the net effect of methylation mediated by the METTL3/METTL14 complex and demethylation mediated by ALKBH5 and FTO. Thus, increased ALKBH5 expression should lead to decreased m⁶A levels in RNA. Exposure of NTC subclones of MDA-MB-231 (Fig. 1F) and MCF-7 (Fig. 1G) to 1% O₂ for 24 h led to a significant decrease (>30%) in the percentage of m⁶A residues among total adenosine residues in RNA. In contrast, there was no significant change in total m⁶A levels in RNA when the DKD subclones were exposed to hypoxia.

To demonstrate that decreased m⁶A modification of RNA in hypoxic cells was caused by increased ALKBH5 expression, we generated subclones of MDA-MB-231 and MCF-7 that were stably transfected with a lentiviral vector encoding either of two different shRNAs targeting ALKBH5 (shALK1#1 and shALK#2). Compared with the NTC subclone, knockdown of ALKBH5 in MDA-MB-231 (Fig. 1H) and MCF-7 (Fig. 1I) cells significantly increased m⁶A levels in total RNA isolated from cells incubated at 20% O₂, and ALKBH5 knockdown blocked hypoxia-induced RNA demethylation. Taken together, the data in Fig. 1 indicate that HIF-dependent ALKBH5 expression mediates m⁶A demethylation in total cellular RNA pools of hypoxic breast cancer cells.

Hypoxia Induces HIF- and ALKBH5-Dependent NANOG Expression.

We next analyzed the effect of hypoxia on expression of the core pluripotency factor NANOG in human breast cancer cells. In NTC subclones of MDA-MB-231 (Fig. 2A) and MCF-7 (Fig. 2B), hypoxia significantly increased NANOG mRNA levels, whereas the hypoxic induction was abrogated in the sh1α, sh2α, and DKD subclones. Hypoxic induction of NANOG protein expression was also abrogated in the HIF knockdown subclones (Fig. 1 D and E).

ALKBH5 knockdown by either of two different shRNAs also significantly impaired the hypoxic induction of NANOG mRNA expression in MDA-MB-231 (Fig. 2C) and MCF-7 (Fig. 2D) cells. Finally, immunoblot assays demonstrated that ALKBH5 knockdown led to decreased NANOG protein expression in MDA-MB-231 (Fig. 2E) and MCF-7 (Fig. 2F) cells. Decreased levels of NANOG mRNA and protein were also observed in ALKBH5-knockdown subclones at 20% O₂, indicating that there is significant ALKBH5 activity under nonhypoxic conditions, which is further increased by hypoxia.

ALKBH5 Enhances NANOG mRNA Stability by Catalyzing m⁶A Demethylation.

To investigate the mechanism by which ALKBH5 regulates NANOG expression, we first explored whether hypoxia-induced ALKBH5 targeted NANOG mRNA for m⁶A demethylation. Total cellular RNA was immunoprecipitated with an antibody that recognizes m⁶A, and the immunoprecipitated RNA was subjected to RT-qPCR to amplify the beginning of the NANOG 3′-UTR, which contains a match to the m⁶A consensus sequence, 5′-RRACU-3′. Hypoxic exposure led to decreased levels of m⁶A⁺ NANOG mRNA in NTC subclones of MDA-MB-231 (Fig. 3A) and MCF-7 (Fig. 3B) cells. In contrast, expression of either of two different shRNAs targeting ALKBH5 led to increased levels of m⁶A⁺ NANOG mRNA under normoxic and hypoxic conditions. HIF DKD also increased m⁶A⁺ NANOG mRNA levels, although to a lesser degree, and blocked the hypoxia-induced decrease in m⁶A⁺ NANOG mRNA levels that was observed in NTC subclones of MDA-MB-231 (Fig. 3C) and MCF-7 (Fig. 3D). These data indicate that, under hypoxic conditions, NANOG mRNA is subject to m⁶A demethylation that is HIF- and ALKBH5-dependent.

Hypoxia induced decreased m⁶A modification of NANOG mRNA and increased total NANOG mRNA levels, which is consistent with increased degradation of m6A⁺ mRNA (29, 33). To measure NANOG mRNA stability, flavopiridol was used to inhibit global mRNA transcription as previously described (29), and the ratio of NANOG mRNA in flavopiridol-treated cells relative to vehicle-treated cells (F/V ratio) was calculated. Exposure of NTC subclones of MDA-MB-231 (Fig. 3E) and MCF-7 (Fig. 3F) to hypoxia increased the NANOG mRNA F/V ratio, indicating decreased degradation of NANOG mRNA. However, this effect of hypoxia was lost in ALKBH5 and HIF knockdown subclones, indicating that hypoxia enhances NANOG mRNA stability through HIF-dependent ALKBH5 expression.

Sequence analysis revealed three matches to the 5′-RRACU-3′ (methylated adenosine residue is underscored) m⁶A consensus sequence within the NANOG 3′-UTR. Reporter plasmids, which contained or lacked the entire NANOG 3′-UTR immediately downstream of luciferase coding sequences, were transiently transfected into MDA-MB-231 (Fig. 3G) and MCF-7 (Fig. 3H) subclones, followed by incubation at 20% or 1% O₂ for 24 h and measurement of luciferase activity. In NTC subclones exposed to 20% O₂, luciferase activity was reduced four- to fivefold by insertion of the NANOG 3′-UTR into the reporter, but hypoxia significantly increased luciferase activity, whereas this effect was lost in the shALK and DKD subclones (Fig. 3 G and H).

Next, the adenine residue embedded within the consensus sequence located closest to the translation termination codon in the NANOG 3′-UTR was mutated (5′-AAACU-3′ to 5′-AAUCU-3′ in mRNA), and luciferase assays were performed comparing WT and mutant reporters. Mutation of the adenosine residue resulted in constitutively increased luciferase activity in parental MDA-MB-231 (Fig. 3I) and MCF-7 (Fig. 3J) cells, suggesting that the mutation prevented methylation and thereby increased the stability of the luciferase-NANOG 3′-UTR fusion mRNA. These results suggest that the mutated adenosine residue was a target for methylation, which was consistent with the identification of this site by others using m⁶A immunoprecipitation and RNA sequencing in ESCs (29). Taken together, the results presented in Fig. 3 indicate that hypoxia-induced and HIF-dependent ALKBH5 expression increases NANOG mRNA demethylation, stabilization, and accumulation.

ALKBH5 Deficiency Impairs Hypoxia-Induced BCSC Enrichment.

We next investigated whether ALKBH5 plays a role in regulation of the BCSC phenotype. Two properties that identify populations of breast cancer cells that are enriched in BCSCs are the ability to generate clusters of daughter cells when cultured on ultra-low adherence plates (mammosphere assay) (39) and increased aldehyde dehydrogenase (ALDH) 1 activity, which can be quantified by flow cytometry using a fluorogenic substrate (40). Analysis of MDA-MB-231 (Fig. 4A) and MCF-7 (Fig. 4B) cells grown as adherent monolayers or as nonadherent mammospheres revealed significantly increased ALKBH5 mRNA expression in mammosphere cultures, which are enriched for BCSCs.

To determine the effect of ALKBH5 depletion on the specification/maintenance of BCSCs, we exposed NTC and shALK subclones to 20% or 1% O₂ for 72 h, then transferred the cells to ultra-low adherence plates, incubated them at 20% O₂ for 1 wk, and counted the number of primary mammospheres that had formed (Fig. 4 C–F). We then dissociated the primary mammospheres, replated the cells on ultra-low adherence plates, incubated them at 20% O₂ for 1 wk, and counted the number of secondary mammospheres (Fig. 4 G and H). Hypoxic exposure of NTC subclones for 72 h dramatically increased primary and secondary mammosphere formation at 1 and 2 wk after the hypoxic exposure was terminated, as previously reported (21). In contrast, ALKBH5 knockdown significantly impaired hypoxia-induced mammosphere formation and, in MCF-7 cells, significantly decreased mammosphere formation even when cells were not exposed to hypoxia (Fig. 4 F and H). Analysis of BCSCs by the ALDH assay gave remarkably similar results: ALKBH5 knockdown significantly impaired the hypoxia-induced enrichment of ALDH⁺ cells in MDA-MB-231 (Fig. 4I) and MCF-7 (Fig. 4J) cells and decreased the percentage of ALDH⁺ cells even when MCF-7 cells were not exposed to hypoxia. Thus, mammosphere and ALDH assays indicate that ALKBH5 expression is necessary for hypoxia-induced BCSC enrichment.

Overexpression of ALKBH5 Is Sufficient to Enhance NANOG Expression and BCSC Enrichment.

To determine whether ALKBH5 induction is sufficient to decrease the methylation of NANOG mRNA and thereby increase the BCSC population, we stably transfected MDA-MB-231 and MCF-7 cells with a lentiviral vector encoding human ALKBH5 or empty vector (EV), and exposed the cells to 20% or 1% O₂. Under nonhypoxic conditions, ALKBH5 overexpression led to significantly decreased m⁶A modification of NANOG mRNA (Fig. 5 A and B), increased levels of NANOG mRNA (Fig. 5 C and D) and protein (Fig. 5 E and F), and increased percentage of ALDH⁺ cells (Fig. 5 G and H). In MCF-7 cells, ALKBH5 overexpression also potentiated the decrease in m6A⁺ NANOG mRNA levels (Fig. 5B) and increase in ALDH⁺ cells (Fig. 5H) that was observed in response to hypoxia. These data indicate that, under nonhypoxic conditions, overexpression of ALKBH5 is sufficient to demethylate NANOG mRNA, increase NANOG expression, and enhance BCSC enrichment, thereby phenocopying the effects of hypoxia.

Knockdown of ALKBH5 Impairs Tumor Formation and Decreases BCSCs in Vivo.

To analyze the effect of ALKBH5 deficiency on the tumor-initiating potential of breast cancer cells, we injected a limiting number of cells (1 × 10³) from MDA-MB-231 subclones into the mammary fat pad of female nonobese diabetic/SCID/IL2Rγ-null (NSG) immunodeficient mice. Ten weeks after injection, 100% of the mice (n = 7) that were injected with NTC cells developed palpable tumors, compared with only 43% (n = 6 of 14) of the mice injected with ALKBH5-deficient cells (Fig. 6A). To further study the effect of ALKBH5 deficiency on the BCSC population in vivo, the tumors that formed after injection of NTC or shALK cells were harvested for analysis of NANOG expression and BCSCs. NANOG mRNA (Fig. 6B) and protein (Fig. 6C) levels were significantly decreased in tumors formed by ALKBH5-knockdown subclones compared with the NTC subclone. Both mammosphere (Fig. 6D) and ALDH (Fig. 6E) assays revealed a significant decrease in the BCSC population in tumors derived from injection of shALK compared with NTC cells. These results indicate that loss of ALKBH5 expression significantly impairs tumor formation and decreases the BCSC population in breast tumors.

Cell Culture.

MCF-7, MDA-MB-231, MDA-MB-435, and T47D cells were cultured in DMEM. BT-474, HCC-1954, and ZR75.1 cells were cultured in RPMI-1640 medium. SUM-149 and SUM-159 cells were cultured in DMEM/F12 (50:50) media supplemented with hydrocortisone and insulin. All culture media were supplemented with 10% (vol/vol) FBS and 1% (vol/vol) penicillin/streptomycin. Cells were maintained in a 5% CO₂ and 95% air incubator [20% (vol/vol) O₂]. For hypoxia exposure, cells were placed in a modular incubator chamber (Billups-Rothenberg), which was flushed for 2 min at 2 psi with a gas mixture containing 1% O₂, 5% CO₂, and 94% N_2.

RT-qPCR.

Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis was performed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). qPCR was performed by using the following primers: ALKBH5, 5′-CGG CGA AGG CTA CAC TTA CG-3′ and 5′-CCA CCA GCT TTT GGA TCA CCA-3′; NANOG, 5′-TTT GTG GGC CTG AAG AAA ACT-3′ and 5′-AGG GCT GTC CTG AAT AAG CAG-3′; and 18S rRNA, 5′-CGG CGA CGA CCC ATT CGA AC-3′ and 5′-GAA TCG AAC CCT GAT TCC CCG TC-3′. For each sample, the ratio of NANOG mRNA:18S rRNA was determined.

Immunoblot Assay.

Whole cell lysates were prepared in modified RIPA lysis buffer (50 mM Tris⋅HCl, pH 7.5, 1 mM β-mercaptoethanol, 150 mM NaCl, 1 mM Na₃VO₄, 1 mM NaF, 1 mM EDTA, 0.25% sodium deoxycholate and 1% Igepal CA-630). Blots were probed with HIF-1α (BD Biosciences), HIF-2α (Novus Biologicals), ALKBH5 (Novus Biologicals), NANOG (Novus Biologicals), and Actin (Santa Cruz) antibodies. HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were used and the chemiluminescent signal was detected by using ECL Plus (GE Healthcare). Densitometric analysis was performed by using ImageJ (NIH).

Lentiviral Vectors and Transduction.

Lentiviral vectors encoding shRNA targeting HIF-1α and HIF-2α were described previously (38). pLKO.1-puro lentiviral vectors encoding shRNA targeting ALKBH5 mRNA (clone ID, NM_017758.2–1625s1c1 and NM_017758.2–1176s1c1) were purchased from Sigma-Aldrich. pLenti-GIII-CMV lentiviral vector containing cDNA encoding human ALKBH5 was purchased from Applied Biological Materials (cat. no. LV073201). All lentiviral vectors were transfected into 293T cells for packaging. Viral supernatant was collected after 48 h and used for transfection as described before (38). Puromycin (0.5 μg/mL) was added to the medium of cells transduced with lentivirus for selection.

Measurement of Total m⁶A and m⁶A⁺ NANOG mRNA Levels.

Total m⁶A content was measured in 200-ng aliquots of total RNA extracted from MDA-MB-231 and MCF-7 subclones using an m⁶A RNA methylation quantification kit (cat. no. P-9005; Epigentek) according to the manufacturer’s instructions. To measure m⁶A⁺ NANOG mRNA levels, m⁶A immunoprecipitation was performed as described before (47). A 1-µg aliquot of m⁶A antibody was conjugated to protein A-agarose slurry (Millipore) overnight at 4 °C. A 100-μg aliquot of total RNA was incubated with the antibody in immunoprecipitation buffer (50 mM Tris⋅HCl, 750 mM NaCl, and 0.5% Igepal CA-630) supplemented with RNase inhibitor for 3 h at 4 °C, RNA was eluted from the beads by incubation in 300 μL of elution buffer (5 mM Tris⋅HCl, 1 mM EDTA, and 0.05% SDS) with 4.2 μL of proteinase K for 1.5 h at 50 °C, and m⁶A⁺ RNA was purified by phenol/chloroform extraction and analyzed by RT-qPCR. The nucleotide sequences of the primers used for detection for m⁶A⁺ NANOG mRNA were as follows: 5′-ATG CAA CCT GAA GAC GTG TG-3′ and 5′-GAG ATT GAC TGG ATG GGC AT-3′.

Measurement of NANOG mRNA Stability.

Cells were exposed to 20% or 1% O₂ for 24 h and then treated with vehicle or flavopiridol (Sigma-Aldrich) at a concentration of 0.8 μΜ for 3 h (MDA-MB-231) or 3.2 μΜ for 6 h (MCF-7), followed by RNA extraction and RT-qPCR as described earlier.

Luciferase Assay.

The luciferase coding sequence NANOG 3′-UTR reporter (cat. no. S807466) and luciferase coding sequence-only reporter (cat. no. S890005) were purchased from SwitchGear Genomics. Cells were seeded in a 96-well plate and transfected with luciferase-NANOG 3′-UTR reporter or luciferase-only reporter, and exposed to 20% or 1% O₂ for 24 h. Luciferase activity was measured using a commercial kit (SwitchGear Genomics) and is presented as the activity ratio of luciferase-NANOG 3′-UTR to luciferase only. Adenine-to-thymine mutation of the luciferase-NANOG 3′-UTR reporter plasmid was performed by using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) according to the manufacturer’s instructions.

Mammosphere Assay.

Cultured cells were trypsinized, whereas tumor tissue was minced and digested with 1 mg/mL of type 1 collagenase (Sigma) at 37 °C for 30 min to prepare single-cell suspensions, which were seeded in six-well ultra-low attachment plates (Corning) at a density of 5,000 cells per milliliter in Complete MammoCult Medium (Stem Cell Technologies). After 7 d, the cells were photographed under an Olympus TH4-100 microscope, and primary mammospheres (diameter ≥70 μm) were counted. For secondary mammosphere formation, primary mammospheres were trypsinized, plated at a density of 5,000 cells per milliliter, incubated for 7 d, and counted.

ALDH Assay.

Cells were exposed to 20% or 1% O₂ for 72 h and harvested for Aldefluor assay (Stem Cell Technologies). Single-cell suspensions (5 × 10⁵ cells in 1 mL of assay buffer) were prepared as described earlier and incubated with BODIPY-aminoacetaldehyde (1 μM) for 45 min, followed by flow cytometry analysis.

Orthotopic Transplantation.

Protocols were approved by the Johns Hopkins University Animal Care and Use Committee and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. A total of 1,000 breast cancer cells were injected into the mammary fat pad of 6–8-wk-old female NSG mice in a 1:1 suspension of Matrigel (BD Biosciences) in PBS solution. At 10 wk after injection, mice were examined for the presence of tumors, which were harvested for analysis.

Statistical Analysis.

Data are presented as mean ± SEM. Differences between two groups or multiple groups were analyzed by Student t test and ANOVA, respectively. Fisher exact test was used for analyzing tumor initiation capacity. P values <0.05 were considered significant.