HIF-1-dependent heme synthesis promotes gemcitabine resistance in human non-small cell lung cancers via enhanced ABCB6 expression

Abstract

Gemcitabine is commonly used to treat various cancer types, including human non-small cell lung cancer (NSCLC). However, even cases that initially respond rapidly commonly develop acquired resistance, limiting our ability to effectively treat advanced NSCLC. To gain insight for developing a strategy to overcome gemcitabine resistance, the present study investigated the mechanism of gemcitabine resistance in NSCLC according to the involvement of ATP-binding cassette subfamily B member 6 (ABCB6) and heme biosynthesis. First, an analysis of ABCB6 expression in human NSCLCs was found to be associated with poor prognosis and gemcitabine resistance in a hypoxia-inducible factor (HIF)-1-dependent manner. Further experiments showed that activation of HIF-1α/ABCB6 signaling led to intracellular heme metabolic reprogramming and a corresponding increase in heme biosynthesis to enhance the activation and accumulation of catalase. Increased catalase levels diminished the effective levels of reactive oxygen species, thereby promoting gemcitabine-based resistance. In a mouse NSCLC model, inhibition of HIF-1α or ABCB6, in combination with gemcitabine, strongly restrained tumor proliferation, increased tumor cell apoptosis, and prolonged animal survival. These results suggest that, in combination with gemcitabine-based chemotherapy, targeting HIF-1α/ABCB6 signaling could result in enhanced tumor chemosensitivity and, thus, may improve outcomes in NSCLC patients.

Keywords: ABCB6; Gemcitabine resistance; Hypoxia-inducible factor-1; Non-small cell lung cancer; Transcriptional activation.

Fig. 1

High ABCB6 expression is associated with gemcitabine resistance and poor prognosis in human NSCLCs. Relative levels of ABCB6 mRNA expression from microarray analysis of primary NSCLC tumor samples compared with adjacent normal lung tissue from NSCLC patients (TCGA database) are shown. A Red, samples from lung squamous cell carcinoma (LUSC, n = 503); blue, samples from adjacent normal lung tissue (n = 52), P < 0.0001. B Red, samples from lung adenocarcinoma (LUAD, n = 515); blue, samples from adjacent normal lung tissue (n = 59), P < 0.0001. Mann–Whitney U test or ANOVA followed by Bonferroni post-test for multiple comparisons was used to determine P values. C Kaplan–Meier curves were constructed to analyze the association between ABCB6 mRNA levels in lung cancer tissues and the probability of overall survival (n = 1926, P = 0.006) using the KM plotter database. Low = ABCB6 mRNA levels less than the median. High = ABCB6 mRNA levels greater than the median. Statistical analysis was performed using log-rank tests. D Representative images of ABCB6 expression in tissue microarrays containing gemcitabine-resistant tumor tissues from 41 cases, gemcitabine-sensitive tumor tissues from 40 cases, and adjacent normal lung tissues from 6 cases are shown. The bottom right corner panel shows higher magnification of ABCB6 staining (brown, scale bar = 100 μm). E Image analysis was performed to determine the staining density of the ABCB6-positive area per field under × 200 magnification based on 5 random fields per section. One-way ANOVA (mean ± SEM) was used to determine the P value, ***P < 0.0001

Fig. 2

ABCB6 expression is induced in GR cells in a HIF-1α-dependent manner. A, B, RT-qPCR was performed to quantify ABCB6 mRNA levels in A549 (A) and H1703 (B) gemcitabine-resistant (GR) and wild-type (WT) cell lines. For each sample, the expression of ABCB6 mRNA was quantified relative to 18S rRNA and then normalized to the result obtained from WT cells. Statistical analysis was performed before normalization. Data are shown as mean ± SEM; n= 3. ***P < 0.001 vs. WT cells (Student’s t test). C Immunoblot analysis was performed to analyze ABCB6 and HIF-1α protein expression in A549 and H1703 GR and WT cell lines. D, E, ABCB6 mRNA expression was analyzed by RT-qPCR in A549 (D) and H1703 (E) GR and WT subclones, which were stably transfected with non-targeting control (NTC) or vector encoding HIF-1α shRNA (sh1α-1 or sh1α-2) (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared to WT-NTC cells; ^###P < 0.001 compared to GR-NTC cells (ANOVA with Bonferroni post-test). F, G, Immunoblot analysis was performed using lysates prepared from A549 (F) and H1703 (G) subclones transfected with NTC or HIF-1α shRNAs. H, I, Immunoblot analysis of HIF-1α and ABCB6 protein expression levels in A549-WT (H) and H1703-WT (I) exposed to hypoxia (1% O₂) for 24 h or 48 h and DMOG (0 µM, DMSO vehicle, 100 µM, 500 µM, 1 mM) for 24 h in normoxia

Fig. 3

The ABCB6 gene is a HIF-1α target gene. A Nucleotide sequence of the hypoxia response element-1 (HRE site 1; 5′-ACGTG-3′ HIF-1-binding site is shown in red) in the 5′-flanking region of the ABCB6 gene, located − 0.1 kb from the transcription start site. ABCB6 exons and HRE are indicated by black bars and arrow, respectively. B Chromatin immunoprecipitation (ChIP) assays were performed using IgG or antibodies against HIF-1α, HIF-1β and HIF-2α in A549-GR and A549-WT cell lines. Primers flanking HRE site 1 were used for qPCR, and results were normalized to lane 1 (mean ± SEM; n = 3). ***P < 0.001 compared to WT (ANOVA with Bonferroni post-test). C ChIP assays were performed using IgG or antibodies against HIF-1α, HIF-1β and HIF-2α in A549-WT cells exposed to normoxia (20% O₂) or hypoxia (1% O₂) for 16 h. ***P < 0.001 compared to normoxia (ANOVA with Bonferroni post-test). D, E, ChIP assays were performed using IgG or antibodies against H3K27ac (D) and H3K4me3 (E), followed by qPCR with primers flanking HIF-1-binding site 1 in the ABCB6 gene in A549-GR and A549-WT cells. **P < 0.01, ***P < 0.001 compared to WT (ANOVA with Bonferroni post-test). F, G ChIP assays were performed using antibodies against H3K27ac (F) and H3K4me3 (G), followed by qPCR with primers flanking HRE site 1 in A549 cells exposed to normoxia (20% O₂) or hypoxia (1% O₂) for 16 h (mean ± SEM; n = 3). ***P < 0.001 compared to normoxia (ANOVA with Bonferroni post-test). H, Nucleotide sequence of HRE site 2 (5′-GCGTG-3′ HIF-1-binding site and a direct repeat 5′-GCGTG-3′ sequence separated by 8 bp are shown in red) within exons 1 of the ABCB6 gene, located + 87 bp from the transcription start site. Exons and intron are not drawn to scale. I, J ChIP assays were performed using IgG or antibodies against HIF-1α, HIF-1β and HIF-2α in A549 WT and A549-GR cells (I), and in A549-WT cells under normoxic or hypoxic conditions (J). Primers flanking the HRE site 2 were used for qPCR, and results were normalized to lane 1 (mean ± SEM; n = 3). I ***P < 0.001 compared to WT; J **P < 0.01, ***P < 0.001 compared to normoxia (ANOVA with Bonferroni post-test). K, L ChIP assays were performed using IgG or antibodies against H3K27ac (K) and H3K4me3 (L), followed by qPCR with primers flanking HIF-1-binding site 2 in the ABCB6 gene in A549-GR and A549-WT cells. ***P < 0.001 compared to WT (ANOVA with Bonferroni post-test). M, N ChIP assays were performed using antibodies against H3K27ac (M) and H3K4me3 (N), followed by qPCR with primers flanking HRE site 2 in A549-WT cells exposed to normoxia (20% O₂) or hypoxia (1% O₂) for 16 h (mean ± SEM; n = 3). ***P < 0.001 compared to normoxia (ANOVA with Bonferroni post-test)

Fig. 4

Effect of HIF-1α/ABCB6 signaling on heme-mediated ROS scavenging in GR cells. A, B, Heme content in A549 (A) and H1703 (B) GR and WT subclones, which were stably transfected with non-targeting control (NTC), shRNA targeting HIF-1α (shHIF-1α), shRNA targeting ABCB6 (shABCB6), or shRNAs targeting both HIF-1α and ABCB6 (shHIF-1α/ABCB6) (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared to WT-NTC cells; ^##P < 0.01, ^###P < 0.001 compared to GR-NTC cells (ANOVA with Bonferroni post-test). C Catalase protein expression levels in A549 and H1703, GR and WT subclones (N: NTC; 1α: shHIF-1α; A6: shABCB6; DKD, double knockdown: shHIF-1α/ABCB6). D, E Catalase activity in A549 (D) and H1703 (E), GR and WT subclones (NTC, shHIF-1α, shABCB6, and shHIF-1α/ABCB6; mean ± SEM; n= 3). *P < 0.05, ***P < 0.001 compared to WT-NTC cells; ^##P < 0.01, ^###P < 0.001 compared to GR-NTC cells (ANOVA with Bonferroni post-test). F, G ROS generation in A549 (F) and H1703 (G), GR and WT subclones (NTC, shHIF-1α, shABCB6, and shHIF-1α/ABCB6). Intracellular hydrogen peroxide was detected by DCF-based measurement (mean ± SEM; n = 3). *P < 0.05, ***P < 0.001 compared to WT-NTC cells; ^###P < 0.001 compared to GR-NTC cells (ANOVA with Bonferroni post-test). H, I Effect of gemcitabine on the growth of A549 (H) and H1703 (I), GR and WT subclones (WT, NTC, shHIF-1α, shABCB6, and shHIF-1α/ABCB6). Cells were cultured in medium with or without gemcitabine (Gem, 1 μM) before measurement of cell viability. Relative survival is plotted as percent of WT control cells cultured in normal medium (mean ± SEM; n = 3). ***P < 0.001 compared to WT or GR-NTC cells with Gem; ^#P < 0.05, ^##P < 0.01 compared to GR-NTC cells without Gem (ANOVA with Bonferroni post-test)

Fig. 5

ABCB6 inhibition by tomatidine reverses gemcitabine resistance in NSCLC cells. A, B Representative immunoblots from A549 (A) and H1703 (B) cells treated with tomatidine at indicated concentrations (0, 2, 6, 8, 10 μmol/l) for 24 h. C, D Heme content in A549 (C) and H1703 (D) GR and WT cells treated with DMSO or 10 μmol/l tomatidine for 24 h. E, F, Catalase activity in A549 (E) and H1703 (F) GR and WT cells treated with DMSO or 10 μmol/l tomatidine for 24 h. G, H Cell survival of A549 (G) and H1703 (H) GR and WT cells incubated with vehicle or 10 μmol/l tomatidine for 24 h, with or without the treatment with 1 μM gemcitabine (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared to WT cells; ^###P < 0.001 compared to GR cells (ANOVA with Bonferroni post-test)

Fig. 6

Inhibition of the HIF-1α/ABCB6 axis sensitizes NSCLCs to gemcitabine therapy in animal models. A549 and H1703, GR and WT subclones (2 × 10⁶ cells) were injected into the groin of 6- to 8-week-old male SCID mice. After palpable tumors had formed (8 days after tumor implantation), mice received intraperitoneal injection of gemcitabine (20 mg/kg) or saline (250 µl) twice per week. A, C Volume of primary tumor formed by A549 (A) or H1703 (C) subclones, as determined twice weekly. ***P < 0.001 vs. NTC by two-way ANOVA with Bonferroni post-test (mean ± SEM; n = 6). B, D Kaplan–Meier survival curves for the 6 treatment groups of A549 (B) and H1703 (D) cell-derived tumors as mentioned above. The log-rank test was used to demonstrate the statistical difference (n = 10; NTC, non-targeting control; GR, gemcitabine resistant; Gem, gemcitabine). E, F After 42 days, primary tumors were harvested, and apoptosis evaluated by TUNEL assay in A549 (E) and H1703 (F) cell-derived tumors (N: NTC; S: saline; G: gemcitabine; sh1α: shHIF-1α). Scale bar = 50 μm. G, H Percentages of TUNEL-positive cells in A549 (G) and H1703 (H) cell-derived tumors. Cells were counted in 10 randomly selected fields in 6 tumor samples from each group (mean ± SEM). ***P < 0.001

Fig. 7

Proposed mechanism by which activation of HIF-1α/ABCB6 signaling leads to intracellular heme metabolic reprogramming and a corresponding increase in heme biosynthesis to enhance the activation and accumulation of catalase. Increased levels of catalase diminish the effective levels of ROS, thereby promoting gemcitabine resistance