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

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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.

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Acknowledgements

We gratefully acknowledge Dr. Feng Wu and Dr. Yong Lin from the Department of Pathophysiology, Third Military Medical University for their kind help in assessing IHC results.

Funding

This work was supported by the Sichuan Science and Technology Program (2020YJ0046, 2020YFS0272 and 2019YJ0041), the Natural Science Foundation of Chongqing (cstc2018jcyjAX0512 and cstc2019jcyj-msxmX0791), and the National Science and Technology Major Project (2017ZX09304023).

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Authors and Affiliations

  1. Clinical Trial Center, National Medical Products Administration Key Laboratory for Clinical Research and Evaluation of Innovative Drugs, West China Hospital, Sichuan University, Chengdu, 610041, China

    Lisha Xiang & Yongsheng Wang

  2. Department of Thoracic Oncology, Department of Radiation Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, China

    Lisha Xiang, Yongsheng Wang, Jie Lan & Feifei Na

  3. Department of Oncology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University (Army Medical University), 29 Gaotanyan Street, Chongqing, 400038, China

    Shuang Wu, Yuzhu Gong & Ganfeng Xie

  4. Department of Neurosurgery, Chongqing University Cancer Hospital, Chongqing Cancer Institute, Chongqing Cancer Hospital, Chongqing, 400030, China

    Hanjian Du

  5. State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Renmin south road 3 section, Chengdu, 610041, China

    Bin Shao

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  1. Lisha Xiang
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  2. Yongsheng Wang
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  3. Jie Lan
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Contributions

LX, BS and GX designed the study and wrote the paper. LX, GX, JL and FN performed the experiments. LX, SW, YG and HD analyzed the data. BS and GX advised on experimental procedures and revision of the paper. YW revised the paper. All the authors contributed to this manuscript. All the authors read and approved the final manuscript.

Corresponding authors

Correspondence to Bin Shao or Ganfeng Xie.

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The experimental protocol was established according to the ethical guidelines of the 1964 Declaration of Helsinki. The protocol for IHC staining of patient tissues was approved by the Ethics Committee of the First Affiliated Hospital (Southwest Hospital), the Third Military Medical University (Army Medical University), and all patients or family members involved provided written informed consent.

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Figure S1.

Establishment of gemcitabine-resistant (GR) NSCLC cell lines. A549 lung adenocarcinoma and H1703 lung squamous cell carcinoma cell lines with acquired gemcitabine resistance were generated by exposing the corresponding wild-type (WT) cells to increasing concentrations of gemcitabine over 7 months. The resistance status was confirmed by cell viability assays and soft agar assays at each step. A-B, NSCLC cells were treated with gemcitabine for 72 h, and cell viability was analyzed using MTS. The viability of A549-WT (A) and H1703-WT (B) cells was respectively suppressed compared to A549-GR (A) and H1703-GR (B) cells upon treatment with gemcitabine in a dosedependent manner. Images of the colonies formed from A549-GR (A) and H1703-GR (B) cells as compared to relevant WT cells treated with 0.5 or 1 μM gemcitabine (as indicated) for 72 h and cultured for 15 days. C-D, Average colony number (C) and colony size (D) for A549-GR cells compared to A549-WT cells, as determined by soft agar assays. E-F, Average colony number (E) and colony size (F) for H1703-GR cells compared to H1703-WT cells. Colony number and size were determined by pictomicrography. The results shown are the average values from three independent experiments (mean±SEM; n=3). Student’s t-test was used to determine the P value, ***P<0.0001. (TIF 1408 KB)

Figure S2.

mRNA expression of genes in the ABC family in A549 subgroups. RT-qPCR was performed to quantify mRNA levels of genes from the ABC family (A, ABCA1; B, ABCA2; C, ABCB1; D, ABCB2; E, ABCB3; F, ABCB6; G, ABCC3; H, ABCC5; I, ABCG2) in A549-WT versus A549-GR cells, and in A549-WT cells exposed to hypoxia (1% O2) for 24 h. For each sample, gene mRNA expression was quantified relative to 18S rRNA and then normalized to the result obtained from WT cells. Statistical analysis was performed before normalization (mean±SEM; n=3). *P<0.05, ***P<0.001 vs. WT cells (Student’s t test). (TIF 1163 KB)

Figure S3.

HIF-binding sites 1 and 2 in the ABCB6 gene. Two sequences (1 and 2) matching the consensus 5′-RCGTG-3′ (R = A or G; shown in red) and located in a DNase I hypersensitive region of chromatin (coordinates given relative to the transcription start site) were tested for binding of HIF-1α and HIF-1β by ChIP assay. HIF-1 was shown to bind selectively to sites 1 and 2 (see Figure 4 and Figure S3C-S3F). H3K4Me1 and H3K4Me3, monomethylation and trimethylation, respectively, of lysine residue 4 of histone H3; H3K27Ac, acetylation of lysine-27 of histone H3. (TIF 1792 KB)

Figure S4.

A, Protein expression levels of HIF-1α and ABCB6 in A549-GR subclones transfected with lentiviral vectors encoding shRNA targeting ABCB6 (shABCB6-1, shABCB6-2), shRNAs targeting both HIF-1α and ABCB6 (sh1α/ABCB6) and non-targeting control (NTC). B, Protein levels of HIF-1α and ABCB6 in H1703-GR subclones transfected with lentiviral vectors encoding shRNA targeting ABCB6 (shABCB6-1, shABCB6-2), shRNAs targeting both HIF-1α and ABCB6 (sh1α/ABCB6) and NTC. C-F, Two HIF-binding sites (HRE site 1 and HRE site 2) of the human ABCB6 gene were identified by ChIP assay as described below. ChIP assays were performed using IgG or antibodies against HIF-1α, HIF-1β and HIF-2α in H1703-WT and H1703-GR cells (C, site 1; E, site 2), and H1703-WT cells under normoxic or hypoxic conditions (D, site 1; F, site 2). Primers flanking the HRE site were used for qPCR, and results were normalized to lane 1 (mean±SEM; n=3). ***P<0.001 vs. WT or normoxia (ANOVA with Bonferroni post-test). (TIF 1005 KB)

Figure S5. Tumor images of A549 (A) and H1703 (B) subgroups at day 42. (TIF 3762 KB)

Figure

S6. A549 NTC, shHIF-1α, shABCB6 subclones (2×106 cells) were implanted into the groin of 6-to 8-week-old male SCID mice. After palpable tumors had formed (26 days after tumor implantation), mice received intraperitoneal injection of gemcitabine (20 mg/kg) or saline (250 µl) twice per week until day 53. A, Volume of primary tumors formed by A549 subclones treated with gemcitabine or saline, as determined twice weekly. **P<0.01, ***P<0.001 vs. NTC + saline by two-way ANOVA with Bonferroni post-test (mean±SEM; n=5). B, Representative images of tumors in A549 subgroups. (TIF 1706 KB)

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Xiang, L., Wang, Y., Lan, J. et al. HIF-1-dependent heme synthesis promotes gemcitabine resistance in human non-small cell lung cancers via enhanced ABCB6 expression. Cell. Mol. Life Sci. 79, 343 (2022). https://doi.org/10.1007/s00018-022-04360-9

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