Effective breast cancer combination therapy targeting BACH1 and mitochondrial metabolism

Abstract

Mitochondrial metabolism is an attractive target for cancer therapy^1,2. Reprogramming metabolic pathways could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options, such as triple-negative breast cancer (TNBC)^1,3. Here we show that BTB and CNC homology1 (BACH1)⁴, a haem-binding transcription factor that is increased in expression in tumours from patients with TNBC, targets mitochondrial metabolism. BACH1 decreases glucose utilization in the tricarboxylic acid cycle and negatively regulates transcription of electron transport chain (ETC) genes. BACH1 depletion by shRNA or degradation by hemin sensitizes cells to ETC inhibitors such as metformin^5,6, suppressing growth of both cell line and patient-derived tumour xenografts. Expression of a haem-resistant BACH1 mutant in cells that express a short hairpin RNA for BACH1 rescues the BACH1 phenotype and restores metformin resistance in hemin-treated cells and tumours⁷. Finally, BACH1 gene expression inversely correlates with ETC gene expression in tumours from patients with breast cancer and in other tumour types, which highlights the clinical relevance of our findings. This study demonstrates that mitochondrial metabolism can be exploited by targeting BACH1 to sensitize breast cancer and potentially other tumour tissues to mitochondrial inhibitors.

Extended Data Fig. 1 |. BACH1 expression is high in patients with TNBC and suppresses expression of ETC genes at their promoter.

a, Left, BACH1 expression levels (determined by RNA-seq) with respect to relative DNA copy-number alterations in TCGA breast cancers (n = 1105). Middle, BACH1 expression (RNA-seq) in TNBC (n = 83) or basal (n = 98) breast cancers compared to non-TNBC (n = 734) or non-basal (n = 424) breast cancers using Pam50 classification of TCGA data. Right, breast cancer subtypes classified by Pam50 (n = 522 total, n = 98 basal, n = 58 ifER2-enriched, n = 231 luminal-A, n = 127 luminal-B, n = 8 normal-like). Two-tailed t-test. b, BACH1 expression levels (by RNA-seq) in patients with TNBC compared to patients that did not have TNBC, using the datasets of patients with breast cancer of METABRIC (n = 2509), GSE2034 (n = 286) and GSE11121 (n = 200). Two-tailed t-test. c, Gene Ontology terms as determined by gene set analysis for cell components that are positively correlated with BACH1 depletion based on microarray analysis of BM1-shBACH1 cell transcripts. n = 3 biologically independent samples, FDR-corrected P < 0.05. d, Left, relative mRNA levels of mitochondrial inner membrane genes in MB436-shBACH1 cells (two shBACH1 vectors, clone 1, clone 2) compared to the wild type control (MB436-shCont). Data are mean ± s.e.m., n = 3 biologically independent samples, two-tailed t-test. Right, representative western blots of mitochondrial genes using MB436-shBACH1 or control cell lysates. Each experiment was repeated independently three times with similar results. Band density quantification is shown below the blots, e, Schematic showing proximal BACH1 binding on the promoter regions of mitochondrial membrane genes. TSS, transcription start site. Arrows, primers used for ChIP-PCR. f, ChIP assays showing relative fold enrichment of BACH1 recruitment to the HMOX1 promoter using BACH1-depleted TNBC (BM1 and MB436) or control cells, g, h, ChIP assays showing fold enrichment of BACH1 and H3K27me3 recruitment to the mitochondrial membrane genes in low-BACH1-expressing MB468 and MB436 cells. For ChIP assays in f-h, data are mean ± s.e.m., n = 3 biologically independent samples, two-tailed t-test. i, KEGG pathways demonstrating the negative correlation between BACH1 expression and oxidative phosphorylation in all patients with breast cancer (n = 1, 105, left) and patients with TNBC (n = 119, right). FDR values (−log₁₀(FDR)) are generated in the R package GOseq using the default program Wallenius P values with Benjamini–Hochberg-corrected P values, j, Expression of ETC genes (COX15, ATP5D and ATP5G2 (also known as ATP5MC2)) in TNBC compared to tumours from patients that did not have TNBC using multiple breast cancer datasets: METABRIC (TNBC n = 319, non-TNBC n = 1661), GSE2034 (TNBC n = 54, non-TNBC n = 232) and GSE11121 (TNBC n = 33, non-TNBC n = 150). P values are determined by two-tailed t-test. k, Co-expression plots of UQCRC1 or ATP5D and BACH1 in TCGA breast cancer (n = 1105) or TNBC (n = 115) dataset. Pearsons and Spearmans correlation coefficients are shown.

Extended Data Fig. 2 |. BACH1 depletion increases mitochondrial metabolism.

a, Measurement of OCR and ECAR in BM1 or MB436 cells expressing control or shBACH1. Data are mean ± s.e.m., n = 6 biologically independent samples, unpaired two-tailed t-test. b, Relative abundance of steady-state metabolites in BM1-shBACH1 or control cells cultured with DMEM (glucose, 10 mM) measured by mass spectrometry. Pyr, pyruvate; Lac, lactate. Data are mean ± s.e.m., n = 5 biologically independent samples, two-tailed t-test. c, d, Fractional isotopic incorporation of [U-¹³C₆]-glucose (c) or [U-¹³C₅]-glutamine (d) into the metabolites in glycolysis and the TCA cycle are shown. Data are mean ± s.e.m., n = 4 biologically independent samples, two-tailed t-test. M indicates number of carbons labelled. Fraction is ratio of isotopologues to sum of all isotopologues. e, Relative mRNA and protein levels of PDH and PDK genes in MB436-shBACH1 cells compared to controls. qRT-PCR data are mean ± s.e.m., n = 3 biologically independent samples, two-tailed t-test. Representative images of western blots are shown. Band density quantification is shown below the blots. Each experiment was repeated independently three times with similar results, f, ChIP assays showing fold enrichment of BACH1 recruitment to promoters of PDK genes using MB436 and MB468 cells. Data are mean ± s.e.m., n = 3 biological replicates per cell line, two-tailed t-test. g, Relative mRNA levels of pyruvate carboxylase (PC) in shBACH1 cells compared to control. Data are mean ± s.e.m., n = 3 biologically independent samples. NS, not significant by two-tailed students t-test.

Extended Data Fig. 3 |. BACH1 levels determine response to ETC inhibitor treatment in breast cancer cells.

a, Cellular growth (per cent confluency) of BACH1-depleted cells (BM1-shBACH1 or MB436-shBACH1) or their controls treated with vehicle (veh), metformin (met), rotenone (rot) or antimycin A (ant), b, c, Relative cell viability (%) of BACH1-depleted cells (BM1-shBACH1 or MB436-shBACH1) or their controls treated with vehicle, metformin, rotenone or antimycin A. d, e, Cellular growth (per cent confluency) (d) or cell viability (%) (e) of low-BACH1 (MB468), medium-BACH1 (MB436) or high-BACH1 (BM1)-expressing TNBC cells treated with vehicle (control), 1 mM metformin, or 1–10 mM metformin, f, Cell viability (%) of non-malignant mammary epithelial cells (MCF10A and 184A1) treated with vehicle, metformin, rotenone or antimycin A. For cell viability and growth assays in a and d, values are mean ± s.e.m., n = 6 biologically independent samples, unpaired two-tailed t-test. Arrow indicates the time at which inhibitors were added. For cell viability assays in b, c, e and f, cells were incubated for 48 h after addition of inhibitors and stained with CaAM for 1 h.

Extended Data Fig. 4 |. Rescue of BACH1-depleted TNBC cells from metformin treatment.

a, Cellular growth (per cent confluency) of BM1-shBACH1 or control cells treated with vehicle or metformin in growth medium containing glucose (1 mM) and supplemented with or without pyruvate (2.5 mM). b, Relative NAD+/NADH ratios in BACH1-depleted BM1 cells treated with pyruvate (2.5 mM) and/or metformin (5 mM) for 24 h. Data are mean ± s.e.m., n = 3 biologically independent samples, two-tailed t-test. c, Representative western blots of COX15, UQCRC1 and a-tubulin using BM1-shBACH1 cell lysates transfected with siCOX15 (150 nM), siUQCRC1 (150 nM), and siControl (150 nM). Each experiment was repeated independently two times with similar results, d, Cellular growth (per cent confluence) of BM1-shBACH1 cells transfected with siRNA for COX15 and/or UQCRC1 and treated with vehicle or metformin (10 mM). For a and d, data are mean ± s.e.m., n = 6 biologically independent samples, unpaired two-tailed t-test between vehicle-treated and metformin (10 mM)-treated group, e, Relative OCT1 (also known as SLC22A1, left), PPARG and PGC1A (also known as PPARGC1A, right) mRNA levels in BM1-shBACH1 cells. Data are mean ± s.e.m., n = 3 biologically independent samples.

Extended Data Fig. 5 |. Hemin treatment of cells expressing wild-type BACH1 or hemin-resistant BACH1(mut).

a, Left, cellular growth (per cent confluence) of BM1 and MB436 cells treated with hemin (10, 20, 40 or 80 μM) as indicated. Right, representative western blots of BACH1 from MB436 cells after treatment with hemin (10–40 μM) for 24 h (see also Fig. 3d). Each experiment was repeated independently three times with similar results, b, Relative mRNA levels of mitochondrial membrane genes in MB436 cells treated with vehicle or hemin (20 μM) for 48 h. and representative western blots. Data are mean ± s.e.m., n = 3 biologically independent samples, two-tailed t-test. Band density quantification is shown below the blots, c, Measurement of OCR or ECAR of BM1 cells treated with vehicle or hemin (full data from Fig. 3b). d, Cell viability (%) of BM1 cells treated with vehicle, hemin (20 μM) or ETC inhibitors (metformin, rotenone or antimycin A) for 48 h. e, Cell viability (%) or cell growth (per cent confluence) of MB436 cells treated with vehicle, hemin (20 μM) or metformin (1 mM). f, Representative western blots from MB436-shBACH1 cells transiently transfected with Bach1^mut (100 ng) and treated with vehicle or hemin (10, 20, 40 or 80 μM) for 48 h. Each experiment was repeated independently three times with similar results. g, Measurement of OCR in BM1 or MB436 cells stably expressing shControl, shBACH1 or shBACH1 + Bach1^mut vectors, h, Relative mRNA levels of mitochondrial genes in BM1-shBACH1 cells, shCont cells, or BM1-shBACH1 cells transfected with BACH1 (mut). Data are mean ± s.e.m., n = 3 biologically independent samples, two-tailed t-test. i, Left, cell viability (%) of BM1-shBACH1 cells transfected with BACH1 (mut) and then treated with hemin (20 μM) or vehicle for 48 h. Right, representative western blots showing BACH1 (mut) from cells treated with vehicle or hemin. j, Measurement of OCR and ECAR of BM1-shBACH1 cells expressing BACH1 (mut) pre-treated with hemin. Conditions for OCR and ECAR, and statistics are the same as in Extended Data Fig. 2a. Data are mean ± s.e.m., n = 6 biologically independent samples, k, Cell viability (%) of MB436 cells stably expressing shRNA-resistant BACH1(WT), BACH1 (mut) or shCont vectors treated with vehicle, hemin (20 μM) or metformin (5 mM) for 48 h. Representative western blots of BACH1 expression are shown. Each experiment was repeated independently three times with similar results. For growth and viability assays in a, d, e, i, j and k, data are mean ± s.e.m., n = 4 biologically independent samples), unpaired two-tailed t-test.

Extended Data Fig. 6 |. Metformin suppresses growth of BACH1-depleted breast tumours.

a, Tumour weights and volumes of mice injected with MB436-shBACH1 or control cells (left, n = 6–7 per group) or BM1-shBACH1 or control cells (right, n = 8–10 per group) and treated with vehicle or metformin. Data are mean ± s.e.m., unpaired two-tailed t-test. b, Tumour images of representative mice in each treatment group of mouse models. Scale bar, 1 cm. c, Primary tumour (%) indicates the ratio of mice with tumours or tumour-free upon metformin treatment compared to the total number of mice per treatment group at the end of experiment, d, Body weights of mice monitored before and after treatment of hemin and metformin. Arrow indicates initiation of hemin (H) or metformin (M) treatment, e, Relative mRNA expression of PDK and PDH mRNAs in tumours from MB436-shBACH1 xenograft mice by qRT-PCR. Data are mean ± s.e.m., n = 2 per group, f, Representative western blots of total PDH, BACH1 and mitochondrial membrane proteins (COX15, SLC25A15, NDUFA9) using MB436-shBACH1 or control tumour lysates. Each experiment was repeated independently three times with similar results, g, Lung metastases from mice with MB436-shBACH1 or control xenograft tumours. Lung tissues sectioned and H & E-stained to visualize and count lung metastases in mice, n = 5 mice per group. Data are mean ± s.e.m., two-tailed unpaired t-test. h, Representative lung metastasis images. Arrow indicates tumour metastases with a scale bar (1000 μm).

Extended Data Fig. 7 |. Combination treatment using hemin and metformin suppresses growth of tumours through BACH1 in multiple TNBC mouse models.

a, Left, monitoring of BACH1 degradation by hemin treatment assayed by western blotting using tumour lysates. Mice (n = 2 per treatment group) injected with BM1 cells (2 × 10⁶ cells) for 4 weeks to form tumours were treated with 25 mg kg⁻¹ or 50 mg kg⁻¹ hemin for the indicated times. Right, representative western blots showing relative BACH1 expression using tumours from mice treated with hemin for the experiments (see Fig. 4a–d). Western blotting experiments were repeated at least twice with similar results, b, Schematic depicting experimental plans with time line for cancer cell injection, hemin treatment (50 mg kg⁻¹ day⁻¹) or metformin treatment (200–300 mg kg⁻¹ day⁻¹) using TNBC mouse models, c, Representative western blots showing relative BACH1 expression from xenograft tumour models using tumours derived from BM1 cells, MB436 cells or two independent patients (n = 2 biologically independent samples). Western blotting experiments were repeated twice with similar results, d, Relative tumour volumes of BM1 or PDX (no. 4195) mouse xenograft monitored weekly during treated with vehicle, hemin or metformin. Tumour volume data are mean ± s.e.m., two-way ANOVA with multiple comparisons. BM1 tumours (vehicle n = 10, hemin n = 10, metformin n = 10, hemin + metformin n = 5) or PDX tumours (vehicle n = 9, hemin n = 10, metformin n = 8, hemin + metformin n = 7). e, Tumour weights, collected and measured at the end of the treatment using hemin and metformin of MB436, BM1-xenograft, or two PDX models (no. 2147 and no. 4195). Data are mean ± s.e.m. with P values using unpaired two-tailed t-test. f, Representative tumour images from each treatment group of MB436, BM1-xenograft or two PDX models are shown. Scale bar. 1 cm. g, Representative western blots of BACH1 using tumour lysates from mice xenografted with BM1-shCont, BM1-shBACH1 expressing BACH1 (mut) or BM1-shBACH1 expressing wild-type BACH1. h, Tumour weights from mice xenografted with MB436-shBACH1 cells expressing BACH1 (mut) and treated with vehicle, hemin or metformin. Data are mean ± s.e.m., two-tailed t-test. i, j, Tumour growth of BM1 BACH1 (mut) (vehicle n = 5, hemin n – 4, metformin n = 5, hemin + metformin n = 5) or wild-type BACH1 xenografts (vehicle n = 5, hemin n = 4, metformin n = 4, hemin + metformin n = 5) treated with vehicle or hemin and representative tumour images from each treatment group of mice. Scale bar, 1 cm. Representative western blots showing BACH1 expression in multiple mouse tumour lysates.

Extended Data Fig. 8 |. BACH1 expression in multiple cancer types.

a, Distribution of BACH1 expression in TNBC. Clinical and RNA-seq data associated with the TCGA cohort of patients with breast cancer were accessed at https://www.cbioportal.org/. Out of all provisional cases (n = 1, 105), breast cancer samples (n = 914) that had clinical information regarding receptor status of ER, PR (also known as PGR) and HER2 (also known as ERBB2) based on immunohistochemistry analysis as well as RNA-seq data for BACH1-related genes were analysed. The TNBC subgroup among these 914 samples were identified as samples that are negative for all three receptors (n = 115). If the immunohistochemistry results were positive, indeterminate or equivocal for any of the three receptors, those samples were grouped in non-TNBC (n = 799). BACH1 status of the samples were based on an arbitrary 0.5 cut-off for the z-score transformed RNA-seq expression values for the BACH1 gene (245 BACH1-high cases with z-score >0.5; 301 BACH1-low cases with z-score <0.5). b, Frequency (%) of patient tumours with overexpression of BACH1 compared to their matched normal tissues across multiple TCGA cancer types. Numbers of patients relative to healthy controls are indicated in the plot, c, Enriched BACH1 expression (RNA-seq) in TCGA provisional cancer datasets. Red bar indicates median BACH1 expression level in breast cancers, d, Extended plots from KEGG pathway analyses in Fig. 4f, carried out using DAVID using Benjamini-corrected P values (FDR), of genes that are negatively correlated with BACH1 expression. The top eight most-significantly enriched pathways with FDR values (−log(FDR)) are shown for each cancer type: colorectal, liver, lung, skin, ovary, pancreas, prostate and TNBC. e, Co-expression plots of UQCRC1 and BACH1 in TCGA cancers such as prostate (n = 497), skin (n = 472), liver (n = 371) and colon (n = 379). Pearsons (<–0.3) and Spearmans (<–0.3) correlation coefficients are shown.

Extended Data Fig. 9 |. OncoPrint analyses of multiple cancer types.

a–d, Heat maps demonstrating upregulation (red) or downregulation (blue) of BACH1 and ETC genes across TCGA tumours (a, prostate carcinoma TCGA provisional, n = 497; b, patients with skin cutaneous cancer TCGA provisional, n = 472; c, patients with liver cancer TCGA provisional, n = 371; d, patients with colorectal cancer TCGA provisional, n = 379).

Fig. 1 |. BACH1 inhibits mitochondrial genes in TNBC.

a, Gene set enrichment analysis of BACH1-regulated genes with normalized enrichment score (NES) and false-discovery rate (FDR) Q value; heat map depicts changes in gene expression levels involved in ‘mitochondrial inner membrane’, based on microarray data from BM1-shBACH1 and control cells (n = 3 biological replicates per cell line). Synonyms shown for: ATP5F1B (ATP5B), ATP5MC1 (ATP5G1), ATP5MC3 (ATP5G3), ATP5PO (ATP50) and ATP5F1D (ATP5D). b, Left, relative mRNA levels of mitochondrial inner membrane genes in BM1-shBACH1 cells (two shBACH1 vectors) compared to the wild-type control (BM1-shCont) measured by qRT-PCR. Mean ± s.e.m., n – 3 biological independent replicates, two-tailed t-test. Right, protein blots of BACH1 and ETC genes in BM1-shBACH1 and control cell lysates. Relative band density shown below the blots, c, Representative BACH1 western blots using lysates of MB468, MB436 or BM1 cells. Each experiment repeated independently more than three times with similar results, d, Recruitment of BACH1 and H3K27Me3 to promoter regions of mitochondrial genes in BM1 cells. Relative fold enrichment compared to IgG binding shown as mean ± s.e.m., n = 3 biologically independent replicates, two-tailed t-test.

Fig. 2 |. BACH1 regulates mitochondrial metabolism.

a, Measurement of OCR (left) and ECAR (right) in control and BM-shBACH1 cells. Mean ± s.e.m., n = 6 biologically independent samples, two-tailed t-test. b, Schematic of simplified ¹³C-metabolic-tracer analysis using uniformly labelled [U-¹³C₆]-glucose. Solid circle, ¹³C; open circle, ¹²C. c, Relative total labelled ion counts derived from ¹³C₆-glucose in BM1-shBACH1 cells compared to control, d, Fractional isotopic incorporation of ¹³C₆-glucose into representative intermediates in TCA cycles as shown. M, number of labelled carbons. In c, d, mean ± s.e.m., n = 4 biologically independent samples, two-tailed t-test. e, Schematic showing BACH1 regulation of PDK and PDH. f, Relative expression of PDK and PDH in BM1-shBACH1 (clone 1) and control cells. Western blots shown are representative images of more than three independent assays. Relative band density shown below the blots, g, Relative BACH1 and RNA polymerase II (Pol II) enrichment in promoter regions of PDK genes compared to IgG binding, in BM1 cells. Mean ± s.e.m., n = 3 biologically independent samples, two-tailed t-test. Three independent experiments repeated with similar results, h, Cell viability of BM1-shBACH1 or BM1 control cells treated with or without metformin for 48 h. Mean ± s.e.m., n = 6 biologically independent samples, two-tailed t-test.

Fig. 3 |. Hemin mimics shBACH1 through BACH1 degradation in TNBC.

a, Relative mRNA levels of mitochondrial inner membrane genes in BM1 cells treated with hemin (20 μM) or vehicle and representative protein blots. Three independent experiments repeated with similar results, b, Measurement of OCR and ECAR in BM1 cells pre-treated with hemin (20 μM) or vehicle. Mean ± s.e.m., n = 6 biologically independent samples, two-tailed t-test. c, Cell growth, measured as confluence area covered by BM1 cells treated with vehicle, hemin or metformin, d, Top, cysteine residues mutated to alanine in haem-binding motifs of BACH1. Bottom, western blots of BACH1 in cell lysates of BM1 and BM1-shBACH1 cells transfected with BACH1 (mut) (100 ng) and treated with hemin (10, 20, 40 and 80 μM) for 48 h. Each experiment repeated independently three times with similar results, e, Cell growth, measured as confluence area of BM1-shBACH1 cells expressing BACH1 (mut), treated with vehicle (veh), hemin (H) or metformin (M). f, Relative mRNA levels of mitochondrial genes in shBACH1 cells stably expressing BACH1 (mut) and treated with hemin (20 μM) for 48 h. For qRT-PCR analyses in a, f, mean ± s.e.m., n = 3 biologically independent samples, two-tailed t-test. For cell growth assays in c, e, mean ± s.e.m., n = 6 biologically independent samples, two-tailed t-test. In c, e, arrow indicates when drugs were added.

Fig. 4 |. Combination treatment with hemin and metformin suppresses tumour growth through BACH1 inhibition.

a-d, Relative tumour volumes in athymic nude mice orthotopically injected with MB436-shBACH1 (a; shCont n = 6, shCont + metformin n = 7, shBACH1 n – 7, shBACH1 + metformin n = 8), MB436 (b; vehicle n = 9, hemin n = 9, metformin n = 9, hemin + metformin n = 8), PDX (no. 2147) (c; vehicle n = 9, hemin n = 9, metformin n = 10, hemin + metformin n = 8) and BACH1 (mut)-expressing MB436-shBACH1 cells (d; vehicle n = 9, hemin n = 5, metformin n = 10, hemin + metformin n = 9), treated with hemin (H, 50 mg kg⁻¹day⁻¹) daily by intraperitoneal injection and/or metformin (M, 200 mg per kg (body weight) per day for MB436 xenograft or 300 mg per kg (body weight) per day for BM1 xenograft and PDXs) or vehicle in drinking water ad libitum until end of experiments. Tumour volumes shown relative to initial volume measured before treatment. Mean ± s.e.m., two-tailed t-test. e, Heat map from OncoPrint analysis demonstrating expression (z-scores) of BACH1 and ETC gene expression for each patient with breast cancer (TCGA provisional dataset, n = 1105). ATP5G2 and ATP5L also known as ATP5MC2 and ATP5MG, respectively f, Heat map showing KEGG pathways that are negatively correlated with BACH1 levels across major TCGA cancers such as breast (n = 1105), TNBC (n = 115), prostate (n = 497), pancreas (n = 186), ovary (n = 606), skin (n = 472), lung (n = 586), liver (n = 371) and colon (n = 379). Values shown as −log(FDR) with Benjamini-Hochberg-corrected P values (FDR) using the GOseq package. Only KEGG pathways commonly enriched in all cancer types studied are on the heat map. g, Proposed model summary of BACH1 regulation of metabolic pathways by inhibiting mitochondrial membrane gene expression and PDH activity; targets of combination therapy by metformin (ETC) and hemin (BACH1) are shown.