Background
Breast cancer is a common cancer worldwide, and its incidence is gradually increasing in Asia. It is a heterogeneous cancer type and can be distinguished into luminal A and B, human epidermal growth factor receptor 2 overexpression, basal-like, unclassified, and various other subtypes [
1‐
3]. These subtypes are associated with specific morphological characteristics, metastatic ability, and chemosensitivity, and yield different clinical outcomes [
2‐
4]. However, metastasis is a major problem resulting in therapy failure and lethality in patients with breast cancer. Therefore, investigating the detailed mechanisms of breast cancer metastasis or developing a favorable prognostic biomarker to predict prognostic outcomes in patients with breast cancer is beneficial.
The isocitrate dehydrogenase (IDH) gene family expresses key functional metabolic enzymes in the Krebs cycle, which can catalyze the conversion of isocitrate to α-ketoglutarate (KG), thus generating nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) from NADP+. In addition, α-KG, a cofactor for the ten–eleven translocation (TET) family, catalyzes the conversion of cytosine-5 methylation to cytosine-5 hydroxymethylation [
5]. Therefore, the physiological function of the NADP-dependent IDH gene has been reported to involve the maintenance of the normal cellular redox status and global DNA methylation status in normal cells [
6,
7]. Studies have identified that somatic heterozygous mutations in IDH1/2 play a crucial role in the development of cancers, including glioma and leukemia [
5,
7,
8]. Zhao et al. reported that IDH1 mutations contribute to tumorigenesis by modulating the stabilization of hypoxia-inducible factor (HIF)-1 [
9]. In addition, IDH1/2 mutations have frequently resulted in the accumulation of D-2-hydroxygluarate, which blocks TET-induced cytosine 5-hydroxymethylation, resulting in increased global DNA hypermethylation. In acute myeloid leukemia, IDH1/2 mutations are closely associated with poor prognosis [
5,
7,
10]. On the other hand, the low frequency of IDH1/2 mutations has been reported in breast cancer [
8,
11,
12]. Furthermore, the biological function and clinical effects of the IDH gene in breast cancer have not been characterized in depth. In this study, we first reported that the IDH1 is downregulated in breast cancer and depletion of IDH1 in breast cancer cells results in accelerating breast cancer migration and invasion activities by activating snail expression. Two novel micro RNAs (miRNAs), miR-32-5p and miR-92b-3p, were identified to directly inhibit IDH1 expression. Clinically, the IDH1–snail axis dysfunction might be a favorable independent marker for predicting breast cancer survival.
Methods
Patients and tissues
This study was approved by the institutional review board (IRB) of Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan (IRB number: VGHKS13-CT10-10). The requirement for written informed consent from patients was waived by the hospital IRB because all the data and specimens were previously collected and anonymously analyzed. Tissue microarrays containing 309 paraffin-embedded samples of breast tissue from invasive ductal carcinoma (IDC) were established, and the pathological information on the patients was summarized in our previous study [
13].
Expression data from The Cancer Genome Atlas
All level-3 expression data for breast cancer were downloaded from The Cancer Genome Atlas (TCGA) database. The transcriptome data on 1102 and 113 breast cancer and adjacent normal tissues, respectively, were downloaded from the TCGA database. In addition, the small RNA profiles of 778 and 87 breast cancer and adjacent normal tissues, respectively, were obtained from TCGA database. The expression levels of protein-coding genes were shown as reads per kilo base million and those of miRNA were shown as transcripts per million.
Immunohistochemistry analysis
A Novolink max polymer detection system (Leica, Newcastle upon Tyne, UK) was used for immunohistochemical (IHC) analysis in this study. The slides were deparaffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was performed by immersing the slides in Tris-ethylenediaminetetraacetic acid (10 mM and pH 9.0) at 125 °C for 10 min in a pressure boiler. Endogenous peroxidase activity was blocked by incubating the slides with 3% hydrogen peroxide in methanol for 30 min. After blocking at room temperature, primary antibodies were immediately applied, and the slides were incubated overnight at 4 °C in a wet chamber. The following primary antibodies were used in this study: rabbit polyclonal anti-IDH1 (1:400; GeneTex, San Antonio, Texas, USA) in Tris-buffered saline solution with 5% bovine serum albumin. After being washed with phosphate-buffered saline, the slides were incubated with horseradish peroxidase-labeled secondary antibody for 10 min at room temperature, and the sections were counterstained with hematoxylin.
IHC analysis and scoring
First, a senior pathologist accompanied a technician to evaluate the slides until all the discrepancies were resolved. Subsequently, the technician independently reviewed all the slides. Finally, 5–20% of core samples at each intensity were randomly selected for re-evaluation by the pathologist. During the evaluation, the pathologist and technician were blinded to the clinical outcomes of the patients. We graded the immunoreactivity using a semiquantitative approach. Marker scores for staining were calculated based on the staining intensity (0, no signal; 1, mild; 2, moderate; and 3, strong) and the proportion of positively stained tumor cells in the 5 high-power field (0, < 5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; and 4, > 75%). The marker score is the sum of the staining intensity score and percentage of the positive tumor cell score. The score was graded as follows: –, 0–1; +, 2–3; ++, 4–5; and +++, 6–7.
Real-time reverse transcription polymerase chain reaction
Two micrograms of total RNA was reverse-transcribed with oligo (dT)
15 primers and SuperScript III Reverse Transcriptase according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The reaction was performed by incubating cells at 42 °C for 1 h; the Reverse Transcriptase was subsequently inactivated by incubation at 85 °C for 5 min. The cDNA was used for real-time polymerase chain reaction (PCR) analysis with gene-specific primers, and gene expression was detected using a SYBR Green I assay (Applied Biosystems, Foster City, CA, USA). To determine their expression levels, the genes were subjected to the following conditions: 94 °C for 10 min, followed by 40 cycles of 94 °C/1 min, 60 °C/1 min, and 72 °C/30 s, with a final extension at 72 °C for 10 min. GAPDH expression was used as an internal control, and the expression levels of epithelial–mesenchymal transition (EMT)-relative genes were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (difference in cycle threshold (ΔCt) = Ct
candidates − Ct
GAPDH). The real-time PCR primers used in this study are listed in Additional file
1: Table S1.
Stem-loop reverse transcription PCR
We reverse-transcribed 1 μg of total RNA with a stem-loop reverse transcription (RT) reaction using microRNA RT primers and SuperScript III Reverse Transcriptase according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The reaction was performed under the following incubation conditions: 30 min at 16 °C, followed by 50 cycles of 20 °C/30 s, 42 °C/30 s, and 50 °C/1 s. The enzyme was subsequently inactivated by incubation at 85 °C for 5 min. Gene expression was detected using the SYBR Green I assay (Applied Biosystems, Foster City, CA, USA), and the expression levels of microRNA were normalized to those of U6 small RNA (ΔCt = target miRNA Ct-U6 Ct).
Ectopic expression of miRNAs
Breast cancer cells were transfected with 10-nM miRNA-32-5p mimics, miR-92b-3p mimics, or the appropriate miRNA mimic control (GenDiscovery Biotechnology Inc., Taiwan) using the Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA). Twenty-four hours after transfection, cells were harvested, and their expression levels were examined using stem-loop RT quantitative PCR.
Knockdown of IDH1 expression
Breast cancer cells were transfected with 10-nM si-IDH1#1 (sense: 5′- CAUUAAAGGUUUACCCAAUtt-3′ and antisense: 5′- AUUGGGUAAACCUUUAAUGca-3′, si-IDH1#2 (sense: 5′- CCAACGACCAAGUCACCAAtt-3′ and antisense: 5′- UUGGUGACUUGGUCGUUGGtg-3′) or scramble control (Invitrogen, Carlsbad, CA, USA) using the Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA). Twenty-four hours after transfection, cells were harvested, and their expression levels were examined through western blotting.
Stable IDH1 knockdown with short hairpin RNA (shRNA)
Stable MDA-MB-231 cells with IDH1 knockdown were generated by infecting MDA-MB-231 cells with lentiviruses expressing sh-IDH1 in the presence of 8 μg/mL of polybrene for 24 h, followed by puromycin (4 μg/mL) selection for 3–5 days. The shLuc vector targeting the luciferase gene provided puromycin resistance and was used as the control. IDH1 expression was confirmed through a western blotting assay. In this study, we designed two shRNA sequences targeting IDH1, and the individual sequences of shRNA used for constructs in this study were as follows: for sh-IDH1 #1, sense: 5′-CCGGCCTATCATCATAGGTCGTCATCTCGAGATGACGACCTATGATGATAGGTTTTT-3′ and antisense: 5′-AAAAACCTATCATCATAGGTCGTCATGAGCTCATGACGACCTATGATGATAGGCCGG-3′; for sh-IDH2#2, sense: 5′-CCGGGCTTTGGAAGAAGTCTCTATTCTCGAGAATAGAGACTTCTTCCAAAGCTTTTT-3′ and antisense: 5′-AAAAAGCTTTGGAAGAAGTCTCTATTGAGCTCAATAGAGACTTCTTCCAAAGCCCGG-3′.
Cell proliferation assays
Breast cancer cells (2.5 × 103 cells/mL) were seeded in a 96-well plate and transfected with siIDH1, shIDH1 or scramble control. After transfection, cell growth was determined at 0, 1, 2, 3, and 4 days using the CellTiter-Glo® One Solution Assay (Promega Corporation, Madison, WI, USA). All the experiments were performed in triplicate.
Wound healing assay
For the wound healing assay, after transfection with si-IDH1 or scramble control for 24 h, cells (1.5 × 106) were seeded on six-well plates. A straight line was scratched on the monolayer in the middle of the well using the tip of a 200-mL pipette. A culture medium of 10% fetal bovine serum (FBS) was replaced with a serum deprivation culture medium, and cells were then incubated at 37 °C. Wound closure was monitored and photographed at different time points under a microscope. Subsequently, the open area was assessed for quantifying cell migration ability.
Invasion assays
Cells were assessed for their invasion ability in vitro using a Transwell assay, according to our previous study [
14]. Briefly, breast cancer cells (4.5 × 10
5) transfected with si-IDH1, miR-32-5p mimics, miR-92b-3p mimics, or scramble control were suspended in 2% FBS and seeded on the upper chamber of the Transwells (Falcon, Corning Incorporated, USA) with a coating of Matrigel (BD Biosciences, MA, USA) for the invasion assay. After incubating in a CO
2 incubator at 37 °C for 12 or 24 h, the remaining cells in the upper chamber were removed with cotton swabs, and cells on the undersurface of the Transwells were fixed with 10% formaldehyde solution. Cells were stained with crystal violet solution, and the numbers of breast cancer cells were calculated by counting three fields under a phase-contrast microscope. All experiments were performed in triplicate.
Candidates of the miRNA target and assay of luciferase activity
The putative miRNAs targeting the 3′-UTR of IDH1 were determined using TargetScan (
http://www.targetscan.org/vert_71/) and microRNA.org (
http://34.236.212.39/microrna/home.do). In this study, we identified that 15 miRNAs were predicted for binding at the 3′-UTR of IDH1. The full-length 3′-UTR of IDH1 was cloned into the PGL3 vector. Subsequently, pGL3–IDH1–3′-UTR vectors were co-transfected with miR-32-5p mimics, miR-92b-3p mimics, or scramble controls in breast cancer cells using the Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA). After transfection for 24 h, cell lysates were used for measuring the luciferase activity by using the Dual-Glo Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA).
Western blotting
Total cell lysates were extracted with radioimmunoprecipation assay (RIPA) buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, and 0.1% sodium dodecyl sulfate). Total proteins were separated through 6–10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose filter membranes (Millipore, Billerica, USA). After the transfer, the membranes were blocked with a blocking buffer for 1 h at room temperature and probed with a primary antibody for the desired molecule overnight at 4 °C, followed by treatment with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the proteins were visualized using WesternBright™ ECL (Advansta Inc., Menlo Park, CA, USA) and detected using the BioSpectrum
TW 500 Imaging System (UVP, USA). The antibodies are shown in Additional file
2: Table S2.
Microarray analysis and pathway enrichment analysis
Total RNA samples were obtained from si-IDH1 and scramble control cells and were subjected to microarray screening. The microarray experiments and data analysis were conducted by Welgene Biotech (Taipei, Taiwan) through the Agilent SurePrint G3 Human V2 GE 8X60K Chip. The differentially expressed genes (fold change > 2) were selected from microarray data, and the candidate genes were mapped onto the Kyoto Encyclopedia of Genes and Genomes pathways based on the enzyme commission numbers by using the R package SubPathwayMiner v.3.1. Subsequently, the hypergeometric test was performed to identify significantly enriched pathways and calculate the false-positive discovery rate in terms of the p value.
α-KG was analyzed through ultra-high performance liquid chromatography (UPLC; Acquity UPLC system, Waters Corporation, MA, US) coupled with a quadrupole time-of-flight mass spectrometer (QTof MS) (Xevo, Waters). Details of the procedures were described in another study [
15]. Raw data on all samples were processed using MassLynx software.
Protein degradation assay
Cells subjected to different treatments were cultured in the presence of 20 μg/mL of cyclohexamide for the indicated time periods and then harvested in RIPA buffer containing protease inhibitors (Roche, CA, USA). Protein lysates were quantified using the bicinchoninic acid (BCA) method for the subsequent western blotting analysis.
Statistical analysis
Many statistical methods were used for data analysis. The chi-squared test, Student t test, analysis of variance (ANOVA), Mann–Whitney U test, and Kruskal–Wallis one-way ANOVA were used to test correlation between the expression levels of each protein and different types of breast tissues or clinicopathological parameters. Studies on breast cancer have typically defined the outcomes as the time from diagnosis or surgery until a particular event of interest (endpoint). Disease-specific survival (DSS) is measured from the time of the initial resection of the primary tumor to the date of cancer-related death or last follow up. Disease-free survival (DFS) is defined as the time from surgery to an event (local recurrence, regional recurrence, and distant metastasis, but not disease-related death). The cumulative survival curves were estimated using the Kaplan–Meier method, and the survival curves were compared using the log-rank test. A Cox proportional hazards model was used to determine the independent predictors of survival using factors significant in univariate analysis as covariates. We considered p < 0.05 (two-sided) as significant.
Discussion
There are reports from studies that IDH genes play crucial roles in the metabolism of glucose, fatty acids, and glutamine in humans [
6,
7]. NADPH is an essential reducing agent for glutathione (GSH) regeneration by GSH reductase and the NADPH-dependent thioredoxin system, both of which are important for protecting cells from oxidative damage. IDH1 and IDH2 can produce cytosolic NADPH in cells [
27], suggesting that they defend against the oxidative stress caused by reactive oxygen species [
27,
28]. Therefore, the disruption of the normal functions of IDH1 and IDH2 may significantly affect the cellular redox balance and result in cancer cell growth dysfunction [
28‐
31]. Studies have revealed that IDH1 and IDH2 mutations promote global hypermethylation with concomitant reductions in 5hmC levels [
32‐
34]. In previous studies, IDH mutations were rarely detected in human solid tumors, except for glioma [
8,
11,
35]. Therefore, IDH repression seems to be a more crucial mechanism for regulating global DNA methylation in solid tumors, rather than IDH mutations, which are more common in gliomas and hematological malignancies [
18].
In our previous work, we reported that the contents of 5mC and 5hmC were significantly lower in breast cancer as compared to those in corresponding adjacent normal tissues [
13]. We also revealed that IDH1 and IDH2 modulate 5hmC levels in human gastric cancer [
36]. However, only IDH2 expression levels were significantly lower in gastric cancer tissues than in adjacent normal tissues. Furthermore, low IDH2 expression levels have been closely associated with the poor survival curve in patients with gastric cancer [
36]. In this study, we provided a novel insight to clarify the role of IDH1 expression in breast cancer. Our results indicated that IDH1 expression levels were significantly lower in breast cancer tissues than in adjacent normal tissues and that IDH1 played a crucial role in modulating breast cancer metastasis. Furthermore, low IDH1 expression was significantly associated with the poor prognosis in breast cancer. A study has shown that the siRNA knockdown of either IDH1 or IDH2 can significantly reduce the proliferative capacity of a glioblastoma cell line expressing both wild-type IDH1 and IDH2 [
30]. Lee et al. suggested that the attenuation of IDH family gene expression may protect the skin from ultraviolet (UV)-B-mediated damage by inducing the apoptosis of UV-damaged cells [
31]. According to these findings, IDH genes seem to play a crucial role in cell growth by maintaining a normal cellular redox status. Although we observed that the low expression levels of IDH1 in IDC were associated with poor clinical pathological features of breast cancer, IDH1 depletion showed no significant effects on the growth of breast cancer cells (Fig.
4b). Furthermore, the siRNA knockdown of IDH1 can promote the invasion ability of breast cancer cells, suggesting that IDH1 acts as a tumor suppressor in breast cancer progression. Wang et al. reported that the expression levels of IDH1 and IDH2 were significantly decreased in breast cancer cells with adriamycin resistance, implying that IDH acts as a tumor suppressor in breast cancer drug resistance [
37]. Altogether, these results implied that the IDH family possibly has distinct biological functions in human cancer.
We first reported that the loss of IDH1 expression resulted from the aberrant overexpression of miR-32-5p and miR-92b-3p in breast cancer. Studies have indicated that miR-32-5p plays an oncogenic role in the growth and invasion ability of breast cancer cells by directly silencing PHLPP2 and FBXW7 [
38,
39]. On the other hand, the biological role of miR-92b-3p is controversial and unknown in human breast cancer. miR-92b-3p has been reported to play a tumor-suppressing role in esophageal cancer by silencing RAB23 or integrin α6 [
40,
41]. Contrastingly, miR-92b-3p has also been reported to play an oncogenic role in the growth and invasion ability by regulating phosphatase and tensin homolog and Smad3 in bladder cancer, hepatocellular carcinoma, and glioblastoma cells [
42‐
44]. Herein, we reported a novel finding that the expression levels of miR-92b-3p significantly increased and promoted breast cancer invasion by silencing the expression of IDH1.
IDH1 mutants can play a dominant negative role in wild-type IDH1 functions, resulting in the increased phosphorylation of MAPK and signal transducer and activator of transcription 3 in melanoma cells [
18]. Zhao et al. reported that IDH1 mutants can activate the hypoxia pathway by preventing HIF-1α degradation through prolyl hydroxylase activation in glioma cells [
9]. Studies have revealed that HIF-1α can promote cancer cell metastasis through EMT by upregulating snail, twist, and vimentin expression [
21‐
23]. In the present study, we demonstrated that IDH1 depletion in breast cancer cells led to an increase in the HIF1α protein level to promote cell migration and invasion. An α-KG-dependent dioxygenase called prolyl hydroxylase 2 (PHD2) regulates the protein stability of HIF1α under normoxic conditions by hydroxylating the proline residues 402 and 405 on the oxygen-dependent degradation domain at its N terminus [
24], and hydroxylated HIF1α is subjected to protein degradation by the 26S proteasome [
45]. Our mechanistic study demonstrated that IDH1 depletion led to a decrease in intracellular α-KG, which in turn stabilized HIF1α (Fig.
7). The addition of 1 mM α-KG reversed the increase of HIF1α in IDH1-depleted cells. Furthermore, we also demonstrated that the depletion of IDH1 did not increase the mRNA level of HIF1α. Therefore, we reasoned that IDH1 depletion caused the low level of intracellular α-KG to impair PHD2 enzyme activity, which stabilized HIF1α. Collectively, these results demonstrated that IDH1 plays a tumor-suppressing role in the progression of breast cancer.