ABCG1 as a potential oncogene in lung cancer.

ATP-binding cassette transporter G1 (ABCG1) is a member of the ABC transporter family and regulates cellular cholesterol homeostasis. It has important roles in cholesterol homeostasis and tumor immunity, which has, however, not been reported in lung cancer. The present study showed that ABCG1 protein is upregulated in lung cancer compared with adjacent normal tissues and furthermore, aberrant ABCG1 expression was detected in lung cancer cell lines. ABCG1 was shown to promote the proliferation of HKULC4 lung cancer cells. Moreover, ABCG1 was found to regulate proliferation-, apoptosis- and cancer stem cell-associated markers in HKULC4 cells, implying that ABCG1 has important roles not only in the regulation of proliferation but also of apoptosis and cancer stem cells. A microRNA microarray analysis showed that ABCG1 significantly downregulated miR-29a, -b and -c expression in HKULC4 cells. Finally, it was demonstrated that ABCG1 promoted the migration and invasion in HKULC4 cells. Thus, ABCG1 may be a novel therapeutic target to improve the treatment of non-small cell lung cancer.

Introduction

Lung cancer is the most common cause of cancer-associated mortality, with >226,000 new cases in the USA. Non-small cell lung cancer (NSCLC) is by far the most common type of lung cancer, accounting for ~80% of cases (1). In spite of marked improvements in radiotherapy and chemotherapy over the past few decades, the prognosis for patients with NSCLC is dismal, with the 5-year survival rate only being slightly above 15% (2). Thus, to improve the treatment of NSCLC, it is urgently required to identify novel markers as well as therapeutic targets and strategies.

ATP-binding cassette transporter G1 (ABCG1) is a member of the ABC transporter family and regulates cellular cholesterol homeostasis (3). Cholesterol homeostasis is crucial for the function and survival of cells (4). ABCG1 engages in reverse cholesterol transport by effluxing excess cholesterol from cells to high-density lipoprotein (HDL) particles, which is the only path for elimination of cholesterol from the body (5,6). Furthermore, ABCG1 is important for intracellular cholesterol transport (7,8). It is ubiquitously expressed in numerous cell types, including endothelial cells, lymphocytes and myeloid cells (3). ABCG1 has been reported to be a mediator of tumor immunity. ABCG1 deficiency in mice dramatically suppressed subcutaneous growth of B16-melanoma and MB49-bladder carcinoma cells and prolonged survival (9). However, to the best of our knowledge, the role of ABCG1 has, so far, not been reported in lung cancer.

MicroRNA (miRNA/miR) are endogenous short non-coding RNA molecules of 18–25 nucleotides in length that regulate gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner (10–12). Abnormal miRNA expression has been implicated in a multitude of cellular processes, including proliferation, apoptosis, migration and differentiation, and linked to various diseases, including cancer (12–18).

The present study showed that ABCG1 protein is upregulated in lung cancer compared with adjacent normal tissues and aberrant ABCG1 expression was detected in lung cancer cell lines. In HKULC4 lung cancer cells, ABCG1 promoted proliferation. Moreover, ABCG1 regulated proliferation-, apoptosis- and cancer stem cell-associated markers in HKULC4 cells, implying that it has important roles not only in the regulation of proliferation, but also of apoptosis and may also be of significance in cancer stem cells. By using a microRNA microarray, ABCG1 was found to significantly downregulate miR-29a, miR-29b and miR-29c expression in HKULC4 cells. Finally, the present study demonstrated that ABCG1 promoted migration and invasion of HKULC4 cells. Thus, ABCG1 is a novel potential therapeutic target to improve the treatment of NSCLC.

Materials and methods

Ethics statement

The present study was approved by the Medical Ethics Committee of the Medical School of Shandong University (Jinan, China). All experiments were monitored and approved by the Yuncheng Second People's Hospital (Yuncheng, China). Written informed consent was provided by all patients enrolled in the present study.

Tissue samples

Lung cancer tissues and adjacent normal tissues were obtained from Shandong Cancer Hospital (Qingzhou, China) between January 2010 and January 2015. All tissues were examined histologically and pathologists confirmed the diagnosis. The medical ethics committee approved and monitored the experiments undertaken. The study also followed the internationally and locally accepted ethical guidelines of the Declaration of Helsinki, as well as the local laws.

Cell culture

The NCSLC cell lines H1650, HKULC2, H1299, HKULC4, HCC827 and H23 were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Western blot analysis

Tissues and cells were dissolved in lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (Beijing Solarbio Biological Technology Co., Ltd., Beijing, China). Protein concentration was determined using a bicinchoninic acid kit (Tiangen Biotech Co., Ltd., Beijing, China). Subsequently, 20 µg of total protein from the different samples was loaded per lane and subjected to 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% skimmed milk and incubated overnight at 4°C with primary antibodies, including rabbit anti-human monoclonal anti-ABCG1 (1:5,000; ab52617), anti-c-Myc (1:5,000; ab32072), anti-p21 (1:5,000; ab109520), anti-p53 (1:5,000; ab32049), anti-Ki-67 (1:5,000; ab15580), anti-retinoblastoma (1:5,000; ab47763), anti-B-cell lymphoma 2 (BCL2; 1:5,000; ab23124), anti-myeloid cell leukemia 1 (MCL1; 1:5,000; ab32087), anti-CD133 (1:5,000; ab32077) and anti-aldehyde dehydrogenase (ALDH; 1:5,000; ab52492; all Abcam, Cambridge, MA, USA). The membranes were washed with PBS containing 0.05% Tween-20 (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000; ab6721; Abcam). The membrane was placed in an enhanced chemiluminescence solution (Tiangen Biotech Co., Ltd.) and exposed to a Gel Doc EZ Imager (Bio-Rad Laboratories, Inc., Hercules, USA). Image Lab v.3.0 software (Bio-Rad Laboratories, Inc.) was used for protein band analysis. The relative content of the target protein was determined as the ratio to the internal reference β-actin.

MTT assay

The cell growth-inhibitory activity was assessed by an MTT assay. HKULC4 cells were seeded in a 96-well plate (2,000 cells/well) and exposed to fresh medium with ABCG1-expressing plasmids or empty vectors (pcDNA 3.1; both Tiangen Biotech Co., Ltd.) for 48 h. Subsequently, 20 µl MTT solution (5 mg/ml; Sigma-Aldrich; Merck-Millipore, Darmstadt, Germany) was added. Following incubation for 4 h at 37°C, the medium was discarded and the formed formazan crystals were dissolved in DMSO (150 µl/well). The absorbance, which is directly proportional to the number of viable cells, was measured at a wavelength of 490 nm using a multilabel counter microplate reader (Safire; Tecan Austria GmbH, Grödig, Austria).

Cell cycle analysis

Cells were seeded into a 6-well culture plate (1×105 cells/well) and transfected with plasmids for 24 h, washed twice with PBS and then centrifuged at 1,000 × g for 5 min at room temperature. The pellet was resuspended and fixed in 70% ethanol for at least 12 h at 4°C. The fixed cells were incubated with DNase-free RNaseA and propidium iodide (PI; Nanjing Kaiji Biological Technology Development Co., Ltd., Nanjing, China) according to the manufacturer's instructions. The stained cells were analyzed using a flow cytometer (BD Accuri™ C6; BD Biosciences, Franklin Lakes, NJ, USA).

5-Bromo-2′-deoxyuriding (BrdU) incorporation assay

BrdU incorporation reflects the rate of DNA synthesis. HKULC4 cells transfected with ABCG1-expressing plasmid or empty vector after 48 h were cultured in 96-well microplates (2,000 cells/well), followed by incubation with bromodeoxyuridine (BrdU; Roche Diagnostics, Indianapolis, IN, USA) for 4 h. After the culture supernatant was removed, the cells were fixed and incubated with anti-BrdU antibody (1:5,000; ab6326; Abcam) and detected and quantified using a flow cytometer (BD Accuri™ C6; BD Biosciences) (19).

miRNA detection

Total RNA from cultured cells, with efficient recovery of small RNA, were isolated using a mirVana™ miRNA Isolation kit (Ambion, Austin, TX, USA). cDNA for each sample was synthesized by using the 3′ IVT Express kit (Affymetrix, Santa Clara, CA, USA) according to the supplier's protocol. The purified cDNA was fragmented by incubation in fragmentation buffer (provided in the 3′IVT Express kit) at 95°C for 35 min and chilled on ice. The fragmented labeled cDNA was subjected to an miRNA 2.0 array (Affymetrix) and hybridized in a Genechip hybridization oven 640 (Affymetrix) at 45°C for 18 h. After washing and staining in a GeneChip Fluidics Station 450 (Affymetrix), the arrays were scanned by using GeneChip Scanner 3000 (Affymetrix). The gene expression levels of samples were normalized and compared by using Partek Genomics Suite 6.5 (Partek Inc., St. Louis, MO, USA). Average-linkage hierarchical clustering of the data was applied by using the Cluster function and the results were displayed by using TreeView (http://genome-www.stanford.edu/clustering/).

In vitro migration and invasion assays

Transwell migration assays were performed in 24-well plates (BD Biosciences) with non-coated membrane, while invasion assays were performed with Matrigel-coated membrane (pore size, 8 mm; BD Biosciences). In each assay, 1×105 cells suspended in 200 µl serum-free RPMI-1640 medium that had been transfected with ABCG1-expressing plasmids or empty vectors were seeded into the upper chamber. A total of 500 µl RPMI-1640 medium supplemented with 10% FBS was added to the lower chamber. Following 24-h incubation, the cells on the upper side of the membrane were gently removed and the cells on the lower surface of the insert were stained with crystal violet and counted as described previously (20).

Statistical analysis

Statistical comparisons were made using a two-tailed Student's t-test. Values were expressed as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were conducted using SAS v.9.3 software (SAS Institute, Inc., Cary, NC, USA).

Results

ABCG1 is upregulated in lung cancer tissues and aberrantly expressed among various lung cancer cell lines

In an attempt to identify ABCG1 expression in lung cancer tissues and adjacent normal tissues, western blot analysis was performed. Protein was isolated from 8 pairs of lung cancer and adjacent normal tissues. ABCG1 protein was revealed to be significantly increased in cancer tissues compared with adjacent normal tissues (Fig. 1A). This implied that ABCG1 may be an oncogene in lung cancer. In order to identify the protein expression of ABCG1 among different lung cancer cell lines, western blot analysis of lysates of lung cancer cell lines (H1650, HKULC2, H1299, HKULC4, HCC827 and H23) was performed. ABCG1 expression was revealed to be lowest in HKULC4 cells (Fig. 1B).

Figure 1.

ABCG1 is upregulated in lung cancer and differentially expressed in lung cancer cell lines. Western blot analysis of ABCG1 protein in (A) eight lung cancer and adjacent normal tissues as well as (B) in lung cancer cell lines (representative images of three experiments are shown). β-actin was used as a loading control. ABCG1, ATP-binding cassette transporter G1; N, normal tissues; C, cancer tissues.

ABCA1 overexpression promotes proliferation of lung cancer HKULC4 cells

To investigate whether ABCG1 promotes the proliferation of lung cancer cells, HKULC4 cells were transfected with ABCG1-expressing plasmid and stable overexpression of ABCG1 protein was assessed by western blot analysis. The results showed that ABCG1 was significantly increased by transfection with ABCG1-expressing plasmid (Fig. 2A). In order to determine the effect of ABCG1 on proliferation, an MTT assay was performed. The results showed that overexpression of ABCG1 significantly promoted the proliferation of HKULC4 cells (Fig. 2B). To further elucidate the effects of ABCG1 on proliferation, cell cycle analysis was performed. The results showed that HKULC4 cells transfected with ABCG1-expressing plasmid had higher S-phase fractions than those transfected with empty vector (Fig. 2C). To further confirm that promotion of DNA synthesis contributed to higher S-phase fractions in HKULC4 cells transfected with ABCG1-expressing plasmids, a BrdU incorporation assay was performed. The results confirmed that compared with the empty vector group, transfection with ABCG1-expressing vector significantly promoted DNA synthesis in the cells, as shown by increased BrdU incorporation in the representative micrographs and quantified bar graph (Fig. 2D).

Figure 2.

ABCA1 overexpression promotes proliferation of HKULC4 lung cancer cells. ABCG1 cells were transfected with ABCG1-expressing plasmid or empty vector. (A) Western blot analysis of ABCG1. β-actin was used as a loading control (representative images of three experiments are shown). (B) The proliferation of HKULC4 cells was assessed using an MTT assay. (C) Cell cycle analysis of HKULC4 cells. Histograms of DNA content obtained by fluorescence-assisted cell sorting analysis are shown. The percentages of cells in each cell cycle stage are shown in the inserts of the histograms. (D) BrdU incorporation assay for HKULC4 cells. Representative micrographs (left) and quantification (right) of BrdU-incorporating cells (magnification, ×100). Values are expressed as the mean ± standard error of the mean (n=3). ABCG1, ATP-binding cassette transporter G1; BrdU, 5-bromo-2′-deoxyuridine.

ABCG1 regulates proliferation-, apoptosis- and cancer stem cell-associated markers in HKULC4 lung cancer cells

In a subsequent experiment, western blot analysis was employed to identify whether the protein levels of proliferative markers were affected by ABCG1 in the cells. The results of the western blot analysis showed that c-Myc expression was upregulated, while p21 and p53 expression were downregulated by ABCG1-expressing plasmids in the cells (Fig. 3). In addition, the levels of apoptosis markers were assessed, revealing that BCL2 and MCL1 were upregulated by ABCG1 in HKULC4 cells (Fig. 3). Furthermore, the cancer stem cell-associated makers CD133 and ALDH were found to be increased in HKULC4 cells with ABCG1 overexpression (Fig. 3).

Figure 3.

ABCG1 regulates proliferation-, apoptosis- and cancer stem cell-associated markers in HKULC4 lung cancer cells. Western blot for c-Myc, p21, p53, Ki67, RB, BCL2, MCL1, CD133 and ALDH1 in HKULC4 cells transfected with ABCG1-expressing plasmid or empty vector. β-actin was used as a loading control (representative images of three experiments are shown). ABCG1, ATP-binding cassette transporter G1; BCL2, B-cell lymphoma 2; MCL1, myeloid cell leukemia 1; ALDH, aldehyde dehydrogenase; RB, retinoblastoma protein.

ABCG1 significantly downregulates miR-29a, miR-29b and miR-29c expression in lung cancer HKULC4 cells

Oncogenes may exert their functions by regulating miRNA expression in lung cancer (21). A number of miRNA are involved in lung cancer pathogenesis, which may either function as tumor suppressor genes or oncogenes (22). Thus, it was reasoned that ABCG1 may function as an oncogene by regulating relevant miRNA. An miRNA microarray analysis was performed by isolating RNA from HKULC4 cells transfected with ABCG1 expression or empty vector and hybridizing them to a custom miRNA microarray platform. After three repetitions of hybridization, quantification and normalization, miR-29a, -b and -c were found to be downregulated by >100-fold in the cells (Fig. 4).

Figure 4.

ABCG1 downregulates miR-29a, miR-29b and miR-29c expression in HKULC4 lung cancer cells. miRNA microarray analysis of HKULC4 cells transfected with ABCG1-expressing plasmid or empty vector. Three independent repetitions were performed. ABCG1, ATP-binding cassette transporter G1; miR, microRNA; hsa, Homo sapiens.

ABCG1 promotes migration and invasion in HKULC4 lung cancer cells

To determine whether ABCG1 overexpression increases the basal levels of cell migration or invasion, HKULC4 cells were subjected to Transwell assays. Ectopic expression of ABCG1 resulted in a 4-fold increase in cell invasion and a 5.5-fold increase in cell migration (Fig. 5). These results indicated that overexpression of ABCG1 promoted migration and invasion in HKULC4 lung cancer cells.

Figure 5.

ABCG1 promotes migration and invasion of HKULC4 lung cancer cells. Invasion and migration assays of HKULC4 cells transfected with ABCG1-expressing plasmid or empty vector. Values are expressed as the mean ± standard error of the mean (n=3). ABCG1, ATP-binding cassette transporter G1.

Discussion

The present study identified a novel role for the cholesterol transporter ABCG1 as a modulator of proliferation, migration, invasion, apoptosis and microRNA regulation in lung cancer. This result is consistent with a previous study reporting that the absence of ABCG1 inhibits tumor growth through the modulation of macrophage survival and phenotype within the tumor (9). The present study found that ABCG1 overexpression not only promoted proliferation, but also upregulated the expression of the anti-apoptotic proteins BCL2 and MCL1 in HKULC4 lung cancer cells. ABCG1 effluxes excess cholesterol from cells to HDL particles for reverse cholesterol transport, which is the only pathway for cholesterol elimination from the body (5,6). Studies have demonstrated that pulmonary macrophages of ABCG1-/- mice accumulate massive levels of cholesterol and sterol esters, consistent with these cells being particularly sensitive to loss of function of this transporter (5,23–25). These lipid-loaded ABCG1-/-pulmonary macrophages also undergo increased apoptosis (26). It is probable that the accumulation of cholesterol that produced specific toxic effects induced apoptosis in lung cancer cells.

Lung cancer contains a rare population of CD133+ cancer stem-like cells with the ability to self-renew, which generates an unlimited progeny of non-tumorigenic cells (27). Highly tumorigenic lung cancer CD133+ cells display stem-like features and are resistant to cisplatin treatment (28). Increased ALDH1 activity has been found in stem cell populations in human brain and breast cancer, acute myeloid leukemia and multiple myeloma (29–36). Therefore, ALDH1 activity may be utilized as a common marker for normal as well as malignant stem cell populations (36). ALDH1 expression has been reported in certain lung cancer cell lines (37), and the increased expression of ALDH1 could result from cigarette smoking and contribute to malignant transformation of lung cells (38). ALDH1 has been suggested to be a tumor stem cell-associated marker in lung cancer (39). The results of the present study showed that ABCG1 upregulated CD133 and ALDH1 expression in lung cancer, implying that ABCG1 is a regulator of lung cancer stem cells.

It has been reported that in lung cancer, expression profiles of miRNA are different from those in normal lungs; however, the significance of this aberrant expression remains poorly understood. Among the miRNA reported to be downregulated in lung cancer, the miR-29 family (miR-29a, -b and -c) target the 3′-untranslated region and downregulate the expression of DNA methyltransferase (DNMT) 3a and -b, two key enzymes involved in DNA methylation, which are frequently upregulated in lung cancer and associated with poor prognosis (22,40). The present study showed that ABCG1 overexpression significantly inhibited miR-29a, -b and -c expression. A future study will detect whether ABCG1 affects DNMT 3a and -b.