AIBP and APOA-I synergistically inhibit intestinal tumor growth and metastasis by promoting cholesterol efflux
The roles played by cholesterol in cancer development and progression represent a popular field in the cancer community. High cholesterol levels are positively correlated with the risk of various types of cancer. APOA-I binding protein (AIBP) promotes the reverse cholesterol transport pathway (RCT) in cooperation with Apolipoprotein A-I (APOA-I) or high-density lipoprotein cholesterol. However, the combined effect of AIBP and APOA-I on intestinal tumor cells is still unclear.
Immunohistochemistry, western blot and qPCR were performed to investigate the expression of AIBP and APOA-I in intestinal tumor tissues and cell lines. The anti-tumor activity of AIBP and APOA-I was evaluated by overexpression or recombinant protein treatment. Cholesterol efflux and localization of lipid raft-related proteins were analyzed by a cholesterol efflux assay and lipid raft fraction assay, respectively.
Here, we reported that both AIBP expression and APOA-I expression were associated with the degree of malignancy in intestinal tumors. Co-overexpression of AIBP and APOA-I more potently inhibited colon cancer cell-mediated tumor growth and metastasis compared to overexpression of each protein individually. Additionally, the recombinant fusion proteins of AIBP and APOA-I exhibited a significant therapeutic effect on tumor growth in Apcmin/+ mice as an inherited intestinal tumor model. The synergistic effect of the two proteins inhibited colon cancer cell migration, invasion and tumor-induced angiogenesis by promoting cholesterol efflux, reducing the membrane raft content, and eventually disrupting the proper localization of migration- and invasion-related proteins on the membrane raft. Moreover, cyclosporine A, a cholesterol efflux inhibitor, rescued the inhibitory effect induced by the combination of AIBP and APOA-I.
These results indicate that the combination of APOA-I and AIBP has an obvious anticancer effect on colorectal cancer by promoting cholesterol efflux.
KeywordsAIBP APOA-I RCT Colorectal cancer Cholesterol efflux
APOA-I binding protein
membrane type I matrix metalloproteinase
Cholesterol is essential for maintaining both animal cell membrane architecture and cell signaling [1, 2]. The intestine is one of the main organs for cholesterol absorption and excretion in mammals, and aberrant regulation of cholesterol metabolism has long been linked to the gastrointestinal cancer risk [3, 4, 5]. Lipid rafts, as cholesterol-enriched plasma membranes, play an active role in the regulation of cell proliferation, apoptosis, migration and invasion, which are important biological processes involved in cancer initiation, development and progression [6, 7, 8]. Thus, many functional responses are probably caused by direct or indirect modulation of the membrane cholesterol content, which may be a potential target for anticancer therapy.
APOA-I, a major protein component of HDL, contributes to the RCT pathway and is considered a potential therapeutic agent for preventing a variety of inflammation-related diseases, including cancer [9, 10]. Clinically, the concentrations of HDL and APOA-I were found to be inversely associated with the risk of colon cancer . Genetic interference with APOA-I levels in vivo exacerbates dextran sulfate sodium (DSS)-induced colitis and colitis-associated carcinogenesis, suggesting that APOA-I plays a protective role in colorectal cancer progression . Recently, AIBP was reported to cooperate with HDL to reduce the lipid raft content of endothelial cells by accelerating cholesterol efflux, leading to restriction of cell migration and angiogenesis in vivo and in vitro [13, 14]. In another study, AIBP promoted APOA-I binding to ATP-binding cassette transporter member 1 (ABCA1) on the cell membranes of macrophages to enhance cholesterol efflux, prevented lipid accumulation and reduced foam cell formation . Early studies reported that treating enterocytes with a polyclonal antibody against AIBP inhibited [125I] HDL degradation and binding to cholesterol-loaded cells, suggesting that the synergy of AIBP and APOA-I/HDL in regulating cholesterol metabolism may be a universal phenomenon in mammalian cells . Therefore, we hypothesized that this synergy affects intestinal epithelial tumor development and cancer cells’ biological behavior.
To test the hypothesis, we first evaluated the correlation between AIBP/APOA-I expression and intestinal malignant tissues. Then, we examined the synergistic effect of AIBP and APOA-I on intestinal tumor growth and metastasis, as well as cell proliferation, viability, apoptosis, migration, and invasion and tumor-induced angiogenesis. Finally, we further explored the underlying mechanism involved. This study not only expands our understanding of AIBP and APOA-I functions but also provides some new ideas for the development of novel anticancer strategies targeting cholesterol metabolism.
All cell lines were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China, 2016). LIM1863, HIECs, HISCs and HUVECs were maintained in RPMI 1640 medium (Invitrogen Gibco, USA). Caco2, HEK293, HCT116, SW480, SW620, HT29, 841, LS174T, RKO and LOVO cells were maintained in DMEM (Invitrogen Gibco, USA). All culture media contained 10% FBS (Invitrogen Gibco, USA). All cell lines were authenticated and tested negative for mycoplasma contamination by the providers. And the information of the cell lines are listed in Additional file 2: Table S1.
Lentiviral vector construction and cell transfection
Lentiviral-based expression vectors containing full-length human AIBP, APOA-I or a negative control (NC) coding DNA sequence (CDS) driven by EF1α were provided by GenePharma (Shanghai, China). Cells were transfected according to the manufacturer’s instructions (GenePharma, China).
Colony formation and CCK-8 assays
Stably transfected cells were seeded at approximately 300 cells/well in a 6-well plate. Two weeks later, colony formation was analyzed as previously described . Moreover, cells were seeded at approximately 1000 cells/well in a 96-well plate, and CCK-8 assays were performed according to the manufacturer’s protocol (7 Sea Biotech, China).
Wound healing assay
The stably transfected cells were seeded in fibronectin-coated 6-well plates and cultured to confluence. Cells were serum-starved for 6 h and then incubated at 37 °C in 5% lipoprotein-deficient serum (AngYuBio, China) and DMEM. The wound healing assay was performed as previously described .
Migration and invasion assay
Serum-starved HCT116 cells (2 × 105 cells) were collected from the plate, washed, re-suspended in 5% LPDS/DMEM and added to the transwell (8.0-μm pore size) or another transwell coated with purified fibronectin at (20 µg/ml, Sigma) for migration and invasion assays, respectively. Subsequently, cells were seeded into transwell chambers in the presence or absence of CsA (10 µM, Sigma-Aldrich, USA). After 48 h of incubation, the cells that transmigrated onto the lower surface of the filter were stained with crystal violet and counted under a microscope (Olympus, Japan). Three independent experiments were conducted, and the data are presented as the mean ± SD.
Mouse xenografts and in vivo studies
The stably transfected cells (5 × 106 cells) were implanted into the flanks of BALB/c male nude mice (Nanjing, China). Because all lentiviral-based cells were marked by GFP, tumor growth was monitored in vivo 21 days after transplantation with a Kodak In-Vivo FX professional imaging system (Connecticut, USA). APOA-I recombinant protein (0.5 mg/kg, Sino Biological) was administered subcutaneously once every 3 days at a site away from the cell implantation location. Tumor size was determined by measuring the tumor length (a) and width (b). Tumor volume (V) was calculated according to the formula V = (ab)2/2. Additionally, we generated a recombinant protein containing AIBP fused to the C-terminal of full-length APOA-I and linked by peptides Pro-Gly-Ser-Gly-Ser-Gly, which was designated as R-AIBP + APOA-I. R-AIBP + APOA-I, recombinant APOA-I proteins (Sino Biological) and the APOA-I mimetic D-4F (Ac-DWFKAFYDKVAEKFKEAF-NH2) (OntoRes), which were used to treat the C57BL/6J-ApcMin/+ mice (Jackson Laboratory). The mice were treated intravenously with BSA (n = 8), D-4F (n = 6), APOA-I (n = 6) or the AIBP + APOA-I combination (n = 7) (0.5 mg/kg) daily until the first tumors from randomly selected mice were detectable, and the mice were then intraperitoneally injected (10 mg/animal) thrice per week until they reached 20 weeks of age. Statistical analysis of the tumor number and size was performed.
Liver metastasis assay
For liver metastasis studies, 8- to 9-week-old male BALB/c nude mice were used. The abovementioned stably transfected colon cancer cells were harvested and mixed with 50% Matrigel at a concentration of 2 × 105 cells per ml. Experimental liver metastases were generated by intrasplenic injections of 1 × 104 cells (50 µl of cell suspension). After 28 days, liver metastatic foci and mouse survival were analyzed.
Cholesterol efflux assay
The stably transfected cells (2 × 105 cells) or HCT116 cells were treated with BSA, APOA-I, AIBP or the AIBP + APOA-I combination for 6 h at 37 °C in 5% LPDS and DMEM or EBM. Then, cholesterol efflux assays were performed using the Cholesterol Efflux Fluorometric assay kit (Biovision, USA) according to the manufacturer’s instructions. The following equation was used to calculate the cholesterol efflux percentage: % Cholesterol efflux = (Fluorescence intensity of the media) / (Fluorescence intensity of the cell lysate + media) × 100.
Cell culture supernatants and mouse sera were collected and assayed using APOA-I (R&D Systems, USA) and AIBP (Aviva Systems Biology, China) ELISA kits according to the manufacturer’s protocol.
The stably transfected cells receiving treatment were plated onto 10-cm dishes at 1 × 106 cells per dish. Cells without any modifications were treated with MβCD (10 mM, Solarbio Life Sciences, China) for 20 min and washed twice with ice-cold PBS; the cytosolic fraction (1 ml) was extracted using the ProteoExtract Subcellular Proteome Extraction kit (Merck, Germany). Triton X-100 soluble materials were extracted with 500 µl of TNE buffer (25 mM Tris-HCl, 0.15 mM NaCl, 5 mM EDTA, and protease inhibitors) containing 1% Triton X-100. Insoluble materials were further extracted with 250 µl of TNE buffer containing 1% SDS. Equal amounts of protein from each fraction were analyzed by western blot.
Western blot analysis was performed with reference to a standard protocol . The following primary antibodies were used: APOA-I (Bioss, Beijing, China); integrin β1, CAV-1 and VEGFR2 (CST, Danvers, MA, USA); and β-actin and GAPDH (Beyotime, Shanghai, China). Other primary antibodies were purchased from Abcam (Cambridge, MA, USA) using the recommended concentrations in accordance with the manufacturer’s instructions.
Visualization of lipid rafts with cholera toxin B
The stably transfected cells were plated on glass coverslips and analyzed by Vybrant® Lipid Raft Labeling Kits (Invitrogen, USA) according to the manufacturer’s instructions.
IHC and immunofluorescence (IF) analysis
For experimental mouse or human intestinal and colorectal tissues, IHC and IF staining was performed as previously described [20, 21]. The study methodologies were approved by the local ethics committee, and the following primary antibodies were used: AIBP (Abcam, ab81907, 1:300 dilution), APOA-I (Bioss, bs-0849R, 1:300 dilution) and CD31 antibody (Abcam, ab28364, 1:300 dilution). The corresponding secondary antibodies were used in accordance with the manufacturer’s instructions.
Total RNA from cells or tissues was extracted using TRIzol reagent (Invitrogen, USA), and cDNA synthesis was performed using the PrimeScript™ RT reagent Kit (Takara, China). Quantitative RT-PCR was carried out using the Bio-Rad CFX96 Touch system with Real-time PCR Master Mix (SYBR Green). The PCR primers are listed in Additional file 2: Table S2.
Statistical analyses between groups were performed by two-tailed Student’s t-test to determine significance when only 2 groups were compared. Statistical comparisons among 3 or more groups were performed using one-way ANOVA followed by the Tukey test or Dunnett’s test. Differences were considered significant at the level P < 0.05 (*P < 0.05, **P < 0.005, ***P < 0.001). The data are expressed as the means ± SDs. Kaplan-Meier curves were used to compare survival times among groups. All statistical analyses was performed using SPSS 16.0.
AIBP expression and APOA-I expression are associated with the malignant degree of intestinal tumors
To further determine the correlation between AIBP/APOA-I and the malignant degree of intestinal tumors, we analyzed their expression in colon cancer cell lines with different malignant degrees. The results showed that the expression patterns of AIBP and APOA-I were inconsistent in cancer cells with low malignancy, such as HT29, SW480, and LS174-T cells, while those in relatively highly malignant cells, such as SW620 and HCT116 cells, were consistently decreased to a minimum (Fig. 1e, f).
Taken together, these results suggested that low expression levels of both AIBP and APOA-I were associated with the malignant degree of intestinal tumors, and such low expression levels may provide a favorable condition for intestinal tumor progression.
AIBP and APOA-I cooperate to inhibit tumor growth and metastasis
To further examine the role of AIBP and/or APOA-I in metastasis of colon cancer cells, the above stably transfected HCT116 cells were injected into the spleens of nude mice (Fig. 2f, g). Fewer liver metastatic foci were observed in the mice injected with HCT116-AIBP + APOA-I cells compared to the other three groups. In parallel experiments, the mean survival time of HCT116-APOA-I + AIBP-implanted mice (35.4 ± 4.0) was prolonged by 7.7 days or 4.1 days compared with that of HCT116-NC or HCT116-APOA-I implanted mice, respectively (Fig. 2h; Additional file 2: Table S5).
Taken together, these results suggested that AIBP and APOA-I synergistically played a significant inhibitory role in colon cancer cell-mediated tumor growth and metastasis.
Evaluation of the therapeutic effect of AIBP + APOA-I in ApcMin/+ mice
AIBP and APOA-I in combination inhibited cell migration, cell invasion and tumor-induced angiogenesis
The cooperative inhibitory role is dependent on accelerating the cholesterol efflux
The integrity of lipid rafts is necessary for proper localization and functioning of migration- and invasion-related proteins . Localization of integrin β1 and FAK [30, 31] or MT1-MMP and CAV1 [32, 33, 34, 35] at lipid rafts is closely related to migration and invasion, respectively. We isolated cell raft fractions, and the blot results showed that in the positive control group, MβCD treatment markedly altered the locations of the above proteins on the lipid rafts. Consistent results were obtained in the cells co-overexpressing AIBP and APOA-I, suggesting that the combination of AIBP and APOA-I simulated the effect of MβCD on membrane rafts and interfered with the migration- and invasion-related signaling pathway (Fig. 5e, f).
Collectively, our data indicate that the anti-tumor effect of AIBP and APOA-I in combination is mainly dependent on promoting the cholesterol efflux.
Recently, disruption of lipid rafts of malignant cells has been considered an essential strategy for the prevention and treatment of cancer . In this study, we found that low expression levels of AIBP and APOA-I are associated with the degree of malignancy in intestinal tumors. Combined with AIBP and APOA-I inhibited tumor growth and metastasis as well as cell migration, invasion and tumor-induced angiogenesis. Mechanically, cholesterol efflux promoted by AIBP + APOA-I interfered with the raft-related signaling pathway.
Prior studies have shown that AIBP often cooperates with APOA-I to regulate the RCT pathway in various types of cells. However, the expression patterns of AIBP and APOA-I in malignant tissues remain poorly understood. In this study, we found that both AIBP expression and APOA-I expression decreased gradually with progression from an adenoma to an advanced tumor. In this process, the decreasing rates of AIBP and APOA-I levels are not entirely synchronized. For example, in low-malignancy adenoma or colon cancer cells, the expression of one of these proteins was always significantly higher or lower than that of the other. However, in advanced tumors or high-malignancy colon cancer cells, the expression of both proteins was consistently decreased to a minimum. These results suggested that simultaneous low expression levels of both proteins likely represent a critical step in the malignant transformation of tumor cells. Therefore, the expression levels of AIBP and APOA-I can be used as an indicator of malignancy of intestinal cancer.
APOA-I was reported to have anti-tumor activity in some xenograft tumor models [10, 36], but its therapeutic effect is limited in the inherited tumor model . Consistently, we found that APOA-I treatment alone can inhibit colon cancer cell-mediated tumor growth to a certain extent but has no apparent inhibitory effect on tumor growth in Apcmin/+ mice. However, the addition of AIBP enhanced the anti-tumor effect of APOA-I both in xenograft and inherited tumor models. Similar to in vitro studies, APOA-I treatment alone can induce cholesterol efflux to a certain degree but was insufficient to produce significant changes in cellular behavior due to a lower rate of cholesterol efflux. In contrast, AIBP hardly promoted cholesterol efflux when APOA-I was absent due to the lack of a cholesterol acceptor. Thus, APOA-I is necessary for cell cholesterol efflux, whereas AIBP serves as an accelerator to augment this effect triggered by APOA-I. These results suggested that the combined anti-tumor effect is closely related to enhanced cholesterol efflux. Whether AIBP in combination with APOA-I promotes other anti-tumor pathways, such as anti-inflammatory or anti-oxidation pathways, requires further study.
Cell migration and invasion are essential processes for cancer metastasis. Assembly of the integrin-based adhesion structure is critical for effective cell movement. Recent evidence suggests that integrin β1 clustering and functioning are regulated by membrane rafts, which provide a large platform for assembling different proteins to facilitate the migration-related signaling pathway . Moreover, MT1-MMP, as one of the raft-affiliated matrix metalloproteinases (MMPs), has been shown to contribute to the invasive abilities of tumor cells by activating pro-MMP-2 [32, 38, 39]. In the present study, AIBP combined with APOA-I promoted cholesterol efflux from the membrane and directly affected the tight packing and stabilization of lipid rafts, thus impairing proper localization of integrin β1 and MT1-MMP and eventually migration- and invasion-related signaling cascades. On the other hand, the combination of AIBP and APOA-I inhibited tumor-induced angiogenesis, which is an essential process for tumor cell growth and metastasis. Therefore, the combination of AIBP and APOA-I not only restricts cancer cell migration and invasion but also blocks hematogenous metastasis.
APOA-I-dependent cholesterol efflux involves a Ca2+-dependent endocytic pathway, followed by recycling and subsequent release of the nascent lipoprotein particle from the cell [40, 41]. Therefore, CsA acts as a potent inhibitor of cholesterol efflux by suppressing the internalization of APOA-I [42, 43]. Here, CsA treatment compromised the inhibitory role of AIBP combined with APOA-I in colon cancer cell-mediated tumor growth as well as cell migration, invasion and tumor-induced angiogenesis. These results suggested that the anti-tumor activity of AIBP combined with APOA-I is dependent on cholesterol efflux.
In this study, we found that the combination of AIBP and APOA-I exerts a significant inhibitory effect on intestinal tumor growth and liver metastasis. This effect was achieved by promoting cholesterol efflux and subsequent suppression of the lipid raft-associated signaling pathway. Taken together, these findings suggest that AIBP and APOA-I in combination serve as a natural cholesterol-depleting agent and could be of therapeutic value in preventing metastasis of human CRC and potentially other cancers.
We thank Mr. Chi Liu at the Department of Nephrology, Xinqiao Hospital for assistance with editing the manuscript. We thank Mr. Xiao Yang at the State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China for useful suggestions.
TZ carried out all experiments, performed the statistical analysis. TZ and GW drafted the manuscript. QW and YW participated in the mouse studies, especially the xenograft model and metastatic model with the indicated cells. FW, JW and YS conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.
This study was partially supported by grants from the National Natural Science Foundation of China (11572064, 31771599), the National Key Research and Development Program of China (2016YFC1102305, 2016YFC1101101), and the Fundamental Research Funds for the Central Universities(106112017CDJZRPY0012, 106112017CDJXY230002) as well as the Public Experiment Center of the state Bioindustrial Base (Chongqing), China.
Ethics approval and consent to participate
The study protocol was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Chongqing Third Military Medical University. Informed consent was received from all patients included in the current study or their direct relatives. The animal study was approved by Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University.
Consent for publication
The authors declare that they have no competing interests.
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