VLDL and LDL, but not HDL, promote breast cancer cell proliferation, metastasis and angiogenesis

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Cancer Letters xxx (2016) 1e9

Contents lists available at ScienceDirect

Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Original Articles

Q4 Q3

VLDL and LDL, but not HDL, promote breast cancer cell proliferation, metastasis and angiogenesis Chun-Wun Lu a, Yi-Hsuan Lo a, Chu-Huang Chen b, c, d, e, Ching-Yi Lin a, Chun-Hao Tsai a, f, Po-Jung Chen a, Yi-Fang Yang a, Chie-Hong Wang a, Chun-Hsiang Tan g, Ming-Feng Hou h, i, j, k, *, Shyng-Shiou F. Yuan a, f, l, ** a

Translational Research Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Lipid Science and Aging Research Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Lipid Science and Aging Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan d Lipid and Glycoimmune Research Center, Changhua Christian Hospital, Changhua, Taiwan e Vascular and Medicinal Research, Texas Heart Institute, Houston, TX 77030, USA f Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan g Department of Neurology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan h Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan i Division of General and Gastroenterological Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan j National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan k Kaohsiung Municipal Siaogang Hospital, Kaohsiung, Taiwan l Department of Obstetrics and Gynecology, Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 September 2016 Received in revised form 24 November 2016 Accepted 24 November 2016

Abnormal lipoprotein profiles are associated with breast cancer progression. However, the mechanisms linking abnormal lipoprotein levels to breast cancer progression, especially metastasis, remain unclear. Herein, we found that L1 and L5 subfractions of LDL and VLDL, but not HDL, enhanced breast cancer cell viability. L1, L5, and VLDL also increased the in vitro tumorigenesis of breast cancer cells in anchorageindependent soft agar assay. In addition, L1, L5, and VLDL, but not HDL, increased the levels of mesenchymal markers Slug, Vimentin, and b-Catenin, and promoted breast cancer cell migration and invasion. L1, L5, and VLDL increased Akt Ser473 phosphorylation and promoted cell migration, which were reversed by the PI3K/Akt inhibitor wortmannin. Further in vitro angiogenesis assay and cytokine array analysis demonstrated that L1, L5, and VLDL enhanced secretion of angiogenic factors in breast cancer cells and promoted angiogenic activity. However, only VLDL reduced anchorage-dependent cell death and promoted lung metastasis in nude mice. In summary, our data suggest that L1, L5, and especially VLDL promote breast cancer progression and metastasis through Akt-induced EMT and angiogenesis, and provide a novel mechanism of how dyslipoproteinemia promotes breast cancer progression. © 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Breast cancer Lipoprotein Metastasis VLDL LDL

Introduction

* Corresponding author. Kaohsiung Municipal Hsiao-Kang Hospital, No. 482, Shanming Rd., Siaogang Dist., Kaohsiung City 812, Taiwan. Fax: þ886 7 8065068. ** Corresponding author. Translational Research Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 100, Shih-Chuan 1st Road, Kaohsiung, 80708, Taiwan. Fax: þ886 7 3112493. E-mail addresses: [email protected] (M.-F. Hou), [email protected] (S.-S.F. Yuan).

Breast cancer is the most common malignancy among women in developed countries and its prevalence is increasing worldwide. Although recent advances in cancer therapy have been remarkable, breast cancer still ranks as the second leading cause of cancer death in developed countries [1]. To provide a basis for new therapeutic strategies, we investigated the mechanisms underlying the roles of different lipoproteins, namely low-density lipoprotein (LDL), very low density lipoprotein (VLDL) and high-density lipoprotein (HDL), in breast cancer growth and metastasis.

http://dx.doi.org/10.1016/j.canlet.2016.11.033 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: C.-W. Lu, et al., VLDL and LDL, but not HDL, promote breast cancer cell proliferation, metastasis and angiogenesis, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.11.033

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Among the known risk factors for breast cancer, obesity is an important risk factor and considered to be strongly correlated with a poor survival rate in breast cancer patients, especially in postmenopausal women [2,3]. Obesity is often accompanied by abnormal lipid and lipoprotein profiles, such as elevated LDL cholesterol (LDL-C) and VLDL cholesterol (VLDL-C) [4,5]. In addition, epidemiological studies have shown a strong relationship between breast cancer and lipid disorders, including elevated total cholesterol [6e11], VLDL-C [10,12,13], LDL-C [8,10], and decreased HDL cholesterol (HDL-C) [6,10,12]. However, the exact mechanisms of how dyslipidemia/dyslipoproteinemia promotes breast cancer development and progression remain elusive. LDL promotes breast cancer progression by inducing cell proliferation, migration and loss of adhesion in breast cancer cells [14,15]. With anion exchange chromatography, LDL lipoprotein complex can be resolved into five charge-based subfractions, L1eL5, with L5 being the most electronegative and atherogenic [16e18]. Previous reports have shown that L1 and L5 have opposite effects on blood vessels. L5, but not L1, induces endothelial cell apoptosis and atherosclerosis, and inhibits the differentiation of endothelial progenitor cells [19,20]. While the roles of L1 and L5 in breast cancer progression remain unclear, a recent report demonstrated that VLDL, but not LDL, promotes mammosphere formation and radiation resistance in inflammatory breast cancer cells [21]. Cancer development and progression involve multiple processes, including proliferation, metastasis, and angiogensis [22]. Obesity and dyslipidemia/dyslipoproteinemia are major contributors to the increased breast cancer maligancy [23e25]. However, the exact roles of lipoproteins in modulating cancer behaviors remain mostly unclear. In this study, we investigated the effects of lipoproteins on breast cancer progression and the underlying mechanisms in vitro and in vivo. Materials and methods Cell culture Breast cancer cell lines MCF7, HS578T, MDA-MB-468, and MDA-MB-231, and human umbilical vein endothelial cells (HUVEC) were all purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). All of them were maintained in Dulbecco's modified eagle medium (DMEM) (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Beit Haemek, Israel), 100 U/mL penicillin (Biological Industries, Beit Haemek, Israel), 100 ug/mL streptomycin (Biological Industries, Beit Haemek, Israel), and 2.5 mg/mL amphotericin B (Biological Industries, Beit Haemek, Israel). HUVECs were maintained in EndoGRO-LS Complete Culture Media Kit (SCME001, Milipore, Billerica, MA, USA). Cells were incubated at 37  C in a humidified 5% CO2 incubator and medium was changed every other day. MDA-MB-231 cells stably expressing luciferase (MDA-MB-231-Luc) were kindly provided by Prof. Wen-Chun Hung at National Health Research Institutes, Taiwan. Lipoproteins L1, L5, VLDL, and HDL were provided by Lipid Science and Aging Research Center, Kaosiung Medical University, Taiwan. All the lipoproteins were isolated from the blood plasma of patients with cardiovascular disease or metabolic syndrome by sequential ultracentrifugation according to density. L1 and L5 were further fractionated by using fast protein liquid chromatography with anion-exchange columns [16,18]. Cell viability assay Cell viability was determined by XTT colorimetric assay according to manufacturer's instructions (SigmaeAldrich, 147 St Louis, MO, USA). In brief, MCF7 and MDAMB-231 cells were treated with L1, L5, VLDL, or HDL for 72 h, and followed by XTT viability assay. For wortmanin study, cells were pretreated with wortmannin (400 nM) for 2 h and followed by lipoproteins treatment.

24-well plate containing DMEM with 10% FBS. After 24 h, cells were fixed and stained by crystal violet solution (0.05% crystal violet, 1% formadehyde, 1% methanol in 1X PBS). Those cells which did not migrate or invade were removed with cotton swabs. The migrated or invasive cells were imaged by a Nikon SMZ745T dissection microscope (Tokyo, Japan) and quantified with the ImageJ software. Western blot Equal amounts of proteins were resolved by SDS-PAGE and transferred to a polyvinyl difluoride (PVDF) membrane. PVDF membrane was blocked with 5% skim milk at room temperature for 1 h, and then incubated with anti-b-catenin (ab16051, abcam, Cambridge, MA), anti-Slug (PA5-20290, ThermoFisher, USA), anti-Vimentin (GTX100619, GeneTex, Irvine, CA), anti-phospho-Akt (S473) (4060S, Cell Signaling Technology; Beverly, MA, USA), anti-Akt (4691, Cell Signaling Technology; Beverly, MA, USA), or anti-b-Actin (GTX110564, GeneTex, Irvine, CA) antibodies at 4  C for 16e18 h. After washing with 1X TBST (Tris Buffered Saline with Tween 20), the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000, PerkinElmer, Boston, MA) at room temperature for 1 h and then exposed by using Enhanced chemiluminescence (ECL, PerkinElmer, Waltham, MA). The images were captured by ChemiDoc XRS þ System (Bio-Rad Laboratories, 164 Hercules, CA, USA) and quantified with the ImageJ software. In vitro matrigel tube formation assay HUVECs were incubated with 25% conditioned medium (CM) from MCF-7 or MDA-MB-231 cells pretreated with L1, L5, VLDL, or HDL for 48 h. The cells were then seeded onto 96-well plates containing 100 ml matrigel (356231, BD Biosciences, CA, USA) and incubated for 2e3 h at 37  C in a humidified 5% CO2 incubator. The images were captured by a Nikon Eclipse TS100 microscope (Tokyo, Japan) and quantified with the ImageJ software. Human angiogenesis antibody array The levels of angiogenesis-related proteins in the CM from MDA-MB-231 cells treated with L1, L5, or VLDL were analyzed by human angiogenesis antibody array (ARY007, R&D Systems Inc., USA). Briefly, array membranes were incubated with CM at 4  C overnight and further processed according to the manufacturer's instructions. Soft agar clonogenic assay A two-layer soft agar system was performed for soft agar clonogenic assay. Briefly, DMEM mixed with 0.6% agar (Sigma, St Louis, MO) was paved onto 6-well plates (Corning Life Sciences) and allowed to solidify (as base agar). Then MDAMB-231 cells, pretreated with L1, L5, VLDL, or HDL for 48 h, were mixed with 0.3% agarose in DMEM with 10% FBS and overlaid onto the base agar. During the 15 days incubation period, medium change with 1 ml of MEM supplemented with 10% FBS was done every 2e3 days. The colonies were then fixed and stained with crystal violet solution (0.05% crystal violet, 1% formadehyde, 1% methanol in 1X PBS), followed by destaining with ddH2O till the background was clear. The colonies were photographed and scored by a Nikon SMZ745T dissection microscope (Tokyo, Japan) and ImageJ software. Only colonies with sizes larger than 1 mm in diameter were counted. Three independent experiments were performed. Anoikis assay MDA-MB-231 cells were treated with L1, L5, or VLDL for 48 h. Then cells were trypsinized into a single cell suspension and seeded onto ultra-low attachment 24well plates (1.25  104 cells/ml) (Corning, NY, USA) in DMEM with 10% FBS. After 48 h incubation at 37  C, cell viability was determined by staining with 0.4% trypan blue in PBS (ratio of 1:1) before examination under a Nikon Eclipse TS100 microscope (Tokyo, Japan). Tail vein injection and in vivo imaging system (IVIS) Six-week old female immunodeficient mice (BALB/cAnN.Cg-Foxn1nu/CrlNarl) were purchased from the National Laboratory Animal Center (NLAC, Taiwan). MDAMB-231 cells with luciferase expression (MDA-MB-231-Luc) were pretreated with L1, L5, or VLDL for 48 h, followed by tail vein injection (1  106 cells/100 mL PBS) into mice. Tumor volume and lung metastasis were analyzed by IVIS-50 optical imaging system (Caliper Life Sciences, Hopkinton, MA, USA) after injection of 150 mg/kg Dluciferin (PerkinElmer, Waltham, MA, USA) periodically. The images were acquired and analyzed by Xenogen Living Image software (Caliper Life Sciences). The animal study was approved by the Institutional Animal Care and Use Committee (IACUC No. 104096) of Kaohsiung Medical University, Taiwan. Immunohistochemistry (IHC)

Transwell migration and invasion assays Breast cancer cells were seeded onto 6-well plates and serum starved for 24 h before lipoprotein treatment. Cells were treated with L1 (5 or 10 mg/ml), L5 (5 or 10 mg/ml), VLDL (10 or 25 mg/ml), or HDL (10 or 25 mg/ml) for 48 h. After treatment, cells were suspended in serum-free DMEM and transferred into migration chambers or matrigel invasion chambers with 8 mm pores (Corning, NY, USA) assembled on a

The IHC procedure was performed as previously described [26]. The primary antibodies used in this study included anti-Slug (GTX30813, GeneTex, CA, USA), antiKi67 (orb247042, Biorbyt, CA, USA), and anti-phospho-Akt (S473) (#3787S, Cell Signaling Technology; Beverly, MA, USA). The IHC images were acquired by a Nikon Eclipse E600 microscope (Tokyo, Japan). For tumor micrometastases in mouse lung tissues, the total number of cell aggregations with positive staining for Ki67 was

Please cite this article in press as: C.-W. Lu, et al., VLDL and LDL, but not HDL, promote breast cancer cell proliferation, metastasis and angiogenesis, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.11.033

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C.-W. Lu et al. / Cancer Letters xxx (2016) 1e9 calculated from every section (S6 sections/mouse). For the scoring of protein expression in mouse lung tissues, the stainings of Slug and phosphorylated Akt (pAkt) were stratified into 4 scores (0, absent; 1, low; 2, intermediate; 3, high) on the basis of intensity (Supplementary Fig. S1). Statistical analysis GraphPad Prism 5 (GraphPad Software, La Jolla, CA) was used for statistical analysis. All the data were shown as mean ± SEM. Student's t-test (for comparing two groups) and one-way ANOVA (for comparing more than two groups) were used to compare significant differences for all the results. Significant difference was set at *P < 0.05; **P < 0.01. ***P < 0.001.

Results

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like cells; more aggressive and metastatic breast cancer subtype) were treated with L1, L5, VLDL, or HDL for 72 h. Then cell viability was determined by the XTT assay. In MCF-7 cells, both L1 and L5 had no effect on cell viability (Fig. 1A, upper panel). On the contrary, VLDL enhanced cell viability, while HDL reduced cell vibility (Fig. 1A, lower panel). In MDA-MB-231 cells, L1, L5, and VLDL enhanced cell viability, while HDL had no effect (Fig. 1B). Soft agar clonogenic assay further demonstrated that L1, L5, and VLDL, but not HDL, enhanced anchorage-independent growth, a hallmark of oncogenic transformation, in MDA-MB-231 cells (Fig. 1C). L1, L5, and VLDL, but not HDL, increased breast cancer cell motility via Akt/Slug pathway

L1, L5, and VLDL, but not HDL, enhanced breast cancer cell viability To investigate the effects of lipoproteins on the viability of breast cancer cells, MCF-7 (luminal-like cells) and MDA-MB-231 (basal-

To investigate the effects of lipoproteins on breast cancer cell motility, MCF-7 and MDA-MB-231 cells were treated with lipoproteins for 48 h, followed by transwell cell migration and invasion

Fig. 1. The effects of lipoproteins on the cell viability of breast cancer cells. MCF7 (A) or MDA-MB-231 cells (B) were treated with lipoproteins followed by the XTT assay. C, MDA-MB231 cells were treated with L1 (10 mg/ml), L5 (10 mg/ml), VLDL (25 mg/ml), or HDL (25 mg/ml) for 48 h, followed by the soft agar assay. Representative figures (left panel) and quantitative results (right panel) from three independent experiments showing the number of colonies formed on the soft agar. Scale bars represent 1 mm. Data were shown as mean ± SEM from three independent experiments. Asterisks indicate significant difference from control (0 mg/ml); *, P < 0.05. **, P < 0.01. ***, P < 0.001.

Please cite this article in press as: C.-W. Lu, et al., VLDL and LDL, but not HDL, promote breast cancer cell proliferation, metastasis and angiogenesis, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.11.033

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assays. In MCF-7 cells, VLDL and L5 increased cell migration by 2fold and 1.5-fold respectively (Fig. 2A, upper panel). In contrast, L1 and HDL had no significant effect on cell migration (Fig. 2A, upper panel). Similar to the cell migration result, VLDL had the highest enhancing effect on cell invasion (4.5-fold and 7-fold increase after 10 mg/ml and 25 mg/ml VLDL treatment respectively), while L1 or L5 had only minor enhancing effects and HDL decreased cell invasion to 0.5-fold (Fig. 2A, lower panel). In MDA-MB-231 cells, both cell migration and invasion increased more than 2fold, when treated with VLDL or L5 (Fig. 2B). On the contrary, there were no significant changes in cell migration or invasion when MDA-MB-231 cells were treated with HDL (Fig. 2B). While L1 increased MDA-MB-231 cell migration 2-fold, it had no effect on cell invasion (Fig. 2B). Furthermore, L1, L5, and VLDL also increased cell migration in two other basal-like breast cancer cell lines, MDAMB-468 and HS578T (Supplementary Fig. S2). Next, we investigated the underlying mechanisms of how these lipoproteins regulate breast cancer cell behaviors. We found that L1,

L5, and VLDL increased the phosphorylation of Akt at S473 in MDAMB-231 cells (Fig. 3A). To investigate whether these lipoproteins regulated breast cancer cell behaviors via Akt phosphorylation, MDA-MB-231 cells were treated with wortmannin, a PI3K inhibitor, to inhibit lipoprotein-induced Akt phosphorylation (Fig. 3B). While wortmannin had no effect on lipoproteins-enhanced cell viability (Fig. 3C), it reversed lipoprotein-induced cell migration in MDAMB-231 cells (Fig. 3D). In addition, three mesenchymal markers, including b-Catenin, Slug, and Vimentin, were increased after L1, L5, or VLDL treatment (Fig. 3E). Wortmannin treatment also reversed L1-, L5-, or VLDL-induced Slug expression (Fig. 3F). Other than Akt, we also studied the phosphorylation status of other kinases after lipoprotein treatment. L1, L5, or VLDL treatment increased the phosphorylation of Stat3 at Y705, but not the phosphorylation of Erk, p38, or NF-kB (Supplementary Fig. S3A, data not shown). However, inhibition of Stat3 phosphorylation by stattic, a Stat3 inhibitor, did not reverse the cell viability and migration induced by the lipoproteins (Supplementary Figs. S3B and S3C).

Fig. 2. The effects of lipoproteins on breast cancer cell migration and invasion. MCF-7 (A) and MDA-MB-231 cells (B) were pretreated with lipoproteins for 48 h, followed by the transwell migration or invasion assay. Scale bars represent 100 mm. Data were shown as mean ± SEM from three independent experiments. Asterisks indicate significant difference from control (0 mg/ml); *, P < 0.05. **, P < 0.01. ***, P < 0.001.

Please cite this article in press as: C.-W. Lu, et al., VLDL and LDL, but not HDL, promote breast cancer cell proliferation, metastasis and angiogenesis, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.11.033

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Fig. 3. Akt phosphorylation and EMT were involved in L1, L5, or VLDL-induced breast cancer cell migration. MDA-MB-231 cells were pretreated with/without a PI3K inhibitor, wortmannin (400 nM), for 1 h, followed by treatment with L1 (10 mg/ml), L5 (10 mg/ml), or VLDL (25 mg/ml) for another 48 h. Then the protein levels, cell viability, and migration were determined by Western blot, XTT assay, and transwell migration assay respectively. A & B, Representative and quantitative results showing the protein levels of phosphorylated Akt at S473 after treatment. C, A representative figure from three dependent experiments showing the cell viability as determined by the XTT assay. D, Representative and quantitative results showing the migratory ability of MDA-MB-231 cells. E & F, Representative and quantitative results showing the levels of b-Catenin, Slug, and Vimentin by using Western blot. b-Actin was used as an internal control. N S 3. Data were shown as mean ± SEM. Asterisks indicate significant difference from control; *, P < 0.05. **, P < 0.01. ***, P < 0.001.

L1, L5, and VLDL enhanced the breast cancer cells-induced angiogenesis Angiogenesis is a critical step for cancer progression. Previous studies have shown that breast cancer cells secrete angiogenic factors to promote metastasis [27]. In this study, we investigated whether lipoproteins regulated the angiogenic effect of breast cancer cells. We first examined the direct effects of lipoproteins on HUVEC endothelial cell viability. In agreement with a previous study, only L5 significantly decreased HUVEC cell viability [28] (Fig. 4A). However, CM from L1-, L5-, or VLDL-treated MDA-MB231 cells increased the cell viability of HUVECs (Fig. 4B). On the contrary, CM from HDL-treated MDA-MB-231 cells decreased the cell viability of HUVECs (Fig. 4B). Moreover, microvessel tube formation increased by 1.5-folds when HUVEC cells were treated with CM from MDA-MB-231 cells that were pretreated with L1-, L5-, or VLDL, but not HDL (Fig. 4C and Supplementary Fig. S4A). Similarly, the cell viability and microvessel tube formation of HUVECs were also increased by CM from MCF-7 cells treated with L1, L5, or VLDL, but not HDL (Fig. 4D and E). To identify the angiogenic factors involved in this CM-induced angiogenesis,

a human angiogenesis antibody array was applied. We found that 1 out of 55 angiogenesis-related proteins increased at least 2-fold in the L1, L5, or VLDL-treated CM from MDA-MB-231 cells compared with the control CM (Fig. 4F and G). The levels of amphiregulin (AREG) increased in the L1-, L5-or VLDL-treated CM compared with the control CM (Fig. 4G). VLDL decreased anoikis in vitro and promoted lung metastasis of breast cancer cells in vivo Anoikis, an anchorage-independent form of cell growth and resistance to cell death induced by loss of contact with extracellular matrix or neighboring cells, is essential for cancer cells to disseminate through the body and form metastases [29]. Herein, we found that anoikis was only decreased following VLDL treatment, but not L1 or L5 treatment (Fig. 5A). To further study the in vivo effects of L1, L5, and VLDL on cancer cell metastasis, MDA-MB-231 cells carrying the luciferase expression gene were preincubated with/without L1, L5, or VLDL, and then injected into the tail vein of nude mice. Two weeks after tail vein injection, we noticed a 1.5-fold increase of luminescence signal in the lungs of mice injected with VLDL-

Please cite this article in press as: C.-W. Lu, et al., VLDL and LDL, but not HDL, promote breast cancer cell proliferation, metastasis and angiogenesis, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.11.033

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Fig. 4. L1, L5, and VLDL increased the angiogenic activity of breast cancer cells. A, HUVEC cells were treated with lipoproteins for 72 h, and cell viability was determined by the XTT assay. B and C, MDA-MB-231 cells were treated with lipoproteins for 48 h and the conditioned media (CM) were collected. HUVEC cells were treated with 25% CM from MDA-MB- Q2 231 cells treated with L1 (10 mg/ml), L5 (10 mg/ml), VLDL (25 mg/ml), or HDL (25 mg/ml), followed by XTT assay after 72 h (B), or in vitro matrigel tube formation assay after 48 h (C). C, Quantitative results for tube formation expressed as length of microvessel-like structures after normalization to control (0 mg/ml) from three independent experiments. D and E, Similar to previously described, CM were collected from MCF-7 cells treated with lipoproteins for 48 h. HUVEC cells were treated with 25% CM from MCF-7 cells treated with L1 (10 mg/ml), L5 (10 mg/ml), VLDL (25 mg/ml), or HDL (25 mg/ml), followed by XTT assay after 72 h (D) or in vitro matrigel tube formation assay after 48 h (E). F, The human angiogenesis array analysis. The CM collected from three independent experiments were used for the analysis, according to the manufacturer's instructions. Each spot in duplicate indicates a different cytokine. G, The quantitative results from human angiogenesis array showed the blot density. 1 & 2: reference spots. 3: amphiregulin (AREG). Data were shown as mean ± SEM. Asterisks indicate significant difference from control (0 mg/ml); *, P < 0.05. **, P < 0.01. ***, P < 0.001.

pretreated, but not L1-or L5-pretreated MDA-MB-231 cells (Fig. 5B). The luminescence signals in the lungs of mice increased in a VLDL dosage-dependent manner (Fig. 5C). In addition, the number of tumor micrometastases in lungs, determined by positive Ki67 staining, was higher in mice injected with MDA-MB-231 cells preincubated

with VLDL (Fig. 5D). The levels of Slug and phosphorylated Akt were also higher in the lung micrometastases of mice injected with MDAMB-231 cells preincubated with VLDL (Fig. 5E and F). Moreover, the level of Slug was positively correlated with the level of phosphorylated Akt in the mouse lung tissue (Fig. 5G).

Please cite this article in press as: C.-W. Lu, et al., VLDL and LDL, but not HDL, promote breast cancer cell proliferation, metastasis and angiogenesis, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.11.033

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Fig. 5. VLDL increased the malignancy and metastasis of breast cancer cells in vitro and in vivo. MDA-MB-231 cells were treated with L1, L5, or VLDL for 48 h, followed by anoikis assay (A) or injection into nude mice through tail vein (B & C). A, Quantitative results from three independent experiments in triplicate showing the percentage of cells that died after suspension culture. B, Representative luminescence images and quantification of nude mice injected with MDA-MB-231 cells pretreated with L1 (10 mg/ml), L5 (10 mg/ml), or VLDL (10 mg/ml) (N S 6). C, Quantitative results showing the relative level of luminescence in the nude mice injected with MDA-MB-231 cells pretreated with VLDL (N S 6). D-G, IHC results showing the number of lung nodules (D) and the levels of Slug and phosphorylated Akt (E & F) in the lungs of nude mice injected with MDA-MB-231 cells pretreated with VLDL via tail vein. Scale bars represent 50 mm. G, The correlation between Slug and phosphorylated Akt levels in the lungs of nude mice injected with MDA-MB-231 cells pretreated with VLDL. Data were shown as mean ± SEM. Asterisks indicate significant difference from control (0 mg/ml); *, P < 0.05. **, P < 0.01. ***, P < 0.001.

Discussion From epidemiological studies, metabolic syndrome, including obesity and dyslipidemia/dyslipoproteinemia, is closely associated with the incidence of various cancers, including breast cancer [28,30]. Highly proliferative cancer cells increase lipid biosynthesis, lipid uptake and abnormal cholesterol accumulation to overcome the requirement for cholesterol [31e33]. However, the underlying mechanisms of how metabolic syndrome regulates cancer development and progression, especially for metastasis, remain largely unclear. In this study, we provide new evidence for the involvement of lipoproteins, especially VLDL, in breast cancer progression by promoting cell proliferation, cell migration/invasion and angiogenesis. The transport of cholesterol in the circulation is mediated by its packaging into lipoproteins. Lipoproteins not only act as transporters, but are also involved in several physiological and pathological processes such as uptake of triacylglycerols or cholesterol into cells, resulting in lipoprotein abnormality and atherosclerosis.

Previous studies have shown abnormal lipoprotein profiles in breast cancer patients, with the levels of LDL-C and VLDL-C increased and the level of HDL-C decreased [10,12,13]. In addition, LDL-C and VLDL-C may promote breast cancer development through increased proliferation, migration, or drug resistance of breast cancer cells [14,15,21], but the underlying mechanisms remain undisclosed. In this study, we provide new insights into how LDL and VLDL regulate breast cancer metastasis. Metastasis not only requires increased cell migration and invasion ability, but also requires the support of blood vessels to facilitate the transportation of cancer cells to other organs. Therefore, angiogenesis is also of crucial importance in metastasis. It has been known that LDL and VLDL are associated with endothelial dysfunction and cardiovascular disease [34,35]. LDL induces endothelial apoptosis through L5, but not L1 [36e38]. VLDL also causes apoptosis in HUVECs but only under the help of THP-1 monocytes [39]. In addition, loss of VLDL receptor promotes angiogenesis in retinal vascular endothelial cells [40]. Therefore, LDL and VLDL are regarded as “bad cholesterol” for the vascular

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system. In this study, we found that conditioned media from breast cancer cells treated with L1, L5, and VLDL significantly increased angiogenesis. L1, L5, and VLDL increased the secretion of an angiogenic factor, AREG, from breast cancer cells (Fig. 4). Knockdown of AREG in human breast cancer cells reduces tumor vascularization and the expression of TGFb1, a proangiogenic factor [41,42]. As a result, the formation of capillary-like structures in HUVECs was enhanced by CM from breast cancer cells pretreated with L1, L5, or VLDL (Fig. 4C and E, and Supplementary Fig. S4). Therefore, these data provide the first evidence for the involvement of AREG in the angiogenic activity of L1, L5 and VLDL. Previous studies have shown that LDL increases cell proliferation in both ERþ and ER breast cancer cells [14,15] and HDL only increases the proliferation of ER cells [43,44], while the effects of L1, L5, and VLDL on the proliferation of breast cancer cells have not been addressed before. In this study, we found that lipoproteins regulate cell proliferation in breast cancer cells. In MCF7 cells (ERþ, PRþ, HER2), only VLDL increased the number of viable cells, while HDL decreased the number of viable cells (Fig. 1A). In MDA-MB-231 cells (ER, PR, HER2), L1, L5 and VLDL all increased the number of viable cells (Fig. 2B). However, the viability of MDA-MB-231 cells was not affected by HDL in our study (Fig. 1B), in disagreement with previous studies [43,44]. These conflicting results may be due to the different experimental settings, working concentrations of lipoproteins, racial differences in the source of lipoproteins, or cell lines used. In this study, we also showed that L1, L5, and VLDL promoted anchorageindependent growth of breast cancer cells on soft agar (Fig. 1C). Together, our data, in accordance with previous reports, suggest that LDL and VLDL promote breast cancer cell proliferation and in vitro tumorigenesis. In summary, our study demonstrated that VLDL, L5 and L1, but not HDL, promoted breast cancer cell aggressiveness through enhancing cell migration/invasion, angiogenic activity, and anchorage-independent growth. However, only VLDL provided survival advantage in anchorage-independent condition and promoted lung metastasis in vivo (Supplementary Fig. S5). This study provides clear evidence linking lipoproteins with breast cancer aggressiveness, and future efforts targeting VLDL are warranted for control of breast cancer metastasis. Acknowledgements This work was supported by grants from Kaohsiung Medical University Hospital (KMUH102-2T07, KMUH103-3R28, KMUH1044R29), Kaohsiung Medical University (KMU-DT103010, KMUDT105008, KMU-DT106004, KMU-TP103D18, KMU-TP104D17), and Ministry of Health and Welfare (MOHW104-TDU-B-212-124-003, MOHW105-TDU-B-212-134007, Health and welfare surcharge of tobacco products) of Taiwan. Conflicts of interest No potential conflicts of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.canlet.2016.11.033. References [1] American Cancer Society, Breast Cancer Facts and Figures, American Cancer Society, 2016.

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