What Is The Role Of Cholesterol In Animal Cell Membranes?

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• Published: 20 February 2019

Cholesterol content in prison cell membrane maintains surface levels of ErbB2 and confers a therapeutic vulnerability in ErbB2-positive chest cancer

• Qiong Li^{one,two  na1},
• Yueguang Wu¹,
• Duchuang Wang¹,
• Lu Xu²,
• Yang Zhang¹,
• Shanshan Wang¹,
• Taishu Wang^one,
• Fang Liu^ane,
• Mohamed Y. Zaky^one,three,
• Shuai Hou¹,
• Shuyan Liu¹,
• Kun Zou⁴,
• Haixin Lei^one,
• Lijuan Zou^two,
• Yingqiu Zhangⁱ &
• Han Liu ORCID: orcid.org/0000-0002-0014-4675 ^1,5

Cell Communication and Signaling volume 17, Commodity number:fifteen (2019) Cite this article

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Abstruse

Background

ErbB2 overexpression identifies a subset of breast cancer as ErbB2-positive and is frequently associated with poor clinical outcomes. As a membrane-embedded receptor tyrosine kinase, cell surface levels of ErbB2 are regulated dynamically past membrane physical properties. The present written report aims to investigate the influence of membrane cholesterol contents on ErbB2 condition and cellular responses to its tyrosine kinase inhibitors.

Methods

The cholesterol abundance was examined in ErbB2-positive breast cancer cells using filipin staining. Cellular ErbB2 localizations were investigated by immunofluorescence with contradistinct membrane cholesterol contents. The inhibitory effects of the cholesterol-lowering drug lovastatin were assessed using cell proliferation, apoptosis, immunoblotting and immunofluorescence assays. The synergistic effects of lovastatin with the ErbB2 inhibitor lapatinib were evaluated using an ErbB2-positive breast cancer xenograft mouse model.

Results

Membrane cholesterol contents positively correlated with prison cell surface distribution of ErbB2 through increasing the rigidity and decreasing the fluidity of jail cell membranes. Reduction in cholesterol abundance assisted the internalization and deposition of ErbB2. The cholesterol-lowering drug lovastatin significantly potentiated the inhibitory furnishings of ErbB2 kinase inhibitors, accompanied with enhanced ErbB2 endocytosis. Lovastatin besides synergized with lapatinib to strongly suppress the in vivo growth of ErbB2-positive chest cancer xenografts.

Conclusion

The cell surface distribution of ErbB2 was closely regulated by membrane concrete properties governed by cholesterol contents. The cholesterol-lowering medications tin can hence be exploited for potential combinatorial therapies with ErbB2 kinase inhibitors in the clinical treatment of ErbB2-positive breast cancer.

Background

ErbB2, which is besides called Her2 or Neu, encodes a receptor tyrosine kinase from the EGFR/ErbB family. Unlike other ErbB members, ErbB2 has no identified cognate ligand, and thus till now is deemed equally an orphan receptor [1]. Nevertheless, due to the unique conformation of its extracellular domain, this receptor tyrosine kinase is considered as a favorable dimerization partner among ErbB family members [2, three]. Distension of ErbB2 factor is frequently observed in cancer patients, which identifies a subgroup of breast cancers called Her2/ErbB2-positive that accounts for xx–30% of chest malignancies. ErbB2 amplification leads to the accumulation of surplus ErbB2 receptors on cell membrane, promoting receptor dimerization and subsequent activation of a broad assortment of downstream oncogenic signaling circuitries [iv, 5]. Hence, the overexpression of ErbB2 inversely correlates with patient prognosis, while ErbB2 has proved as a top therapeutic target in breast cancer handling with multiple ErbB2-targeted therapies received FDA approvals [vi,7,8].

ErbB2 is a unmarried pass transmembrane receptor embedded in the plasma membrane, a circuitous construction composed of primarily lipids and proteins [nine,10,11]. Among its many essential physiological functions, cell membrane plays an important role to maintain the homeodynamics of cell surface proteins including the receptor tyrosine kinase ErbB2 [12,13,14]. On average, nigh half of the weight of eukaryotic plasma membranes can be attributed to lipids, which form the bilayer membrane structures incorporating 3 types of amphipathic lipids: phospholipids, sterols, and glycolipids [15, 16]. The majority of the lipid bilayer is composed of phospholipids and sterols, while glycolipids but make upwards a small fraction of less than 5% in general.

Cholesterol is the major sterol component of animate being cell membranes, which makes up about 30% of the lipid bilayer on average. Acting equally essential building blocks of the plasma membranes, cholesterol plays pivotal roles in maintaining the structural integrity and regulating the fluidity of prison cell membranes [17,18,19,20], therefore contributing to the homeodynamics of various membrane proteins on the prison cell surface. For example, alterations in membrane microviscosity and lipid fluidity mediated by cholesterol depletion or enrichment were revealed to significantly affect the prison cell surface distribution of membrane proteins in human erythrocytes [21, 22]. Furthermore, regarding its jail cell membrane-associated functions, cholesterol is likewise implicated in the modulation of cellular bespeak transmission and intracellular trafficking through contributing to lipid raft assembly and assisting the formation of endocytic pits [23, 24]. Although the oncogenic backdrop of ErbB2 in breast cancer has been extensively investigated, the connection between its expression levels and the physical properties of breast cancer prison cell membranes is obscure. Several proteins including HSP90, flotillin, and caveolin have been shown to regulate the jail cell surface distribution of ErbB2, simply how cholesterol content in cell membrane regulates the overall surface presence of this cancer-driving receptor tyrosine kinase remains elusive and then far [25,26,27,28]. In the nowadays study, we report that cholesterol content modulates the rigidity and fluidity of plasma membranes to maintain the surface levels of ErbB2 in chest cancer cells, while the reduction in cholesterol affluence in plasma membrane facilitates the endocytic degradation of ErbB2 and thus synergizes with the tyrosine kinase inhibitors against ErbB2 to suppress ErbB2-positive chest cancer growth.

Methods

Prison cell lines

Breast cancer SKBR3, AU565, and HCC1954 cell lines were purchased from the American Type Civilization Collection (ATCC). SKBR3 cells were cultured with McCoy'south 5A, while AU565 and HCC1954 cells were cultured with RPMI-1640 media, both supplemented with fetal bovine serum (10%, ExCell Bio, Shanghai) and antibiotics (1% penicillin/streptomycin, Gibco). Cells were maintained in a humidified atmosphere in the incubator (Thermo) at 37 °C with 5% CO₂.

Antibodies and other reagents

Mouse anti-ErbB2 (A-2), anti-ErbB2 (9G6), anti-Vinculin antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Rabbit anti-PARP antibody was purchased from Proteintech (Wuhan, Prc). Rabbit anti-phospho-Akt (Ser473) antibody was purchased from Cell Signaling Technology. Secondary goat anti-mouse and anti-rabbit, donkey anti-goat antibodies were obtained from LICOR. Neratinib (HKI-272) and lapatinib (GW572016) were purchased from Selleck. Oleic acrid (OA) and lovastatin were obtained from MeilunBio (Dalian, Mainland china). Filipin was obtained from Sigma.

Prison cell lysis and immunoblottings

Cells were lysed with the RIPA buffer (10 mM Tris-HCl pH seven.5, 150 mM NaCl, 1% (w/v) TritonX-100, 0.1% (w/v) SDS, 1% sodium deoxycholate) supplemented with mammalian protease and phosphatase inhibitor cocktails (Sigma) equally described previously [29]. Protein concentrations were determined via BCA protein assays (Takara). In general, 20–thirty μg of protein samples were loaded per lane onto SDS-PAGE gels to exist resolved, before transferred to nitrocellulose membranes (Merck Millipore, USA). Membranes were then blocked with 4% non-fat-milk in PBS at room temperature for an hour, followed by incubation with primary antibodies at 4 °C overnight. The blots were washed with PBS for three times before incubation with secondary antibodies. Membranes were finally washed with PBS and detected on a LICOR Odyssey arrangement. Captured images were quantitated using Image Studio software (Version four.0) as per manufacturer's instructions.

MTT assay

Ii thousand chest cancer cells were seeded into each well of 96-well plate and incubated overnight. Side by side day, cells were treated with indicated inhibitors for 24 h, before MTT (3-(4, 5-dimethylthiazol-2-yl)-2,v diphenyltetrazolium bromide) incubation for another 3 h. DMSO was added to dissolve formazan and absorbance was recorded at 570 nm using a spectrometer (Perkin Elmer, USA).

Wound healing assay

Cells were seeded onto 6-well plates to form a confluent monolayer. A wound was generated by scratching with a 200 μl pipette tip. Detached cells were cleaned past PBS washes. Mitomycin was added into growth media to a final concentration of ii.5 μg/ml to cease jail cell proliferation. Images of wound recovery were captured every 24 h using a phase contrast microscope (Leica, DMI4000B).

Flow cytometry

For apoptosis assays, cultured cells were processed for Annexin 5 and PI double staining using an apoptosis analysis kit (KeyGEN Biotech, China) according to manufacturer's instructions. The samples were finally analyzed using a benchtop menstruation cytometer (Accuri C6, BD Biosciences) and acquired data were analyzed using FlowJo software version 7.vi.1 (FlowJo, LLC, USA).

Cholesterol staining

AU565, SKBR3, and HCC1954 cells were seeded onto coverslips placed in six-well plates with full media. Next day, cells were done with PBS for three times and fixed with 4% paraformaldehyde for 15 min at room temperature. Following PBS washes, cells were incubated with 50 μg/ml of filipin for 1 h at room temperature, as described previously [30]. Then coverslips were washed and mounted onto a slide using Mowiol (Sigma). Cholesterol staining was examined with ultraviolet excitation using a fluorescence microscope (Olympus, BX63).

Immunofluorescence

Cultured SKBR3, AU565, and HCC1954 cells were treated as indicated. Subsequently PBS washes, cells were stock-still with four% paraformaldehyde for fifteen min and so permeabilized with 0.two% Triton X100. Samples were blocked with ii% BSA in PBS for half an hour, before incubation with primary antibodies for twenty min at room temperature. Coverslips were washed with PBS for three times and incubated with Alexa Fluor® 488-conjugated caprine animal anti-mouse secondary antibodies for 20 min at room temperature. Finally coverslips were washed and mounted onto a slide using Mowiol supplemented with DAPI (for visualization of cell nucleus). Antibody staining was observed nether a fluorescence microscope (Olympus, BX63).

Xenograft mouse model

Animal experiments were performed in accordance with the 1996 National Institutes of Health Guide for the Intendance and utilise of Laboratory Animals, and the procedures were canonical past the Institutional Animal Intendance and Apply Committee of Dalian Medical University. Female nude mice (4–6 weeks, Balb/c background) were obtained from Vital River Laboratories (Beijing, China) and maintained nether sterile conditions during the unabridged experiments. To generate tumor xenografts, cultured HCC1954 cells (6 1000000/mouse) were implanted subcutaneously into the correct flank of each mouse. The sizes of the xenograft tumors were measured using a vernier caliper every 3 days. After the average size of xenografts reached about 150 mm^three, the mice were randomly divided into 3 groups (v mice per grouping) to receive lapatinib, lovastatin and lapatinib, or vehicle command treatment. Lapatinib was dissolved in 2% DMSO, thirty% PEG 300, and 5% Tween lxxx in distilled water, while lovastatin was dissolved in xxx% PEG 400, 0.5% Tween 80, and 5% propylene glycol in distilled water. Lapatinib and lovastatin were administered by daily oral gavage for 17 sequent days at dosages of 100 mg/kg and xl mg/kg, respectively. Tumor volumes were calculated according to the following formula: tumor volume (mm³) = (length) × (width)^two × 0.52. Tumor xenografts were finally excised from the mice later on cede, and processed for Western blotting analyses.

Statistics

To determine statistical significance, experiments were conducted three times with biological repeats. Experimental results were presented as the ways ± standard error of the mean (SEM). Statistical differences between experimental groups were examined by performing Student'due south t-test in the GraphPad Prism software (version 7), and a p value less than 0.05 was considered every bit statistically significant.

Results

Cholesterol content in cell membrane correlates with ErbB2 localization and cell migration

Through immunofluorescence examination of ErbB2 localization in ErbB2-positive breast cancer cells, nosotros observed that, in SKBR3 and AU565 cells that possessed round shapes, ErbB2 was almost exclusively distributed to the cell membrane; while in HCC1954 cells that showed flattened and all-inclusive configurations, ErbB2 besides formed many intracellular punctae besides surface localizations (Fig. 1a). This influence of cell shapes on ErbB2 distribution prompted united states to speculate that the physical properties of cell membranes might play a role in the regulation of subcellular distribution of ErbB2, i.e. the round and more rigid cell surfaces in SKBR3 and AU565 cells tended to maintain ErbB2 in the cell membrane, while the floppy and more fluid prison cell membrane of HCC1954 facilitated the internalization of this receptor. Considering the essential roles of cholesterol in regulating cell membrane rigidity and fluidity [31, 32], nosotros examined the cholesterol content in prison cell membranes from these 3 ErbB2-positive breast cancer cell lines. Results from fluorescence microscopy using the cholesterol-specific stain filipin revealed that the cell membrane cholesterol in SKBR3 and AU565 cells was considerably more abundant than that in HCC1954 cells (Fig. 1b).

Fig. 1

Cell membrane cholesterol content correlates with ErbB2 localization and cell migration in ErbB2-positive breast cancer cells. a ErbB2-positive SKBR3, AU565, and HCC1954 cells were cultured on glass coverslips and processed for immunofluorescence analyses with anti-ErbB2 antibody. Nucleus was stained with DAPI. Representative images show confocal sections. Calibration bar = 10 μm. b indicated cells were cultured and processed for filipin staining to examine cholesterol amounts. Fluorescence images were captured under identical intensity settings. Calibration bar = 10 μm. c, d, e and f, indicated breast cancer cells were grown to course confluent monolayers and candy for wound healing assays. Mitomycin (2.5 μg/ml) was added during recovery. Cells were either treated with DMSO (control), oleic acid (OA) at 200 nM, or filipin at 2 μg/ml. Images of the wounds were recorded at indicated times under a phase contrast microscope. Calibration bar = 50 μm. Adjacent column charts show the quantification of relative wound recovery from each status. Non significant, n.southward., * and ** indicate p < 0.05 and p < 0.01, respectively

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As cellular cholesterol levels were shown to regulate metastatic phenotypes of cancer cells by altering the fluidity of cell membranes, we investigated whether the differential cholesterol amounts in ErbB2-positive breast cancer cells correlated with their migratory abilities [33]. Indeed, very limited jail cell migration was observed in SKBR3 and AU565 cells bearing high cholesterol contents, indicative of increased rigidity and reduced fluidity of jail cell membranes (Fig. 1c and d). Even in the presence of oleic acid that could increase membrane fluidity to some extent, the migration of SKBR3 and AU565 cells was still restricted, probable owing to the relatively high rigidity and low fluidity of the plasma membranes incurred past the abundance of cholesterol (Fig. 1c and d). On the contrary, in HCC1954 cells that independent relatively low levels of cholesterol, cell migration was readily observable, which was further enhanced by treatments with oleic acid or the cholesterol-interfering drug filipin, suggesting that increased membrane fluidity with reduced cholesterol content in cell membrane assisted prison cell migration (Fig. 1e and f).

Cholesterol abundance maintains ErbB2 levels on cell surface

To further investigate the influence of cholesterol content on the intracellular distribution of ErbB2, we examined the localizations of this receptor in SKBR3, AU565, and HCC1954 cells exposed to the cholesterol-interfering drug filipin. Interestingly, the usually round-shaped SKBR3 and AU565 became more flattened under filipin treatment, and intracellular ErbB2 punctae were readily discernible, suggesting that filipin handling induced receptor internalization (Fig. 2a and b). In improver, we treated SKBR3 and AU565 cells with the combination of oleic acid and filipin to further increase membrane fluidity and reduce surface rigidity. In this scenario, both SKBR3 and AU565 cells became more spread-out and irregular-shaped, while intracellular ErbB2 staining was greatly enhanced (Fig. 2a and b). On the other paw, although ErbB2 internalization was already evident in HCC1954 cells nether normal culturing atmospheric condition, the addition of filipin and oleic acrid significantly increased the formation of intracellular ErbB2 punctae (Fig. 2c). Taken together, these observations bespeak that the cholesterol content in cell membrane regulates membrane rigidity and fluidity to maintain ErbB2 receptors on the prison cell surface, while the subtract of cholesterol abundance in plasma membrane leads to reduced rigidity and increased fluidity of cell membrane that aid ErbB2 internalization.

Fig. two

The cholesterol-interfering drug filipin facilitates ErbB2 internalization. a, b and c, ErbB2-positive SKBR3, AU565, and HCC1954 cells were cultured on glass coverslips and treated with filipin (3 μg/ml) with or without oleic acid (OA, 200 nM) for 12 h. ErbB2 location was examined by immunofluorescence. DAPI stains the nucleus. Images were captured using a fluorescent microscope (Olympus BX63, 40X objective). Representative intracellular ErbB2 punctae are indicated with yellow triangles. Scale bar = 10 μm

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Cholesterol-lowering drug potentiates ErbB2-targeting agents to suppress breast cancer growth

Having disclosed the correlation between cholesterol content in the cell membrane and the surface levels of ErbB2 receptors, nosotros next sought to investigate the influence of the cholesterol-lowering drug lovastatin on the in vitro growth of ErbB2-positive breast cancer cell lines. In doing this, chest cancer cells overexpressing ErbB2 (SKBR3, AU565, and HCC1954) were treated with lovastatin, the FDA-approved tyrosine kinase inhibitors against ErbB2 (lapatinib and neratinib), or the combinations as indicated (Fig. 3a). Results from jail cell viability assays revealed that lovastatin significantly inhibited cell growth, although less potent than lapatinib and neratinib, but also potentiated the suppression incurred by these two tyrosine kinase inhibitors (Fig. 3a). Furthermore, flow cytometric information from Annexin V and propidium iodide double staining showed that the addition of lovastatin dramatically enhanced the pro-apoptotic effects of the ErbB2 inhibitors, with increases on averages of 3.03 and 2.74 folds in SKBR3, 2.26 and 1.99 folds in AU565, and 1.7 and one.75 folds in HCC1954 cells observed for lapatinib and neratinib, respectively (Fig. 3b and c). Therefore, these findings advise that the cholesterol-lowering drug lovastatin is capable of sensitizing the ErbB2-positive breast cancer cells to both lapatinib and neratinib.

Fig. 3

Lovastatin potentiates ErbB2 kinase inhibitors to suppress ErbB2-positive breast cancer cells. a SKBR3, AU565, and HCC1954 cells were treated with lovastatin (lova, 40 μM), lapatinib (lap, 200 nM), neratinib (ner, 200 nM), or combinations as indicated for 24 h. Jail cell viability was and so examined via MTT assays. Column charts testify data from 3 contained experiments and fault bars represent the standard error of the mean (SEM), with * and ** indicating p < 0.05 and p < 0.01, respectively. b chest cancer cells were treated as above and candy for double staining with Annexin V and propidium iodide (PI) to inspect apoptotic populations. c cavalcade charts show the quantitation of apoptotic cells with positive Annexin V staining (right two quadrants) from b

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Lovastatin treatment leads to increased endocytic degradation of ErbB2

Given that cholesterol abundance in the cell membrane could maintain the surface levels of ErbB2, we speculated that the inhibitory effects of the cholesterol-lowering drug lovastatin against ErbB2-positive breast cancer cells might exist associated with compromised cell membrane expression of ErbB2 and the resultant attenuation of downstream signal transmission. As expected, results from immunoblotting assays revealed that lovastatin treatment led to reduced ErbB2 expression in serum-starved SKBR3, AU565, and HCC1954 cells (Fig. 4a). In accord with our previous findings, treatments with the ErbB2 inhibitors lapatinib and neratinib resulted in increased and decreased cellular ErbB2 levels, respectively [34]. Notwithstanding, lovastatin addition caused pregnant reductions in ErbB2 expression nether the combined conditions compared to inhibitor unmarried treatments, with concomitantly enhanced PARP cleavage and decreased activation of AKT (Fig. 4a). Consistently, in the subsequent immunofluorescence assays to examine the cellular localizations of ErbB2, elevated intracellular ErbB2 staining was readily detected in HCC1954 cells treated with lovastatin (Fig. 4b and c). Fifty-fifty in neratinib-treated cells where ErbB2 endocytosis was induced, the combined handling with lovastatin farther augmented the formation of intracellular ErbB2 punctae (Fig. 4b and c). These immunoblotting and immunofluorescence information collectively suggest that lovastatin facilitated the internalization and intracellular deposition of ErbB2, leading to reduced ErbB2 expression levels and repressed downstream oncogenic signaling.

Fig. four

Lovastatin add-on results in enhanced internalization and degradation of ErbB2. a SKBR3, AU565, and HCC1954 cells were treated with lovastatin (xl μM), lapatinib (200 nM), neratinib (200 nM), or combinations equally indicated for 24 h. Jail cell lysates were analyzed by immunoblotting with indicated antibodies. Column graphs below prove the quantification of relative ErbB2 affluence compared to control samples. All mistake bars represent the standard mistake of the mean (n = 3), with * and ** indicating p < 0.05 and p < 0.01, respectively. b HCC1954 cells were treated with lapatinib (200 nM) or neratinib (200 nM) earlier immunofluorescence analysis to inspect ErbB2 staining. DAPI stains the nucleus. Scale bar = 10 μm. Images were captured using a fluorescent microscope (Olympus BX63, 40X objective). c cultured HCC1954 cells were treated with lovastatin (40 μM) either solitary or in the presence of lapatinib or neratinib (both at 200 nM). Immunofluorescence assays were then carried out as in b

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Lovastatin synergizes with lapatinib to suppress the in vivo growth of ErbB2-positive chest cancer

Although about all types of creature cells generate cholesterol, the majority (80%) of cholesterol synthesis at the organism level is accomplished in the liver and intestines. To investigate the synergistic effects of ErbB2 inhibitor and cholesterol-lowering drug in vivo, we generated ErbB2-positive breast cancer xenograft mouse models by implanting HCC1954 cells into athymic nude mice. Considering that lapatinib is more widely administered than neratinib in the clinical treatment of ErbB2-positive breast cancer, nosotros treated the xenograft-bearing mice with lapatinib or the combination of lapatinib and lovastatin. Every bit illustrated in Fig. 5a-c, lapatinib significantly suppressed HCC1954 xenograft growth in vivo, while the combo treatment with lapatinib and lovastatin farther decreased the sizes and weights of xenograft tumors formed in athymic nude mice, suggesting a synergistic effect of lovastatin with lapatinib to restrain the in vivo growth of ErbB2-positive breast cancer. After, nosotros examined the expression levels of ErbB2 in xenograft tissues resected from mice with different treatments using immunoblotting assays. Consistent with our data from cell line studies, the ErbB2 levels were observed to be elevated in the samples from lapatinib handling grouping compared to those of command group; while the combined treatment with lovastatin and lapatinib impeded the lapatinib-induced upregulation of ErbB2, confirming the negative regulatory effect of lovastatin on ErbB2 expression (Fig. 5d).

Fig. 5

Lovastatin synergizes with lapatinib to suppress HCC1954 xenograft growth. a HCC1954 cells were inoculated into female nude mice to generate xenograft mouse models. Mice were randomized into iii groups to receive control vehicle, lapatinib, or the combination of lovastatin and lapatinib every bit described in the methods section. The sizes of xenografts were measured every three days and calculated tumor volumes were plotted. Presented graph shows data from 5 mice per group. Error bars show the standard fault of the mean (SEM), with ** indicating p < 0.01. b afterward 17 days of oral gavage handling, the mice were sacrificed and xenograft tumors were resected. c tumor weight was measured and plotted. Presented graph shows information from 5 mice per group. Error bars bear witness the standard error of the hateful (SEM), with * and ** indicating p < 0.05 and p < 0.01, respectively. d protein samples were prepared from xenograft tumor tissues (3 per treatment group) and analyzed past immunoblotting using indicated antibodies. Vinculin was probed to bear witness equal loading. Bar nautical chart below shows the relative quantification of ErbB2

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Give-and-take

Breast cancer occurs with the highest incidence among all women malignancies and accounts for about 30% of newly diagnosed women cancer cases annually in the United States [35]. This leading deadly disease is generally divided into luminal A, luminal B, HER2/ErbB2-positive, and triple-negative chest cancer subtypes according to the expression status of estrogen receptor, progesterone receptor, and HER2/ErbB2 [36]. ErbB2 overexpression in breast cancer is always associated with poor patient prognosis [37, 38], but ErbB2 targeted therapies accept significantly improved patient survivals, hence confirming this receptor tyrosine kinase as the pivotal therapeutic target for intervention in the treatment of ErbB2-positive breast cancers.

Through the conscientious observations of distinct patterns in ErbB2 distribution of ErbB2-positive breast cancer cells, we plant a correlation between cell shape and ErbB2 localizations wherein ErbB2 remained mainly on the surface in cells with circular shapes merely became internalized in those with irregular forms. We speculated that the physical features of cell membranes, including rigidity and fluidity, might play a role to influence the cellular ErbB2 localizations. It was therefore intriguing to discover the remarkable difference in cholesterol contents betwixt SKBR3/AU565 and HCC1954 cells. Cholesterol is a major component of animal jail cell membranes, which is required to maintain the integrity and regulate the fluidity of plasma membranes [23]. Cholesterol contains a tetracyclic ring construction and adopts a trans conformation, rendering the molecule a planar and rigid characteristics that increases membrane packing and contributes to the integrity and rigidity of cell membranes. Cholesterol is also a key determinant of membrane fluidity: at loftier temperatures, cholesterol acts to stabilize the cell membrane and increment its melting point; while at low temperatures, it inserts into phospholipids and prevents them from interfering with each other to avoid aggregation [39]. Consequent with our hypothesis, the cholesterol affluence conferred the cell membranes of SKBR3 and AU565 cells increased rigidity and reduced fluidity that collectively limited jail cell mobility. In addition, by manipulating the cholesterol content in jail cell membranes using oleic acid and the cholesterol-interfering drug filipin, the cell shapes of SKBR3 and AU565 could be effectively altered and subsequent ErbB2 internalization was apparently enhanced.

These findings inspired us to investigate the therapeutic effects of cholesterol lowering drugs from the statin course of the HMG-CoA reductase inhibitors, such as lovastatin, on the in vivo and in vitro growth of ErbB2-positive chest cancers. Through a series of cell phenotypic experiments including cell viability and apoptosis assays, we demonstrated that lovastatin significantly potentiated the inhibitory effects of the FDA-canonical ErbB2 kinase inhibitors, lapatinib and neratinib. From the mechanistic aspect, we revealed that lovastatin improver upon lapatinib and neratinib treatment led to reduced cellular ErbB2 levels compared to inhibitor monotherapies, likely due to elevated internalization and degradation of ErbB2 incurred past contradistinct membrane physical backdrop. More than importantly, using ErbB2-positive breast cancer xenograft mouse models, we confirmed the potent synergistic effects of lovastatin and lapatinib to suppress the in vivo growth of HCC1954 xenografts.

It is noteworthy that several statins that received FDA approvals every bit lipid-lowering medications accept been reported to suppress chest cancer growth. For case, simvastatin was shown to induce PTEN transcription by interfering with NFκB activeness to inhibit the proliferation of chest cancer cells [40]. This statin was also observed to inhibit the mammosphere formation and migration of triple-negative breast cancer cells through the regulation of FOXO3a [41]. Lovastatin was reported to suppress the tumor growth and metastasis in a mouse breast cancer animal model by causing increased apoptosis and decreased Dna synthesis [42]. Lovastatin was too shown to suppress the glycolytic and citric acrid cycle activeness to inhibit the proliferation of breast cancer MDAMB231 and MDAMB468 cells [43]. Furthermore, a recent written report proposed that lovastatin in a cerasome-encapsulated nanohybrid class effectively suppressed the stemness backdrop of triple-negative breast cancer [44]. In addition, pitavastatin was recently reported to sensitize breast cancer to radiation by delaying DNA Repair and promoting senescence [45]. Therefore, it seemed that the statin class of medications exhibited pleiotropic effects to suppress the growth of breast cancer, while our findings revealed a novel aspect of the anti-cancer properties of the statins. Lovastatin, and likely other statins, through compromising HMG-CoA reductase activeness, reduced cellular cholesterol abundance and led to altered rigidity and fluidity of the cell membranes. The resultant membrane concrete properties facilitated the internalization and subsequent degradation of jail cell surface ErbB2, which synergized with lapatinib or neratinib to elicit strong inhibitory effects on ErbB2-positive breast cancer growth both in vitro and in vivo (Fig. half-dozen).

Fig. half-dozen

Schematic diagram depicting the synergistic effects of lovastatin with lapatinib. Through inhibiting HMGCR activeness, lovastatin reduces cellular cholesterol contents and hence alters the concrete properties of the cell membrane, conferring increased fluidity but decreased rigidity. These changes facilitates the internalization of cell surface ErbB2, which leads to its intracellular degradation through endosome to lysosome sorting. Reduced cellular ErbB2 expression sensitizes ErbB2-positive chest cancer cells to ErbB2 kinase inhibitors exemplified by lapatinib and neratinib, which collectively elicit more potent anti-cancer effects

Full size paradigm

Conclusions

ErbB2 has proven as a top therapeutic target in the clinical treatment of chest cancer. In the nowadays written report, we report that the cholesterol content in cell membranes regulates the surface levels of ErbB2 via affecting membrane rigidity and fluidity. Cholesterol-lowering drugs exemplified by lovastatin potentiated the inhibitory effects of ErbB2 kinase inhibitors by down regulating ErbB2 expression. Therefore, our findings warrant further investigations to reveal the synergistic effects of the statin class of medications with tyrosine kinase inhibitors against ErbB2 in the clinical management of ErbB2-positive chest cancers.

Abbreviations

EGFR:

Epidermal Growth Factor Receptor

HMGCR:

HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase

Lap:

Lapatinib

Lova:

Lovastatin

Ner:

Neratinib

OA:

Oleic Acrid

PBS:

Phosphate-Buffered Saline

SEM:

Standard Error of the Mean

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Acknowledgements

The authors are grateful to the National Natural Science Foundation of China for financial supports.

Funding

This piece of work was supported by the National Natural Science Foundation of China (No. 81702628 to YZ and No. 81301901 to HL).

Availability of data and materials

All information were generated or analyzed during this study are included in this published commodity.

Author information

Author notes

1. Jinrui Zhang and Qiong Li contributed as to this work.

Affiliations

1. Institute of Cancer Stem Prison cell, Dalian Medical Academy, Dalian, Communist china

   Jinrui Zhang, Qiong Li, Yueguang Wu, Duchuang Wang, Yang Zhang, Shanshan Wang, Taishu Wang, Fang Liu, Mohamed Y. Zaky, Shuai Hou, Shuyan Liu, Haixin Lei, Yingqiu Zhang & Han Liu

2. The 2d Affiliated Infirmary, Dalian Medical Academy, Dalian, China

   Qiong Li, Lu Xu & Lijuan Zou

3. Molecular Physiology Division, Department of Zoology, Faculty of Scientific discipline, Beni-Suef University, Beni Suef, Egypt

   Mohamed Y. Zaky

4. Department of Radiotherapy Oncology, the First Affiliated Hospital of Dalian Medical Academy, Dalian, China

   Kun Zou

5. Cancer Biotherapy & Translational Medicine Center of Liaoning Province, Dalian Medical University, Dalian, China

   Han Liu

Contributions

Study pattern: YZ and HL; Data Collection: JZ, QL, YG, DW, LX, YZ, SW, TW, FL, and MYZ; Data assay: JZ, QL, SH, HL, LZ, YZ, SL, KZ; Manuscript grooming: JZ and HL. All authors read and approved the final manuscript.

Respective authors

Correspondence to Yingqiu Zhang or Han Liu.

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Ethics approval

Animal experiments were performed in accordance with the 1996 National Institutes of Health Guide for the Care and utilise of Laboratory Animals, and the procedures were approved past the Institutional Animal Intendance and Utilize Committee of Dalian Medical Academy.

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Competing interests

The authors declare that they have no competing interests.

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Zhang, J., Li, Q., Wu, Y. et al. Cholesterol content in cell membrane maintains surface levels of ErbB2 and confers a therapeutic vulnerability in ErbB2-positive chest cancer. Cell Commun Signal 17, xv (2019). https://doi.org/10.1186/s12964-019-0328-four

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• Received: 19 November 2018

• Accustomed: 13 February 2019

• Published: xx February 2019

• DOI : https://doi.org/ten.1186/s12964-019-0328-4

Keywords

• ErbB2
• Chest cancer
• Cholesterol
• Membrane fluidity
• Membrane rigidity
• Lovastatin

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