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. 2017 Nov 16;36(46):6462-6471.
doi: 10.1038/onc.2017.247. Epub 2017 Jul 31.

Elevated tumor LDLR expression accelerates LDL cholesterol-mediated breast cancer growth in mouse models of hyperlipidemia

E J Gallagher  1 , Z Zelenko  1 , B A Neel  1 , I M Antoniou  1 , L Rajan  1 , N Kase  1 , D LeRoith  1
Affiliations

Elevated tumor LDLR expression accelerates LDL cholesterol-mediated breast cancer growth in mouse models of hyperlipidemia

E J Gallagher et al. Oncogene. .

Abstract

Obesity is associated with an increase in cancer-specific mortality in women with breast cancer. Elevated cholesterol, particularly low-density lipoprotein cholesterol (LDL-C), is frequently seen in obese women. Here, we aimed to determine the importance of elevated circulating LDL, and LDL receptor (LDLR) expression in tumor cells, on the growth of breast cancer using mouse models of hyperlipidemia. We describe two novel immunodeficient mouse models of hyperlipidemia (Rag1-/-/LDLR-/- and Rag1-/-/ApoE (apolipoprotein E)-/- mice) in addition to established immunocompetent LDLR-/- and ApoE-/- mice. The mice were used to study the effects of elevated LDL-C in human triple-negative (MDA-MB-231) and mouse Her2/Neu-overexpressing (MCNeuA) breast cancers. Tumors derived from MCNeuA and MDA-MB-231 cells had high LDLR expression and formed larger tumors in mice with high circulating LDL-C concentrations than in mice with lower LDL-C. Silencing the LDLR in the tumor cells led to decreased growth of Her2/Neu-overexpressing tumors in LDLR-/- and ApoE-/- mice, with increased Caspase 3 cleavage. Additionally, in vitro, silencing the LDLR led to decreased cell survival in serum-starved conditions, associated with Caspase 3 cleavage. Examining publically available human data sets, we found that high LDLR expression in human breast cancers was associated with decreased recurrence-free survival, particularly in patients treated with systemic therapies. Overall, our results highlight the importance of the LDLR in the growth of triple-negative and HER2-overexpressing breast cancers in the setting of elevated circulating LDL-C, which may be important contributing factors to the increased recurrence and mortality in obese women with breast cancer.

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Conflict of interest statement

Conflicts of Interest: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
(A) Non-fasting serum total cholesterol and (B) serum low density and very low-density lipoprotein levels from wild type (WT) control, LDLR knockout (LDLR−/−) and ApoE knockout (ApoE−/−) mice on western diet. (C) Non-fasting serum total cholesterol and (D) low density and very low-density lipoprotein) levels from Rag1−/− control, Rag1−/−/LDLR−/− and Rag1−/−/ApoE−/− mice on western diet (n=4 (Rag1−/−/ApoE−/−), n=10 Rag1−/− and Rag1−/−/LDLR−/− per group). Graphs represent the mean of each group, error bars are SEM, *p value <0.05, ** p value <0.01 between groups as indicated.
Figure 2
Figure 2
(A) Body weights of WT, LDLR−/− and ApoE−/− mice after 4 weeks of western diet feeding at start of tumor study (n=15 per group). (B, D) Insulin tolerance test performed on mice that were fasted for 2 hours prior to the injection of 0.75units/kg of regular human insulin (n=4 WT, LDLR−/−, ApoE−/− per group, n=5 Rag1−/−, Rag1−/−/LDLR−/− per group). (D) Body weights of Rag1−/−, Rag1−/−/LDLR−/− and Rag1−/−/ApoE−/− mice after 4 weeks of postpubertal western diet feeding, prior to tumor cell injection (n=Rag1−/−/ApoE−/−:4, Rag1−/− and Rag1−/−/LDLR−/−:10 per group). * p value <0.05, **p value <0.01 between groups as indicated. Graphs represent the mean of each group, error bars are SEM.
Figure 3
Figure 3
(A) Western blot of LDLR expression in protein lysates from human MDA-MB-231, MDA-MB-468 and MCF7 cells after growth in DMEM with 10% FBS and 1% penicillin streptomycin (FBS) or cells serum starved in DMEM with 0.1% BSA and 1% penicillin/streptomycin overnight (SFM). Beta actin was used as loading control (B) Western blot of LDLR expression in protein lysates from murine MCNeuA and MVT1 cell lines. (C) MCNeuA tumor volume in WT, LDLR−/− and ApoE−/− mice after orthotopic injection of 2 × 106 cells on day 0 (n=7 (WT), n=10 per group (LDLR−/−, ApoE−/−)). (D) MDA-MB-231 tumor volume in Rag1−/−, Rag1−/−/LDLR−/− and Rag1−/−/ApoE−/− mice after tumor cell injection into the 4th mammary fat pad on day 0 (n=4 Rag1−/−/ApoE−/−, n=9 Rag1−/− and Rag1−/−/LDLR−/− per group). Graphs are the mean of each group, error bars represent SEM. * p value <0.05, ** p value <0.01 between groups as indicated.
Figure 4
Figure 4
(A) MDA-MB-468 tumor volume in Rag1−/− and Rag1−/−/ApoE−/− mice after tumor cell injection into the 4th mammary fat pad on day 0 (n=5 per group). Graphs are the mean of each group, error bars represent SEM. * p value <0.05 between groups. (B) Representative micro ultrasound images of tumors from Rag1−/− and Rag1−/−/ApoE−/− with 3D reconstruction of tumors for volume measurement. (C) Western blot analysis of LDLR expression in protein lysates from MDA-MB-468 tumors from Rag1−/−/ApoE−/− mice, and MDA-MB-468 cells grown in complete growth media. Protein lysates from WT and LDLR knockout (LDLR−/−) liver were used as positive and negative controls for the LDLR, respectively. (D) Representative immunohistochemistry staining of MDA-MB-468 tumor xenografts from Rag1−/− and Rag1−/−/ApoE−/− mice. Positive staining is in the upper panels, negative controls (without primary antibody) are in the lower panels. Positive and negative images were taken at 40× objective. Inserts are cropped magnified images from upper panels.
Figure 5
Figure 5
(A) Representative western blot comparing LDLR protein expression in MCNeuA control (Ctrl) cells, and cells transduced LDLR shRNA M1 and M2 sequences in serum starved medium (SSM) for 48 hours compared with cells grown in complete medium (CM). (B) Quantification of western blot of control, and M1, and M2 LDLR expression/beta actin by densitometry (n=3 per group). Expression is quantified relative to the LDLR/beta actin expression in the control cells. (C) The volume of MCNeuA tumors with control shRNA, or LDLR targeting shRNA (M1, M2) grown in hyperlipidemic LDLR−/− mice (n=10 control shRNA and M1, n=8 M2 shRNA per group). (D) The volume of MCNeuA tumors with control shRNA, or LDLR targeting shRNA (M1, M2) grown in hyperlipidemic ApoE−/− mice (n=10 per group). (E) Densitmetry quantification of cleaved Caspase 3/beta actin expression in control shRNA, M1, and M2 LDLR shRNA from tumor protein lysates from LDLR−/− mice. (F) Densitometry quantification of cleaved Caspase 3/beta actin expression in control shRNA, M1, and M2 LDLR shRNA in tumor protein lysates from LDLR−/− mice. All graphs are the mean of each group, error bars represent SEM. * p<0.05, ** p<0.01 between groups as indicated.
Figure 6
Figure 6
(A) Western blot comparing LDLR protein expression in MDA-MB-231 control (Ctrl) cells, and cells transduced LDLR shRNA H1 and H2 sequences in serum starved medium (SSM) for 48 hours compared with cells grown in complete medium (CM). (B) Quantification of western blot of control, and M1, and M2 LDLR expression/beta actin by densitometry (n=3 per group). (C) Live cell count of MDA-MB-231 cells after 72 hours of proliferation in complete medium. White bars represent 15,000 cells that were plated on day 0. Black bars represent the mean cell count for each group after 72 hours. (D) Live cell count of MCNeuA cells after 96 hours of proliferation in complete medium. White bars represent 15,000 cells that were plated on day 0. Black bars represent the mean cell count for each group after 96 hours. (E) Representative images of 3D “on top” cultures of MDA-MB-231 cells and (F) of MCNeuA cells after 5 days. (G) MDA-MB-231 live cell counts after trypan blue exclusion after 96 hours of cell growth in serum deficient medium (DMEM with 0.3% charcoal stripped FBS). White bars represent the number of cells seeded per well in a 12 well plate black bars represent the number of cells present after 96 hours. (H) Representative Western blot analysis of cleaved Caspase 3 in MDA-MB-231 cells with control shRNA (Ctrl), and LDLR targeting shRNA (H1, H2) after 48 hours of serum starvation in DMEM, 0.3% charcoal stripped FBS, 1% penicillin/streptomycin. (I Densitometry quantification of cleaved Caspase 3/beta actin in MDA-MB-231 cells and (J) MCNeuA cells from Western blots of protein lysates from control (Ctrl) and LDLR shRNA infected cells (H1, H2; M1, M2, respectively) after 48 hours of serum starvation in DMEM 0.3% charcoal stripped FBS, 1% penicillin/streptomycin. Densitometry graphs are the mean of each group relative to the expression level of the control (Ctrl) group. Graphs are the mean of each group, error bars represent SEM. * p<0.05 between groups as indicated.
Figure 7
Figure 7
Overall survival and recurrence free survival (RFS) Kaplan Meier plots generated using PROGgene V2 (A–D) and KMplot (E). LDLR expression was divided into high and low expression by the median of the population. Red lines represent the group with high LDLR expression, green (A–D) or black (E) lines represent the group with low LDLR expression. Hazard ratios (HR), 95% confidence intervals and p values are shown below the graphs. GSE numbers are the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) accession numbers.
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