Hypercholesterolemia induces angiogenesis and accelerates growth of breast tumors in vivo

Abstract

Obesity and metabolic syndrome are linked to an increased prevalence of breast cancer among postmenopausal women. A common feature of obesity, metabolic syndrome, and a Western diet rich in saturated fat is a high level of circulating cholesterol. Epidemiological reports investigating the relationship between high circulating cholesterol levels, cholesterol-lowering drugs, and breast cancer are conflicting. Here, we modeled this complex condition in a well-controlled, preclinical animal model using innovative isocaloric diets. Female severe combined immunodeficient mice were fed a low-fat/no-cholesterol diet and then randomized to four isocaloric diet groups: low-fat/no-cholesterol diet, with or without ezetimibe (cholesterol-lowering drug), and high-fat/high-cholesterol diet, with or without ezetimibe. Mice were implanted orthotopically with MDA-MB-231 cells. Breast tumors from animals fed the high-fat/high-cholesterol diet exhibited the fastest progression. Significant differences in serum cholesterol level between groups were achieved and maintained throughout the study; however, no differences were observed in intratumoral cholesterol levels. To determine the mechanism of cholesterol-induced tumor progression, we analyzed tumor proliferation, apoptosis, and angiogenesis and found a significantly greater percentage of proliferating cells from mice fed the high-fat/high-cholesterol diet. Tumors from hypercholesterolemic animals displayed significantly less apoptosis compared with the other groups. Tumors from high-fat/high-cholesterol mice had significantly higher microvessel density compared with tumors from the other groups. These results demonstrate that hypercholesterolemia induces angiogenesis and accelerates breast tumor growth in vivo.

Figure 1

Study design. A and B: A total of 120 female SCID mice (A) were maintained on a LFNC diet (B) for 3 weeks. C: Blood was drawn to measure serum cholesterol concentration. Any mouse with serum cholesterol levels that did not normalize (means ± SD) was removed from the study. D: Mice were then randomized into four different diet/treatment groups: LFNC, with or without ezetimibe, and high fat/high cholesterol, with or without ezetimibe. E: Mice were maintained on these diets for 4 weeks, and their serum cholesterol levels were monitored biweekly, at which point 2 × 10⁶ MDA-MB-231 cells were orthotopically injected into the fourth mammary fat pads (two tumors per mouse, approximately 28 to 30 mice per group). F: Mice were maintained on their respective diets and were checked daily for tumor formation, and their blood was drawn every several weeks for serum cholesterol determination. After 4 weeks, when tumors appeared, tumor volume was measured three to four times per week until reaching maximum tumor volume. G: Mice were sacrificed, and tumors were harvested and processed for analysis.

Figure 2

Increased serum cholesterol levels are associated with accelerated tumor growth. A: Serum cholesterol levels. Mice were fed either an HFHC or an LFNC diet, with or without ezetimibe (Z), and bled for cholesterol determination every 3 to 4 weeks throughout the course of the study. Serum cholesterol levels just before the first measurable tumors appeared are presented as serum cholesterol (mg/dL) versus diet cohort ± SEM. All groups were statistically different from one another (F = 72.70, P < 0.0001), and this difference was maintained throughout the study. An analysis of variance was used to determine statistical significance. Values <0.01 were considered significant (n = 28 per group). B: Serum insulin levels. Serum insulin levels are plotted as insulin levels (ng/mL) ± SEM versus diet group. There was no difference in serum insulin levels between diet and ezetimibe (Z) treatment groups at sacrifice (P = 0.278, analysis of variance; n = 15 per group). C: Serum estradiol levels. Serum estradiol levels are plotted as estradiol levels (ng/mL) ± SEM versus diet group. There was no difference in serum estradiol levels between groups at sacrifice (P = 0.332, analysis of variance; n = 15 per group). D: Body mass. There was no effect of diet or drug on body weight. Average body mass is plotted as mass (g) ± SEM versus diet group. There was no difference in animal body mass at the time of tumor implantation (P = 0.135, analysis of variance), and no differences in body weight between groups were observed throughout the study, as indicated by the average body weight at tumor end point presacrifice (P = 0.206, analysis of variance; n = 27 to 30 animals per group). E–G: Tumor growth. Line graphs show mean tumor volume for each of the four groups after first appearance. E: Significant mean differences were observed between tumor growth in mice fed an HFHC compared with the LFNC diet (asterisks). The mice in the LFNC group had a reduced rate of tumor growth (slope test: F = 2.05, P = 0.04). F: Significant differences were also measured between tumor growth in mice fed an HFHC diet, with and without ezetimibe (asterisks), where the ezetimibe-treated group demonstrated a reduced rate of tumor growth (slope test: F = 6.97, P < 0.001). G: No significant differences were measured between tumor growth in the mice fed the LFNC versus LFNC + ezetimibe diets. Data are presented as mean volume (mm³) ± SEM versus days. Statistical significance was determined by analysis of variance. Two-tailed values of P < 0.05 were considered statistically significant. H: Tumor cholesterol. Cholesterol was extracted from tumor tissue and assayed (as described in Materials and Methods). Tumor cholesterol is plotted as the average cholesterol (mg) tumor tissue (g) ± SEM versus diet group. Analysis of variance indicated no significant difference in the intratumoral cholesterol levels between groups (P = 0.825) (n = 11 tumors per group).

Figure 3

Hypercholesterolemia, independent of diet/drug, is associated with increased tumor volume. A: Bar graph shows the average final tumor volume (mm³) ± SEM versus serum cholesterol group. Hypercholesterolemic mice, blinded to drug/diet group, were defined as animals possessing serum cholesterol levels of 2 SDs higher than the mean of age-matched, sex-matched mice receiving a normal diet (a value of ≥124 mg/dL cholesterol). These hypercholesterolemic mice demonstrated a significantly greater average terminal tumor volume than animals with cholesterol levels <124 mg/dL cholesterol (751 ± 372 mm³ versus 583 ± 22 mm³; P = 0.007, t-test) (n = 25 mice in ≥124 mg/dL cholesterol group and 81 mice in <124 mg/dL cholesterol group). The Pearson correlation, blinded to treatment, between serum cholesterol and total tumor volume is positive (Pearson r = 0.23, P = 0.018) and clearly reveals an association that animals with higher serum cholesterol tend to have high tumor volumes. B: Hypercholesterolemic mice do not have high serum insulin levels. Bar graph shows the average serum insulin levels ± SEM versus cholesterol. Mice with the highest serum cholesterol levels (≥124 mg/dL), independent of treatment group, do not have increased serum insulin levels. Mean serum insulin levels did not differ between mice with hypercholesterolemia (≥124 mg/dL) and mice with normal serum cholesterol levels (<124 mg/dL) (0.67 ± 0.33 versus 0.73 ± 0.31 ng/mL; P = 0.48, t-test) (n = 15 to 43 per group). No relationship between serum cholesterol and serum insulin exists (Pearson r = −0.01, P = 0.93). C: Serum insulin levels are not associated with tumor volume. Animals with the highest serum insulin levels, defined as serum insulin levels 2 SDs above the mean, a value of ≥1.0 ng/mL, had a final average tumor volume that was the same tumor volume of animals with normal serum insulin levels of <1.0 ng/mL (P = 0.377, t-test) (n = 8 to 49 animals per group). A Pearson correlation analysis blinded to treatment group showed that no association exists between serum insulin levels and tumor volume (Pearson r = −0.140, P = 0.30).

Figure 4

Tumor apoptosis is significantly reduced in mice fed the HFHC diet. A: TUNEL staining. Top row: Representative tumor sections for each group with TUNEL staining (green). Bottom row: Merged image of tumor section with TUNEL stain (green) and nuclei counterstained with DAPI (blue). B: Quantification of tumor cell apoptosis. The level of tumor apoptosis plotted as average percentage of TUNEL-positive cells ± SEM versus diet/drug group. Tumors from animals fed an HFHC diet had less apoptotic tumor cells compared with the other diet/drug groups (F = 9.67, P < 0.001, repeated-measures analysis of variance). ^∗P ≤ 0.007 (Bonferroni's multiple-comparison test), significant difference between the HFHC group and the HFHC + ezetimibe (Z), LFNC, and LFNC + Z groups; ^†P = 0.012 (Bonferroni's multiple-comparison test), significant difference between the HFHC + Z and LFNC + Z groups. No difference in tumor apoptosis was seen between the LFNC + Z and LFNC groups (P = 0.357) (n = 70 images per group).

Figure 5

Tumor cell proliferation is significantly increased in mice fed the HFHC diet. A: Ki-67 staining. Top row: Representative images of tumor sections for each group stained for Ki-67 (red), a proliferation marker. Bottom row: Images of Ki-67 staining (red) merged with nuclei counterstained with DAPI (blue). B: Quantification of tumor cell proliferation. The level of tumor cell proliferation plotted as average percentage of Ki-67–positive cells ± SEM versus diet/drug group. Tumors from the animals fed the HFHC have a significantly greater percentage of proliferating cells compared with tumors from all other groups (F = 17.62, P < 0.001, analysis of variance and Bonferroni's multiple-comparison tests, P < 0.001). The asterisk indicates significant difference compared with the HFHC group, no difference in tumor proliferation was seen between HFHC + ezetimibe (Z), LFNC, and LFNC + Z groups (Bonferroni's multiple-comparison tests, P ≥ 0.665) (n = 50 images per group).

Figure 6

Tumor angiogenesis is significantly greater in tumors from mice fed an HFHC diet. A: CD31 staining. Representative immunofluorescent images of tumor sections from each group stained for the endothelial cell marker, CD31 (green). Images of the CD31-stained panel (green) merged with nuclei counterstained with DAPI (blue). B: Quantification of MVD. Data are plotted as relative level of CD31 staining versus diet/ezetimibe (Z) group ± SEM. Data were analyzed by mixed-model analysis of variance, which indicates a significantly greater vessel area in tumors from mice fed an HFHC diet compared with animals receiving ezetimibe (Z) and/or receiving an LFLC diet (F = 18.06, P < 0.001). ^∗∗P < 0.01 (Bonferroni's multiple-comparison test), significant difference compared with HFHC group, whereas no difference in MVD was observed between LFNC + Z and HFHC + Z groups (Bonferroni's multiple comparison test, P = 0.502) (n = 80 images per group).

Figure 7

Tumors from animals treated with ezetimibe have significantly greater perivascular cell coverage. A: SMA staining. Representative immunofluorescent images of tumor sections from each group stained for the endothelial cell marker, CD31 (green), and for the perivascular cell marker SMA (red). Images of CD31 (green) and SMA (red) stained tumors merged with the nuclei counterstained with DAPI (blue). B: Quantification of perivascular cell coverage. Data are plotted as the average percentage of SMA coverage of CD31-positive microvessels ± SEM versus diet/drug group and demonstrate that ezetimibe (Z)–treated tumors had significantly greater SMA staining (greater vessel stability) compared with untreated tumors (F = 5.916, P = 0.001, analysis of variance). There was no effect of diet on vessel stability (no difference in % SMA staining), mixed-model analysis of variance, P = 0.842. ^∗P ≤ 0.01 (Bonferroni's multiple-comparison test), significant difference from the HFHC group; ^†P ≤ 0.018 (Bonferroni's multiple-comparison test), significant difference from the LFNC group (n = 50 images per group).