Mammary tumor growth and pulmonary metastasis are enhanced in a hyperlipidemic mouse model

Abstract

Dyslipidemia has been associated with an increased risk for developing cancer. However, the implicated mechanisms are largely unknown. To explore the role of dyslipidemia in breast cancer growth and metastasis, we used the apolipoprotein E (ApoE) knockout mice (ApoE(-/-)), which exhibit marked dyslipidemia, with elevated circulating cholesterol and triglyceride levels in the setting of normal glucose homeostasis and insulin sensitivity. Non-metastatic Met-1 and metastatic Mvt-1 mammary cancer cells derived from MMTV-PyVmT/FVB-N transgenic mice and c-Myc/vegf tumor explants respectively, were injected into the mammary fat pad of ApoE(-/-) and wild-type (WT) females consuming a high-fat/high-cholesterol diet and tumor growth was evaluated. ApoE(-/-) mice exhibited increased tumor growth and displayed a greater number of spontaneous metastases to the lungs. Furthermore, intravenous injection of Mvt-1 cells resulted in a greater number of pulmonary metastases in the lungs of ApoE(-/-) mice compared with WT controls. To unravel the molecular mechanism involved in enhanced tumor growth in ApoE(-/-) mice, we studied the response of Mvt-1 cells to cholesterol in vitro. We found that cholesterol increased Akt(S473) phosphorylation in Mvt-1 cells as well as cellular proliferation, whereas cholesterol depletion in the cell membrane abrogated Akt(S473) phosphorylation induced by exogenously added cholesterol. Furthermore, in vivo administration of BKM120, a small-molecule inhibitor of phosphatidylinositol 3-kinase (PI3K), alleviated dyslipidemia-induced tumor growth and metastasis in Mvt-1 model with a concomitant decrease in PI3K/Akt signaling. Collectively, we suggest that the hypercholesterolemic milieu in the ApoE(-/-) mice is a favorable setting for mammary tumor growth and metastasis.

Figure 1

High cholesterol diet significantly increased the body weight (A) as well as the cholesterol and triglycerides levels in the plasma (D,E) of ApoE^−/− mice with no increase in body adiposity (B,C). ApoE^−/− mice had lower blood glucose levels (F). Statistically significant difference between groups is indicated. *, P < 0.05. Student’s t test.

Figure 1

High cholesterol diet significantly increased the body weight (A) as well as the cholesterol and triglycerides levels in the plasma (D,E) of ApoE^−/− mice with no increase in body adiposity (B,C). ApoE^−/− mice had lower blood glucose levels (F). Statistically significant difference between groups is indicated. *, P < 0.05. Student’s t test.

Figure 2

Orthotopically injected mammary tumor Met-1 and Mvt-1 cells (500 000 and 50 000 cells per mammary fat pad, respectively) resulted in larger tumors in ApoE^−/− mice as demonstrated by tumor volume (A,C) and tumor weight (B,D) (n= 8-10 per group). Statistically significant difference is indicated. *, P < 0.05. Student’s t test.

Figure 3

Orthotopic inoculation of Mvt-1 cells resulted in more numerous pulmonary macrometastases in ApoE^−/− mice compared to WT as demonstrated by H & E staining of lung sections (A) (arrows indicate metastases) and the number of macrometastatic lesions detected in the lungs (B) (n= 8-10 per group). Injection of Mvt-1 cells intravenously (12 000 cells) led to a greater level of pulmonary metastases in the lungs of ApoE ^−/− mice in comparison to WT (n= 10-17 per group) (C). Statistically significant difference is indicated. *, P < 0.05. Student’s t test.

Figure 4

100 nM (equivalent to 4 μg/dl) cholesterol significantly increased the number of Mvt-1 cells in vitro (A). Cholesterol stimulated Akt^S473 phosphorylation in Mvt-1 cells in a dose dependent manner (1-10 mM equivalent to 40-400 mg/dl) (B). 1mM of cholesterol activated Akt during a shorter time period (20 min and 40 min) (C). MBCD is known to deplete cholesterol from plasma membrane leading to disruption of lipid rafts. The presence of MBCD (5 mM) inhibited cholesterol-induced Akt^S473 phosphorylation (D). Densitometric analysis of Akt phosphorylation is presented as levels of phosphorylation compared to total Akt protein. Statistically significant differenced is indicated as *, P<0.05, Student’s t test. (*, P<0.05 untreated vs cholesterol treated Mvt-1 cells and/or cholesterol treated vs cholesterol and inhibitor treated Mvt-1 cells). Statistical analyses were determined using the student’s t test where 2 groups were present and ANOVA for comparing more than 2 groups.

Figure 4

100 nM (equivalent to 4 μg/dl) cholesterol significantly increased the number of Mvt-1 cells in vitro (A). Cholesterol stimulated Akt^S473 phosphorylation in Mvt-1 cells in a dose dependent manner (1-10 mM equivalent to 40-400 mg/dl) (B). 1mM of cholesterol activated Akt during a shorter time period (20 min and 40 min) (C). MBCD is known to deplete cholesterol from plasma membrane leading to disruption of lipid rafts. The presence of MBCD (5 mM) inhibited cholesterol-induced Akt^S473 phosphorylation (D). Densitometric analysis of Akt phosphorylation is presented as levels of phosphorylation compared to total Akt protein. Statistically significant differenced is indicated as *, P<0.05, Student’s t test. (*, P<0.05 untreated vs cholesterol treated Mvt-1 cells and/or cholesterol treated vs cholesterol and inhibitor treated Mvt-1 cells). Statistical analyses were determined using the student’s t test where 2 groups were present and ANOVA for comparing more than 2 groups.

Figure 5

Inhibition of the PI3K/Akt pathway by BKM120 significantly reduced growth of Mvt-1 orthograft tumors (A) (**, P<0.001 for ApoE^−/− -BKM120-treated vs ApoE^−/− vehicle) as well as tumor weight (B) in ApoE^−/− mice (**, P<0.001 for ApoE^−/− -BKM120-treated vs ApoE^−/− vehicle, #, P<0.05 WT-BKM120-treated vs WT vehicle). Two weeks after tumor inoculation (50 000 Mvt-1 cells), the ApoE^−/− and WT were treated with the PI3K inhibitor, BKM120 (50mg/kg/d) or vehicle (10% NMP, 90% PEG300) by oral gavage for 13 days. The number of metastases was significantly decreased in BKM120-treated ApoE^−/− mice in comparison to vehicle-treated mice (C) (*, P<0.05 for ApoE^−/− -BKM120-treated vs ApoE^−/− vehicle and #, P<0.05 ApoE^−/− vehicle vs WT vehicle) (n=5-7 per group). A reduced level of phosphorylated Akt^S473 was found in the BKM120-treated ApoE^−/−and WT mice in comparison to vehicle-treated controls. Densitometric analysis of Akt phosphorylation is presented as levels of phosphorylation compared to total Akt protein (D) (*, P<0.05 for ApoE^−/− -BKM120-treated vs ApoE^−/− vehicle). Statistical analyses were determined using the student’s t test where 2 groups were present and ANOVA for comparing more than 2 groups.

Figure 5

Inhibition of the PI3K/Akt pathway by BKM120 significantly reduced growth of Mvt-1 orthograft tumors (A) (**, P<0.001 for ApoE^−/− -BKM120-treated vs ApoE^−/− vehicle) as well as tumor weight (B) in ApoE^−/− mice (**, P<0.001 for ApoE^−/− -BKM120-treated vs ApoE^−/− vehicle, #, P<0.05 WT-BKM120-treated vs WT vehicle). Two weeks after tumor inoculation (50 000 Mvt-1 cells), the ApoE^−/− and WT were treated with the PI3K inhibitor, BKM120 (50mg/kg/d) or vehicle (10% NMP, 90% PEG300) by oral gavage for 13 days. The number of metastases was significantly decreased in BKM120-treated ApoE^−/− mice in comparison to vehicle-treated mice (C) (*, P<0.05 for ApoE^−/− -BKM120-treated vs ApoE^−/− vehicle and #, P<0.05 ApoE^−/− vehicle vs WT vehicle) (n=5-7 per group). A reduced level of phosphorylated Akt^S473 was found in the BKM120-treated ApoE^−/−and WT mice in comparison to vehicle-treated controls. Densitometric analysis of Akt phosphorylation is presented as levels of phosphorylation compared to total Akt protein (D) (*, P<0.05 for ApoE^−/− -BKM120-treated vs ApoE^−/− vehicle). Statistical analyses were determined using the student’s t test where 2 groups were present and ANOVA for comparing more than 2 groups.