Role of cholesterol in the development and progression of breast cancer

Abstract

Diet and obesity are important risk factors for cancer development. Many studies have suggested an important role for several dietary nutrients in the progression and development of breast cancer. However, few studies have specifically addressed the role of components of a Western diet as important factors involved in breast cancer initiation and progression. The present study examined the role of cholesterol in the regulation of tumor progression in a mouse model of mammary tumor formation. The results suggest that cholesterol accelerates and enhances tumor formation. In addition, tumors were more aggressive, and tumor angiogenesis was enhanced. Metabolism of cholesterol was also examined in this mouse model. It was observed that plasma cholesterol levels were reduced during tumor development but not prior to its initiation. These data provide new evidence for an increased utilization of cholesterol by tumors and for its role in tumor formation. Taken together, these results imply that an increase in plasma cholesterol levels accelerates the development of tumors and exacerbates their aggressiveness.

Figure 1

Increased tumor incidence and burden in PyMTTg mice fed a Western diet. A: Female mice were palpated at 8 weeks of age for development of tumors in mammary glands. Mice were examined in a genotype-blinded fashion and palpated in each of the 10 mammary glands. The presence of tumors was examined in mice from each group (PyMTTg fed a chow or Western diet for 4 weeks). The number of tumors per mouse was determined. Results are given as mean ± SE (n = 15). Significant difference at *P < 0.05. B: Tumors were isolated from mice in each group (PyMTTg fed a chow or Western diet for 8 weeks) and weighed. Results are given as mean ± SE (n = 15). Significant difference at *P < 0.05. C: Representative images of tumors obtained from 12-week-old female PyMTTg mice fed a chow or a Western-type diet.

Figure 2

PyMTTg mice fed a Western-type diet exhibit increased lung metastasis. Lungs obtained from 12-week-old female PyMTTg mice were obtained as described in the Materials and Methods section. They were sectioned at 50-μm intervals and stained with H&E. Lung metastasis was scored as the total number of metastatic foci (defined as a cluster of at least 10 cells) per lung. Nine animals were analyzed for each diet. A: Incidence of metastasis in PyMTTg mice fed a chow or Western-type diet. B: Distribution of lung metastases in PyMTTg mice fed a chow or Western-type diet.

Figure 3

PyMTTg mice fed a Western-type diet demonstrate accelerated development of multifocal dysplastic lesions in the mammary gland. Whole mounts of mammary glands were obtained from 3-week-old (A) and 4-week-old (B) virgin female mice whose mothers had received either a chow or a Western diet during pregnancy and the lactation period. Right inguinal mammary glands were fixed in ethanol and acetic acid for 2 to 4 hours and stained overnight with carmine dye. Representative images are shown for each age and diet group. A mammary whole-mount preparation obtained from a wild-type mouse is included for comparison. The primary duct (PD), which originates from the nipple area, is visible in the top left corner of all images. The subiliac lymph node (LN) is visible to the right of the images. Arrows indicate a few examples of dysplastic foci. C: Quantification of the number of hyperplastic lesions per gland in 3-week-old female mice. Results are given as the mean ± SE (n = 8 and 6 for chow and Western-diet groups, respectively; **P < 0.01). D: Quantification of the total area occupied by multifocal dysplastic lesions in 3-week-old female PyMTTg mice. Data are given as mean ± SE (n = 8 and 6 for chow and Western diet groups, respectively; **P < 0.01). E: Quantification of total lesion area in 4-week-old female PyMTTg mice. Results are given as mean ± SE (n = 6 and 9 for chow and Western diet groups, respectively; ***P < 0.001).

Figure 4

PyMT mice fed a Western diet exhibit more advanced mammary carcinogenic lesions. Right inguinal mammary glands were dissected from 8-week-old PyMT transgenic mice fed a regular chow diet or a Western-type diet, fixed, embedded in paraffin, sectioned, and stained with H&E. Using the lymph node as a reference, the proximal (secondary lesions) and distal (primary tumors) areas were analyzed for each mouse. Each tissue section was classified as being normal (N), hyperplastic (HP), well-differentiated carcinoma (WD), moderately differentiated carcinoma (MD), or poorly differentiated carcinoma (PD). The percentage of mice with mammary glands in a given histopathological stage in the proximal (A) and distal (B) areas is represented. Representative histological images are shown for each diet and each tissue section examined. Eight animals were analyzed for each diet. Proximal and distal indications refer to lymph node location. Original magnification, ×40.

Figure 5

PyMT mice fed a Western diet exhibit expression patterns of progression markers consistent with more advanced mammary gland carcinoma. A: Left inguinal mammary glands isolated from 8-week-old female PyMTTg mice were lysed and subjected to immunoblot analysis for ERα, cyclin D1, and caveolin-1. GDI and cytokeratin were used as controls for equal protein loading and epithelial cell content, respectively. Two representative images of the 6 animals examined in each dietary group are shown. B–D, Right inguinal mammary glands from 8-week-old female PyMT mice were excised, formalin-fixed, paraffin-embedded, sectioned at 5-μm intervals, and subjected to immunohistochemical analysis. Representative images are shown for ERα (B), cyclin D1 (C), and caveolin-1 (D) immunohistochemistry in each of the dietary conditions. Six animals were analyzed for each diet. Original magnification, ×60.

Figure 6

Increased plasma cholesterol is associated with increased tumor angiogenesis and lipoprotein receptor in PyMTTg mice. Right inguinal mammary glands from 8-week-old female PyMTTg mice were excised, formalin-fixed, paraffin-embedded, cut to obtain 5-μm sections, and subjected to immunohistochemical analysis. A: Representative images are shown for CD31 immunohistochemistry in each of the dietary conditions. Three animals were analyzed for each diet. Original magnification, ×40. B: Microvessel quantification was performed by counting the number of CD-31–positive vessels in 6 representative fields for each section. Results are given as mean ± SE. Significant difference at *P < 0.05. C: Left inguinal mammary glands isolated from 8-week-old female PyMTTg mice were lysed and subjected to immunoblot analysis for SR-BI and LDL-R. GDI and cytokeratin were used as controls for equal protein loading and epithelial cell content, respectively. Three representative images of the 6 animals examined in each dietary group are shown. D: Right inguinal mammary glands from 8-week-old female PyMTTg mice were excised, formalin-fixed, paraffin-embedded, cut to obtain 5-μm sections, and subjected to immunohistochemical analysis for SR-BI. Representative images are shown for each dietary condition. Six animals were analyzed for both chow and Western-type diet groups. Original magnification, ×60.

Figure 7

PyMT transgenic mice show decreased plasma cholesterol levels after tumor formation. Fasting plasma samples isolated from 10 mice from each group were pooled and loaded on two Superose 6 columns. Fractions were collected and analyzed for cholesterol content using a colorimetric assay. Lipoprotein profiles obtained from 12-week-old wild-type and PyMTTg female mice fed a chow diet (A) or a Western-type diet (B) for 8 weeks. Note the reduction in HDL-cholesterol levels and HDL size (peak shifted to the right) observed in PyMTTg mice compared with wild-type mice that did not develop any tumors.

Figure 8

Mammary tumor cholesterol content is not affected by a Western-type diet in PyMTTg mice. Total cholesterol content of mammary tumors was determined with tissues obtained from 12-week-old PyMTTg mice fed a chow or a Western-type diet for 8 weeks. Portions of the tumors were homogenized, lipids were extracted, and total cholesterol content was determined using a colorimetric assay. Results are given as mean ± SE (n = 10 tumors analyzed for each dietary group). No statistical differences were observed between groups.