27HC, CYP27A1, and Breast Cancer

Myself and Erik Christensen are investigating the relationship between breast cancer and cholesterol for our Cancer Project. In our presentation we are going to give you a brief overview about what 27HC was, but I wanted to blog a little more in-depth about it. In addition, I wanted to introduce CYP27A1 and its effect on 27HC and breast cancer as we won't be discussing that aspect of our topic in our presentation.

The Cholesterol Metabolite 27-hydroxycholestorol (27HC) promotes cell proliferation. Specifically, in this case we are looking at its promotion of ER positive breast tumors. What makes 27HC different is the fact that it is an abundant primary metabolite of cholesterol. Because of its relationship with cholesterol it can attach to and actually stimulate estrogen receptors in the body. Since 27HC is a cholesterol metabolite transported in the same lipoprotein particles as cholesterol, there was a positive association between lipoprotein particles and cholesterol. Patients with ER+ breast cancer have higher amounts of 27HC in breast tissue than women who have not been specifically diagnosed with ER+ breast cancer. Below you can see the process of cholesterol synthesis and where 27HC stands in comparison to cholesterol.

The precedent behind 27HC that we wanted to try and see was if it really did have an effect on breast cancer. In order to determine if 27HC was indeed a promoter of ER positive breast cancer study was conducted with mice. The goal was to determine if 27HC promoted MCF-7 cell growth (Nelson, et. al. 2013). As we can see in Figure 1A, 27HC promotes MCF-7 cell growth. Due to unesterified plasma levels approximating to 10−8M, the threshold concentration for activation of MCF-7 cell proliferation was 10−8M. This can be seen in figure 1B.

Figure 1. 27HC promotes MCF-7 cell and Ishikawa cell proliferation, and in vivo 27HC stimulates MCF-7 cell xenograft growth and a uterotrophic response. A–D. Cell proliferation was evaluated by quantifying BrdU (A) or 3H-thymidine incorporation (B–D), n= 4–8. A. Growth responses of MCF-7 cells to E2 (10−8M) or 27HC treatment (10−8 to 10−6M) for 24h were compared. B. The dose-response of MCF-7 cells to 27HC (10−9 to 10−6M, for 24h) was determined. C. The requirement for ERα in the growth response of MCF-7 cells to E2 (10−8M) or 27HC (10−6M) was evaluated in cells treated with methyl-piperidino-pyrazole (MPP, 10uM) for 24h. D.

So with this information it would be wise to see the potential impacts of 27HC on ER+ breast cancer. Since 27HC is a cholesterol metabolite transported in the same lipoprotein particles as cholesterol, there was a predictable positive association between serum 27HC and cholesterol in both controls and cancer patients. We can see that this was eventually proven to be true in Figure 2C and 2D. In addition, compared with controls, there was a 3 fold greater 27HC concentration in normal breast tissue from cancer patients than controls. 27HC levels were 2.3 fold higher in the breast tumor itself than in the breast tissue.

27HC content is increased in normal breast tissue and tumors from ER+ breast cancer patients, and it is locally modulated. A,B. Serum 27HC (A) and total cholesterol concentration (B) in control and breast cancer patients (n= 17 and 58, respectively). Values for 10 cancer patients with serum 27HC greater than 2SD above the mean value for controls are shown in red. C,D. Relationship of serum 27HC to serum cholesterol concentration in controls (C, n=17) and cancer patients (D, n=58). E. 27HC content in normal breast tissue from controls (n=17) and cancer patients (n=48), and in tumors (n=32). *p<0.05 vs control, †p<0.05 vs cancer patient normal breast. F,G. Relationship of normal breast 27HC content to serum 27HC in controls (F, n=17) and cancer patients (G, n=40). H,I. Relationship of tumor 27HC content to serum 27HC (H) or normal breast 27HC content (I) in cancer patients (n=27).

Along the same lines, CYP27A1 is a gene sometimes referred as sterol 27-hydroxylase. Generally, it is expressed in the macrophages of the cell. We are interested in it because it is required for the conversion of Cholesterol into the metabolite 27HC. Interestingly enough, no matter whether macrophages of the breast tissue were malignant or benign, they consistently stained positively and strongly for the CYP27A1 protein. With an already established connection between macrophage infiltration and breast cancer (Wu, et al. 2013), we clamored to try and find other data and studies that incorporated this connection between the macrophage and breast cancer.

There were a couple of important findings from the next set of data we found research. The first was that (bone derived) macrophages were unable to support MCF7 cell proliferation when treated with CYP27A1 inhibitors. More importantly, introducing 27HC could reverse the effects of the CYP27A1 inhibitors. With the reversal process so effective, this indicated that local production of 27HC by macrophages in the tumor has a high impact on tumor pathology. The second observation was that CYP27A1 was expressed in the cancer cells themselves. From the data, the determination can be made that the expression was based on the size of the tumor. This can be seen below.

Table 1. Overexpression of CYP27A1 increases the likelihood of a higher tumor grade. Results of immunohistochemical analysis of CYP27A1 expression in human breast cancer tissue microarrays are shown. CYP27A1 expression was determined to be low or high and correlated with tumor grade. A Fisher’s exact test was used to determine P values for the likelihood of association. Ordinal logistic regression was used to estimate the odds ratio. N, sample number; N/A, not applicable (because sample number is too small).

The second main area studied was CYP27A1’s relation to a high cholesterol diet. In order to do this, researchers performed a study on mice where a group of mice was fed a high cholesterol diet from birth and another group on a control diet from birth. In addition to the different diet, both CYP27A1 positive and negative mice were tested on either one diet or another. The results show that CYP27A1 positive mice on a high cholesterol diet developed larger tumors faster and earlier than the mice on the control diet. Furthermore, the tumors were tested and those mice with the high cholesterol diet were found to high much higher levels of 27HC in the tumor than those mice that ate a controlled diet. Most surprisingly, the CYP27A1 negative mice had undetectable levels of 27HC in tumors and tumors formed much later and grew lower. Lastly, if the CYP27A1 negative mice that were fed the control diet were injected with 27HC, they started to produce the same exact results as the CYP27A1 positive mice on the high cholesterol diet. 

Genetic or pharmacological inhibition of 27HC production attenuates hypercholesterolemia- promoted tumor growth in mice. The latency and growth of tumors in the MMTV-PyMT mouse model of breast cancer were evaluated in mice in which the conversion of cholesterol into 27HC was inhibited by disruption of the CYP27A1 gene (CYP27A1−/−). For this study, MMTV-PyMT mice were bred onto a CYP27A1+/+ or a CYP27A1−/− background. (A) Tumor latency and (B) tumor growth were measured in mice on a control diet (CD) or a high-cholesterol diet (HCD) from weaning. Note that in the tumor growth studies, daily injection of 27HC overcame the inhibitory effect of CYP27A1 deletion. Significance between curves is indicated by a connecting black line and an asterisk (P < 0.05, n = 9 to 25).

In my opinion, this is the study’s most significant finding because it indicates that 27HC is the direct breast tumor promoter. In addition, the d at a hints that cholesterol metabolite 27HC stimulates MCF-7 cell growth in mice and lastly, in ER+ breast cancer patients, 27HC content in normal breast tissue is increased compared to cancer-free controls, and tumor 27HC abundance is further increased.

References:

1. Nelson, E. R., S. E. Wardell, J. S. Jasper, S. Park, S. Suchindran, M. K. Howe, N. J. Carver, R. V. Pillai, P. M. Sullivan, V. Sondhi, M. Umetani, J. Geradts, and D. P. Mcdonnell. "27-Hydroxycholesterol Links Hypercholesterolemia and Breast Cancer Pathophysiology." Science 342.6162 (2013): 1094-098. Web.
2. Wu, Qian, Tomonori Ishikawa, Rosa Sirianni, Hao Tang, Jeffrey G. Mcdonald, Ivan S. Yuhanna, Bonne Thompson, Luc Girard, Chieko Mineo, Rolf A. Brekken, Michihisa Umetani, David M. Euhus, Yang Xie, and Philip W. Shaul. "27-Hydroxycholesterol Promotes Cell-Autonomous, ER-Positive Breast Cancer Growth." Cell Reports 5.3 (2013): 637-45. Web.
3. Umetani, Michihisa, and Philip W. Shaul. "27-Hydroxycholesterol: The First Identified Endogenous SERM." NIH (2011): 1-10. National Institute of Health. Web.
4. Bianchi F, Kaaks R, Viano H. Overweight, obesity, and cancer risk. Lancet Oncol. 2002; 3:565-574. Web