Saturday, May 31, 2014

A closer look into CYP7B1

As our project progresses, Matt and I are starting to delve deeper into many of the topics associated with cholesterol and cancer.  One of these very important topics is 7ɑ-hydroxylase (usually referred to by its gene name CYP7B1), a very important enzyme that works to hydrolyze the infamous 27-hydroxycholesterol (27HC).  27HC is a cholesterol metabolite that has recently been shown to promote ER positive breast cancer.  This is really all you need to know about this molecule.  However, for more information on 27HC, you can search the blog for my earlier post Cholesterol and its Promotion of ER+ Breast Cancer.  If you are unable to find my post, follow this link to another information page for 27HC.

Physiological roles of CYP7B1

Bile salt synthesis
25-Hydroxycholesterol, 27-hydroxycholesterol
Steroid hormone metabolism
Pregnenolone, dehydroepiandrosterone
Metabolism of estrogen receptor ligands
Immunoglobulin production
    Immune cells

CYP7B1  is an enzyme that works to hydrolyze many different molecules of cholesterol synthesis, including 27HC.  It is found in many parts of the body, as shown in the below table (Table 1).  The different physiological functions of this enzyme are very interesting, such as its role in the creation of stomach bile.  However, many functions of CYP7B1 are unknown, especially when it comes to the brain.  What is clear is the effect that CYP7B1 has on 27HC and the effect this function seems to have on ER + breast cancer.
Fig. 1.
Morphological phenotype of 2-week-old mice. (A and B) Whole-mount staining of mammary glands. In 2-week-old WT mice (A), there was no elongated side branching of ducts (red arrows), as is seen in CYP7B1–/– mice (B). (C and D) BrdUrd staining of mammary glands after 2-h BrdUrd treatment. BrdUrd-labeled DNA was visualized with an anti-BrdUrd antibody. In WT mice, very little BrdUrd incorporation could be detected (C), but in CYP7B1–/– mice, there were many BrdUrd-positive nuclei (D), suggesting that mammary gland growth in CYP7B1–/– mice started earlier than in WT mice. The red arrow signifies a BrdUrd labeled cell. (E and F) Hematoxylin and eosin staining of uterus from WT (E) and CYP7B1–/–(F) mice. There are more glands in the uteri of CYP7B1–/– mice than in WT mice. (GI) BrdUrd staining of uterus after 2-h BrdUrd treatment. There was more incorporation of BrdUrd into DNA in the uteri of CYP7B1–/– mice (H) than there was in WT mice (G). The percentage of total epithelial cells, which were BrdUrd stained in WT and CYP7B1–/– mice is shown in I.(J and K) ERα staining of mammary glands. There are more ERα-positive epithelial cells and staining is stronger in CYP7B1–/– mice (K) than WT mice (J).

Fig. 2.
Whole-mount examination of mammary glands from 2- to 4-week-old mice. Two-week-old WT (A) and CYP7B1–/– (B) mice, 3-week-old WT (C) and CYP7B1–/– (D) mice, and 4-week-old WT (E) and CYP7B1–/– (F) mice are shown. CYP7B1–/– mice show more ductal elongation and branching than WT mice at every age. Mammary ducts are encircled in red.
Before the link between ER positive breast cancer and 27HC was attained, a paper was written discussing the effects of limiting CYP7B1 on breast and ovarian growth in mice.  In this paper they discuss their experiment.  In this experiment they took two types of mice; one group was just normal lab mice who had CYP7B1 expressed (control) and the other group had CYP7B1 knocked out (experimental).  The second group was referred to as CYP7B1 -/-.  The mice were kept in the same conditions and after a certain amount of time, were killed so that their cells could be analyzed.  What they found was that the mice who did not have CYP7B1 showed much more cell proliferation in the mammary glands, also showing growth much earlier in life as well.  In figure 1, we see the greater cell proliferation, illustrated through the greater branching of the mammary ducts of the CYP7B1 -/- mice after 2 weeks compared to the control.  To solidify this finding, the researchers used BrdUrd to better monitor the cell proliferation and to put the results into graphical form (figure 1I).  BrdUrd is used to analyze cell proliferation, with greater amounts of BrdUrd illustrating greater cell proliferation.   Later on in the paper they show that this increased cell proliferation continues for 4 weeks, with the CYP7B1 -/- mice showing greater ducal elongation and branching when compared to the control mice at all time points (Fig.2).  Finally, they looked over the long term, going from 6 months to 15 months of age (fig. 3 A-F).  At these time points they monitored the mammary growth, see that for 6 month old mice, we see the increased branching of the CYP7B1 -/- mice still being expressed.  However, at 15 months, the researchers saw involution of the mammary glands in CYP7B1 -/- mice as opposed to normal function of the mammary glands in the control mice.  This illustrates how the early onset of growth in the CYP7B1-/- mice caused for the earlier involution of the mammary glands.  This experiment was very important in determining the role of CYP7B1 and its effects on cell proliferation in breast tissue, specifically the mammary glands. 

Fig. 3.
Morphological phenotype of adult CYP7B1–/– mice. Comparison of mammary glands from 6-month-old WT (A) and CYP7B1–/– (B and C) mice showed a much more extensively branched ductal tree in CYP7B1–/– mice. (C) Higher magnification of the ducts in B. There was no difference between the two genotypes in lobular formation. The mammary glands from 15-month-old mice (DF) show that, in CYP7B1–/– mice (E and F), there are signs of involution (F, higher magnification of the ducts in E), whereas ducts appear still to be active in WT mice (D). (GL) Uteri at the ages of 6 (G and H),8(I andJ), and 15 (K and L) months. Uteri of 6-month-old CYP7B1–/– mice (H) had larger lumina than did WT mice (G). Enlarged lumina of the uteri were evident in 8-month-old CYP7B1–/– mice (J) but not in WT mice (I). By 15 months of age, the lumina of the CYP7B1–/– mice (L) were extremely enlarged with sclerotic and atrophic glands, whereas the uterine morphology in the age matched WT mice (K) was essentially similar to that of 6-month-old WT mice (G).

What this paper finds overall is that the absence of CYP7B1 allows for greater cell proliferation.  This larger amount of cell proliferation is due to the fact that CYP7B1 is a limiter of 3beta-Adiol, which is and androgen metabolite that is an inhibitor of ER-alpha and ER-beta (estrogen receptors alpha and beta) in the presence of CYP7B1.  However, when CYP7B1 is no longer present, 3beta-Adiol becomes estrogenic for both the ER receptor types and begins to cause increased cell proliferation and early onset puberty.  This is despite the fact that ER-beta is usually used in cell growth limiting.  However, for a reason still unknown to researchers, the function of the ER-beta, 3beta-Adiol binding becomes growth promoting in the absence of CYP7B1.  What this does is it causes for increased risk for breast cancer due to the large increase in cell proliferation, both in the mammary tissue as well as ovarian tissue.  For the purposes of our research, I have focused the above analysis on mammary tissue.  This would explain the reason behind the better outcomes of ER positive breast cancer patients showing higher levels of CYP7B1.  This fact is supported by what we learned in class, that areas of the body with more cell proliferation tend to have higher rates of breast cancer.  This is due to the increase in the number of chances for DNA damage.  In organs such as the breast that already have high cell proliferation, this loss of CYP7B1 can be devastating.  It can increase the chances of cancer in an already cancer susceptible organ.  As I stated in my previous blog, this knowledge can be very helpful in developing treatments that work to promote CYP7B1.  With treatments that are using native body enzymes, there is less chance of adverse side effects while still gaining a much better outcome. 

Works Consulted
  1. Omoto, Y. "Early Onset of Puberty and Early Ovarian Failure in CYP7B1 Knockout Mice." Proceedings of the National Academy of Sciences102.8 (2005): 2814-819. Web.   
  2. 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.