Normal Mammary Biology of Phosphorylated Prolactin

Institution: University of California, Riverside
Investigator(s): Ameae Walker, Ph.D. -
Award Cycle: 2004 (Cycle 10) Grant #: 10PB-0127 Award: $541,444
Award Type: Request for Applications
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point



Initial Award Abstract (2004)
Prolactin is a hormone that enhances two almost opposite activities in the breast. It stimulates breast cells to grow during pregnancy and then causes them to mature and produce milk production while nursing an infant. Stimulation of growth means promotion of cell division and therefore you would expect prolactin to promote breast cancer. Stimulation of milk production would inhibit cell division and therefore you would expect prolactin to inhibit breast cancer. These two very different activities of prolactin have in the past been attributed to interactions with other hormones whose concentrations differ markedly between pregnancy and lactation. Using animal models and animal and human cells in culture, however, we have demonstrated that two different forms of prolactin are responsible for the apparently opposite activities. The growth promoting form increases in the blood during pregnancy in response to rising estrogen levels, while the growth inhibiting form interestingly accumulates to very high concentrations in the milk. Cell surface molecules that respond to prolactin are present in highest concentrations on the side of the mammary epithelial cells that faces the milk.

We hypothesize that the growth inhibitory form of prolactin is important to normal breast health and that it has major effects when present in the milk. We will use animal models to determine how the very large proportion of inhibitory prolactin accumulates in milk. We will use tissue culture models to test whether milk prolactin has effects on mammary epithelial cells.

The novel idea is the possibility that the inhibitory milk prolactin is active on the milk side of the breast cells. If milk prolactin is locally active, this suggests future investigation of a) its role in the protective effect of an early first pregnancy, and b) the potential for prophylactic or even therapeutic application of this form of the hormone via nipple ducts. Knowledge of the mechanism of milk accumulation will allow us to examine ways to increase production of the growth inhibitory form without pregnancy.


Progress Report 1 (2006)
When cells are expending a lot of energy to divide, they usually do this at the expense of other functions such as the production of secretory products. Prolactin is a hormone credited with stimulating both cell proliferation and the production of a secretory product in breast cells. These two very different, and essentially opposing, activities of the hormone have in the past been attributed to differences in the co-existing steroid hormone environment. However, work from our lab has provided evidence that slight differences in the prolactin molecule are also important. This slight difference concerns the addition of a phosphate group to prolactin. A physiologically-regulated proportion of circulating prolactin is phosphorylated, and the high amounts of prolactin in milk are of mostly phosphorylated hormone. While this latter fact may be of some significance to infants, the presence of cell surface receptors that recognize prolactin on the milk face of breast cells also suggests that phosphorylated prolactin may be of importance to normal mammary biology and health.

In the past year:
標e have published an article where we used a normal mouse mammary cell line cultured in such a way as to behave similarly to normal breast epithelium. In these cultures, where the cells show minimal ability to proliferate and most receptors are on the milk face, we have shown that unmodified prolactin promotes the expression and activity of cell proliferation-promoting proteins. By contrast, a molecular mimic of phosphorylated prolactin (S179D prolactin) promotes expression of cell cycle inhibiting proteins. This suggests that unmodified prolactin primes the cells to proliferate, perhaps overcoming other normal constraints and certainly contributing to promotion of breast cancer. At the same time, it suggests that phosphorylated prolactin in the milk may help to keep cell proliferation in check.
標e have also produced evidence that phosphorylated milk prolactin is derived from circulating prolactin and that prolactin enters the milk as a complex with a kind of antibody molecule which has long been known to make the trip across breast cells.
標e have discovered a new, naturally-produced, intracellular molecule that when placed in breast cancer cells, inhibits their proliferation.
標e have discovered a new form of the prolactin receptor expressed by cancer cells which can stimulate proliferation of the cells in the absence of prolactin.
標e have learned a lot about the different prolactin receptors and their ability to respond to each form of prolactin.
標e have learned that unmodified prolactin also contributes to cell proliferation by collaborating with estrogen, and that it may contribute to the progression of breast cancer by decreasing expression of the estrogen receptor.

In the next project period, we will apply what we have learned so far to unequivocally demonstrate that milk prolactin can signal from the milk face of breast cells to bring about its anti-proliferative (and pro-specific cell function) activities. Thus, we aim to determine whether the presence of phosphorylated prolactin on the milk face contributes to the pregnancy-protection against breast cancer. In addition, this opens up possibilities for intraductal prophylactic treatment of high risk groups/ treatment of those with early in situ disease. Work on the signaling from different receptors and the newly-identified breast cancer inhibiting molecule, and breast cancer promoting form of the receptor, should also open up new avenues for intervention at all stages of the disease.

Publications:
Blockade of estrogen-stimulated proliferation by a constitutively-active prolactin receptor having lower expression in invasive ductal carcinoma
Prolactin inhibits a major tumor-suppressive function of wild type BRCA1




Symposium Abstract (2010)

We and others have provided evidence that links increased exposure to the hormone prolactin (PRL) to the development of breast cancer. However, PRL comes in two major forms, unmodified (U) and phosphorylated (P*). The CBCRP project was designed to determine the different functions of U- and P*-PRL in the mammary gland. What we found is that U-PRL increases cell proliferation (both positively and by hampering the effects of proteins that normally inhibit cell proliferation) and that P*-PRL essentially does the opposite. In addition, P*-PRL promotes cell-specific function. In other words, P*-PRL has anti-cancer activities. To take advantage of these activities, we have developed a stable, synthetic version of P*-PRL, called S179D PRL. S179D PRL not only inhibits basal and U-PRL-stimulated growth of breast cancer cells, but also and very importantly it entirely blocks estrogen-stimulated growth. Since a greater proportion of breast cancers have PRL receptors (molecules that would allow them to respond to S179D PRL) than estrogen receptors, the potential therefore exists for S179D PRL to be a more widely useful therapeutic than either estrogen receptor antagonists or aromatase inhibitors.

To increase our understanding of how both types of PRL work in the intact gland, we determined the distribution of PRL receptors, and obtained several unexpected results: First, except during lactation, almost all PRL receptors on duct-lining mammary cells were found on the side facing the duct lumen and not on the side facing the blood. In order for either form of PRL in the blood to interact with these receptors it is therefore necessary for it to pass between the cells to first gain access to the duct. Second, we found two binding proteins in ductal fluids, one that alters the availability of both forms of PRL (type 1) and one that alters the way U-PRL interacts with the PRL receptor (type 2). We have determined that the type 2 binding protein is produced in much lower amounts by breast cancer versus normal tissue from the same patients. Together, these results suggest that ductal content of U-PRL versus P*-PRL and the binding proteins may be more relevant to development of disease than circulating PRL, although it is clear that blood PRL does gain access to the ducts. Third, we have shown that the cells underneath the duct lining cells, known as myoepithelial cells, have lots of PRL receptors, suggesting they are a heretofore unrecognized important target of PRL. These cells proliferate, migrate and invade underlying connective tissue during development of the gland in pregnancy, and are thought to be the source of a particularly aggressive, but less common form of breast cancer. Follow up on this finding will determine whether this form of breast cancer might also be responsive to S179D PRL.

Thus, funding by CBCRP has markedly developed our understanding of the vastly different roles of the two forms of PRL in normal mammary gland biology and the development of breast cancer. Furthermore, the work has identified S179D PRL as a potential novel therapeutic for breast cancer.



Blockade of estrogen-stimulated proliferation by a constitutively-active prolactin receptor having lower expression in invasive ductal carcinoma
Periodical:Cancer Letters
Index Medicus: Cancer Lett
Authors: Kuang-Tzu Huang, Dunyong Tan, Kuan-Hui E. Chen, Ameae M. Walker
Yr: 2015 Vol: 358 Nbr: 2 Abs: Pg:152-160