Genetic Aspects of Physiological Response During Lactation

Institution: University of California, San Diego
Investigator(s): Randall Johnson, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7PB-0108 Award: $593,000
Award Type: Request for Applications
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2001)
As tumors grow, they require new blood vessels. This also holds true for normal tissues as they expand. The stimulus for this new blood vessel growth is thought to be the sensation of lowered oxygen levels (hypoxia) by the tissue as it grows too large for its existing blood vessel network. In response to hypoxia, the activity of a transcription factor called HIF-10 increases rapidly, resulting in activation of the genes that control new blood vessel growth. Mouse embryos that lack HIF-10 die at mid-gestation, and our laboratory has also shown that HIF-10 helps tumors grow, since tumors without the gene grow more slowly, and recruit fewer blood vessels, than tumors with the gene intact. Recently, HIF-10 has been observed at high levels in a variety of human tumors, especially breast tumors that exhibit high rates of cellular division.

The goal of this project is to determine the role of lowered oxygen levels and blood vessel development in mammary gland during pregnancy and lactation. We hypothesize that the response to lowered oxygen contributes to mammary gland development. Since angiogenesis accompanies pregnancy prior to lactation, we predict that loss of HIF-10 will decrease cellular proliferation within the normal mammary gland, subsequently impairing the production of milk. We will dissect the function of HIF-10 during normal mammary development by making mice that are missing HIF-10 in their mammary glands and other mice that have HIF-10 constantly present in their mammary glands. We will determine the effect of these different genetic backgrounds on the proliferative abilities and oxygenation levels of breast cells as well as the rate of new blood vessel growth and correlate them to the development of a functional mammary gland. We have recruited Drs. Jeffrey Rosen and Darryl Hadsell, both specialists in normal mammary gland development and active breast cancer researchers, to help us analyze defects in our model system. In own laboratory, Dr. Tiffany Seagroves has been trained as a mammary gland specialist. These experiments should demonstrate how a regulated response to hypoxia creates a functional breast.

By analyzing mice that lack HIF-10 specifically in the mammary gland, it may be possible to discover mechanisms to block blood vessel growth in breast tumors, potentially evolving into new therapies to treat breast disease. Although it has been known for several years that tumors have pockets of hypoxic regions that are correlated with decreased efficacy of radiation treatment, the role that hypoxia plays during normal mammary gland development has not been investigated. Our hope is to generate background information on the function of HIF-10 to better target therapies to hypoxic regions of tumors, which are often otherwise resistant to conventional modes of treatment.

Final Report (2005)
As part of their selection for a growth advantage, most solid tumors have bypassed the normal cellular control of metabolism and angiogenesis, the formation of new blood vessels. Adaptation to cellular stress is necessary for tumor progression since compared to normal tissues, nutrients and oxygen are rapidly depleted in tumors. In response to hypoxia, or the exposure to low levels of oxygen, tissues try to restore homeostasis by regulating cellular metabolism and by inducing angiogenesis. The expression, activity and protein stability of the transcription factor Hypoxia-Inducible Factor (HIF)1-alpha is increased rapidly under hypoxic conditions, resulting in the induction of several genes implicated in metabolism, angiogenesis and cell death. Recently, HIF-1 alpha has been demonstrated to be up-regulated in poor grade, highly proliferating, breast tumors.

To address whether HIF-1 alpha is a critical regulator of mammary gland development, we have compared mammary gland development and physiology in two conditional gene-deletion mouse models in which either HIF-1alpha, or a protein that regulates HIF-1alpha protein stability, von Hippel-Lindau (VHL), is deleted following activation of a mammary-specific transgene, the Cre recombinase protein. Mice that lack the Cre transgene are used as controls. Mice that lack HIF-1alpha function do not develop sufficient numbers of alveoli, the differentiated, secretory units of the gland, by lactation and fail to properly nourish their litters. Deletion of HIF-1 alpha also decreased the quality of milk produced, which contained less water, less lactose, the primary milk sugar, and increased sodium and chloride levels, perhaps due to leakiness of the junctions that normally seal off the mammary epithelial cells from each other. As a result, pups born to Cre-positive dams weigh 33-50% less than pups nursing Cre-negative control dams. In contrast, when VHL is deleted, HIF-1alpha is over-expressed, leading to over-expression of HIF-1 target genes such as vascular endothelial growth factor (VEGF), a key regulator of angiogenesis. Upon multiple rounds of breeding, loss of VHL function causes the mammary glands to become hyper-vascularized, however, the glands lack fully differentiated alveoli. With each round of breeding, the alveoli become more de-differentiated and microvessel density increases. However, deletion of VHL alone is not sufficient to result in mammary tumors.

The results obtained from analysis of our mouse models, the first to directly address the role of the hypoxic response to normal mammary gland development, suggest that either deletion or over-expression of HIF-1alpha (accomplished via deletion of VHL) impairs the developmentally-regulated metabolic and angiogenic pathways necessary for successful lactation. Through continued analysis of the mechanisms that are defective in these mouse models, it may be possible to discover therapies to block angiogenesis and/or proliferation of mammary cells, potentially evolving into new therapies to treat breast disease.

HIF-1alpha is a critical regulator of secretory differentiation and activation, but not vascular expansion, in the mouse mammary gland.
Index Medicus:
Authors: Seagroves TN, Hadsell D, McManaman J, Palmer C, Liao D, McNulty W, Welm B, Wagner, et al.
Yr: 2003 Vol: 130 Nbr: 8 Abs: Pg:1713-24