Role of IKKa in Mammary Gland Development

Institution: University of California, San Diego
Investigator(s): Michael Karin, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7PB-0071 Award: $563,696
Award Type: Request for Applications
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2001)
Understanding the mechanisms involved in development of normal breast (mammary glands in mice) tissue will allow us to better understand what goes wrong when the epithelial cells of the breast grow in a deregulated fashion and form a malignant tumor. Clear understanding of such basic mechanisms should provide us the means to design much more specific and effective therapies for breast cancer. The principal investigator had identified an enzyme, a specific protein kinase called IKKa, whose inactivation specifically prevents the extensive proliferation of the mammary gland epithelium that normally occurs during pregnancy. Circumstantial evidence suggests that IKKa may also be involved in stimulating the proliferation of breast cancer cells and in protecting them from killing by anti-cancer drugs. This proposal is to study how IKKa controls the proliferation of normal mammary epithelial cells, which make up the milk forming apparatus of the breast. Preliminary evidence suggests that IKKa is a specific regulator of breast cell proliferation, whose inhibition may not result in adverse effects on other tissues or physiological processes.

The major question that needs to be answered is how IKKa acts to control the development of the mammary gland, especially during pregnancy. We also would like to understand why the enzymatic activity of IKKa is so important for proliferation of mammary epithelial cells but is dispensable for most other physiological processes. The central hypothesis is that IKKa , which is a subunit of a larger enzyme called IKK that contains another enzymatic subunit related to IKKa becomes specifically active during pregnancy. Once IKKa is active it generates a signal that increases the production of a protein called cyclin D1 that stimulates the activity of the cell division machinery of mammary epithelial cells. Inhibition of cyclin D1 expression interferes with proliferation of breast epithelial cells and many breast cancers exhibit high levels of cyclin D1. Curiously, IKKa either becomes active or exerts its growth stimulating activity only in mammary epithelial cells. We would therefore like to understand why IKKa has such a restricted activity. The proposed experiments will be mostly carried out in vivo, i.e. in intact experimental animals and therefore will provide information that is of high physiological relevance. As an experimental animal, we will use the laboratory mouse because the development of the mammary gland of female mice is very similar to the development of the human breast. To determine the involvement of specific genes in this process, we will generate mutant mice with specific genetic defects known as knockout or knockin mice. To examine the development of mammary glands in these mice, we will use a variety of histological and biochemical techniques.

The most exciting aspect of this project is that inhibition of the enzymatic activity of IKKa only interferes with the development of the mammary gland during pregnancy but has no adverse effect on development of other tissues and organs, viability, metabolism, fertility, behavior or general well being. Thus IKKa may be a highly specific regulator of breast development and may provide an outstanding target for development of drugs that inhibit the proliferation and increase the killing of breast cancer cells with hardly any side effects.

Final Report (2005)
The female breast, or the mammary gland as it is called in other mammals, is made of different types of cells, the major of which is the mammary epithelial cell. This cell type is also most important for breast cancer as its uncontrolled division is responsible for this type of cancer. Normally, the mammary epithelial cell divides in response to hormonal signals with most of the cell proliferation occurring during pregnancy, a period during which the mammary gland becomes a milk-producing organ.

We were interested in learning more about the regulation of mammary epithelial cell proliferation during pregnancy and in deciphering the molecular mechanisms underlying this process. We were most interested in determining which of the components of the normal developmental mechanism play an important role in the development of breast cancer. We postulated that the same mechanisms that underlie the normal control of mammary epithelial cell proliferation can go awry and give rise to breast cancer. Based on previous results we focused our efforts or studying the role of a protein that regulates gene expression called NF-κb in both normal cell proliferation and in breast cancer. Our studies were performed in mice which provide an excellent model system amenable to genetic manipulation that is of relevance to human breast cancer. The activity of NF-κb becomes deregulated and is constantly turned-on in human breast cancer, where it is known to prevent programmed cell death.

We have identified a biochemical pathway in which signals are transmitted from specific cell surface receptors to NF-κb in the nucleus leading to increased expression of genes whose protein products directly stimulate the division of mammary epithelial cells. We have proven the physiological importance of various components of this pathway, through the construction of mutant mouse strains that exhibit specific defects in mammary epithelial cell proliferation. These mice were used to examine the involvement of this biochemical pathway in the generation of breast cancer. Most importantly, we found that inactivation of a key component of the pathway that we have identified, a protein kinase called IKKα (a protein kinase is an enzyme that transfers phosphate groups to proteins), delays and interferes with the development of mammary gland cancer in mice using two distinct models. One of the models is similar to human breast cancer in that it is based on overexpression of a protein called ErbB2, that is also overexpressed in about 50% of human breast cancers. The second model is based on treatment of mice with a chemical carcinogen and the hormone progesterone. In both cases, inactivation of IKKα resulted in a dramatic decrease in tumor incidence and a significant delay in tumor emergence.

In the future we plan to investigate the involvement of IKKα in human breast cancer that is either positive or negative for ErbB2. We also plan to investigate the contribution of a related protein called IKKα to the carcinogenic process.

We found that inhibition of IKKα activity prevents the proliferation of mammary epithelial cells without any life threatening side effects. Importantly, our results indicate that IKKα activity is important for mammary gland carcinogenesis in mice in two different models of mammary cancer. Thus, inhibition of IKKα may be a relevant and revolutionary approach to the treatment of human breast cancer.

Symposium Abstract (2003)
NF-kB includes a group of proteins that have critical functions in inflammation and immune responses. Recent evidence also suggests that elevated NF-kB activity is a common marker for a variety of cancers, including breast cancer. We have previously generated a mutant mouse strain that contains mutation in the IKKagene, an important activator of NF-kB. This mutation renders IKKa unable to activate NF-kB, and subsequently, activation of cyclin D1 is impaired. As a result of it, female mice carrying this mutation display a severe lactation defect due to defective proliferation of mammary gland during pregnancy. Cyclin D1, another marker for breast cancer, is the major factor promoting cell proliferation in mammary gland. Given the importance of NF-kB and cyclin D1 in breast cancer, IKKa mutant mice provide us an ideal animal model to study mammary tumor associated with high NF-kB activity or cyclin D1 overexpression.

As in many types of cancer, tumor cells originate from the normal cells and often retain tissue-specific pathways but in a dysregulated way, we hypothesize that “upregulation” of IKKa/NF-kB/cyclin D1 pathway might underlie the pathogenesis of tumor formation in a subset of breast cancer cases. Possible mechanisms include overexpression of any of these components or captivation of the pathway by certain oncogenes leading to constitutive activation. We intend to determine whether IkkaAA mutation protects mice against breast cancer induced by several oncogenes.

To address this question, we have crossed the IkkaAA/AA mice with breast cancer mouse model carrying the MMTV-c-neu, which have been shown to depend on cyclin D1 to transform mammary epithe-lia. C-neu is the rat equivalent of human ErbB2/ HER2 gene, which is increased in up to 40% of breast cancer and is associated with poor prognosis. Littermates of Ikka+/+/MMTV-c-neu and IkkaAA/AA/ MMTV-c-neu were observed for breast cancer incidence, and our preliminary results suggest that Ikk_AA mutation reduces tumor incidence by MMTV-c-neu oncogene. In an approximate 6- to 9- month observation period, 17 of 19 Ikka+/+/MMTV-c-neu females have developed tumors, developing a total of 88 tumors (average 4.6 tumor per mouse). In contrast, 6 of 14 females developed tumors in the Ikk_AA/AA/MMTV-c-neu group, developing a total of 14 tumors (average 1.0 tumor per mouse). We have also noticed that the onset is delayed and growth rate is reduced in Ikk_AA/AA/MMTV-c-neu mice than wt mice.

These results suggest that IKKa/NF-kB pathway is activated during tumorigenesis, and this is induced by overexpression of HER-2/neu oncogene. IKKa, therefore, will be a promising drug target for breast cancer treatment in the future.

Grant Numbers: 7PB-007(Karin) and 6FB-0039 (Cao)

NF-kappaB in cancer: from innocent bystander to major culprit.
Periodical:Nature Reviews. Cancer
Index Medicus: Nat Rev Cancer
Authors: Karin M, Cao Y, Greten FR, Li ZW
Yr: 2002 Vol: 2 Nbr: 4 Abs: Pg:301-310