A Novel Signal Transduction Pathway in Breast Cancer

Institution: University of California, San Diego
Investigator(s): Yixue Cao, Ph.D., M.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6FB-0039 Award: $79,981
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2000)
Breast cancer cells differ from normal cells in both their lack of control for growth and their ability to respond to immune surveillance. Our interest is the response of cancer to factors associated with the immune response, such as tumor necrosis factor (TNF) and interleukin 1 (IL-1). In normal cells these immune factors will cause cell death through a process called apoptosis. However, cancer cells have specific ways to avoid cell death and persist in the face of immune attack. And, these same survival strategies serve to limit the effectiveness of chemotherapy and radiation treatments for cancer.

We have previously found that a type of intracellular protein complex, called NF-kB, is a key player in allowing cancer cell survival. This complex, upon removal of endogenous inhibitors, will translocate to the cell nucleus and allow gene activation associated with cell survival. The inhibitory proteins that regulate NF-kB appear to be essential to this survival pathway.

In this project we are looking for protein ‘partners’ of a NF-kB controlling factor, called IKKg. It appears that IKKg is the critical regulatory element and is distinguished from its ‘sisters,’ IKKa and IKKb, by lacking protein phoshorylation functions. However, we believe that IKKg is itself regulated by unknown additional proteins. I plan to use the technique of two-hybrid screening in yeast to find the IKKg binding proteins. Yeast is an ideal system to find this novel factor. We plan to clone, analyze the protein structure, prepare antibodies, and understand this portion of the NF-kB pathway. The end result, we believe, will provide a new understanding of how breast cancer survives in the face of immune surveillance and failed therapies. This will put us closer to controlling this aspect of breast cancer pathogenesis in either a prevention or treatment approach.

Final Report (2002)
The epithelial cells of the mammary gland undergo tremendous proliferation during pregnancy in a regulated fashion. The cell number increases over ten times comparing to the state before pregnancy. After the weaning of newborns, these cells will undergo a process called "apoptosis" (programmed cell death), which reduces the cell number back to non-pregnant state. The regulation of the balance between these two processes, cell division vs. apoptosis, by many genes is critical for the physiology and biology of mammary gland. This balance is critical for carcinognesis, since the underlying regulatory genes and proteins might become deregulated. The end result would be that the capacity of mammary epithelial cells proliferate might predispose them to cancer.

This project involves the detailed study of a gene-regulatory transcription factor, called NFκ- B. Normally, NFκ- B is localized in the cytoplasm of the cell where it is held in an "inactive state" by its association with an inhibitory complex, called IκB. A second group of proteins, called IKK serves to release the effect of IκB. This allows the NFκ- B complex to move to the nucleus where it can serve to regulate gene activity. In terms of cancer, it appears that NFκ- B is a central "player" in adjusting the balance between cell division and cell death. In fact, previous work in our laboratory (Dr. Michael Karin- http://medicine.ucsd.edu/pharmaco/mkarin.html) has established that tumor cells, including breast cancer, have the ability to "self-activate" NFκ- B and avoid cell death responses by immune factors and drug treatment. Thus, we are working towards a complete understanding of how this critical pathway is controlled and catalog all the proteins involved in its regulation. We often use mice for these studies, where we can selectively introduce and deleted genes of interest and observe the physiological effect.

In this project we focused on a protein kinase, called IKKα, which we believe is essential for mammary gland development during pregnancy. We studied this in mice and generated an Ikka AA knockin allele-- containing alanines instead of serines in the IKK activation loop. Ikkα AA/AA mice are healthy and fertile but females display a severe lactation defect due to retarded proliferation of mammary epithelial cells. IKKa activity is required for NF?- B activation in mammary epithelial cells during pregnancy and in response to RANK ligand, but not to TNF . IKKα and NFκ- B activation are also required for cyclin D1 induction. Interference with RANK signaling (i.e., TNF-alpha receptor Receptor Activator of NF-kB) or cyclin D1 ablation cause the same mammary gland defect as the Ikka AA mutation, which is completely suppressed by a mammary-specific cyclin D1 transgene. Thus, IKKa is a critical and specific intermediate in a signaling pathway that controls mammary epithelial proliferation in response to RANK signaling via cyclin D1. This pathway is responsible for proliferation of lobuloalveolar cells during pregnancy.

In the future, we intend to determine whether NFκ- B plays a causative role in breast cancer by mouse genetics. We hypothesize that IKKα /NFκ- B/cyclin D1 pathway, which controls cell proliferation in normal mammary gland, might have been deregulated in some cells resulting in tumor formation in a subset of breast cancer cases. We will test whether IKKα mutation will protect mice against breast cancer induced by several oncogenes, which are genes known to induce breast cancer when overexpressed.

As elevated cyclin D1 is involved in breast carcinogenesis and Ikkα AA/AA mice do not exhibit the severe sensitivity to apoptosis and infections, our results suggest that IKKa kinase inhibition may function as a specific breast cancer therapy that is relatively free of toxic side effects.

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)

IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development
Index Medicus: Cell
Authors: Cao Y, Bonizzi G, Seagroves TN, Greten FR, Johnson R, Schmidt EV, Karin M
Yr: 2001 Vol: 107 Nbr: 6 Abs: Pg:763-75