Rac/STAT5 Signaling

Institution: The Burnham Institute for Medical Research
Investigator(s): Hee Kwang Choi, Ph.D. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8FB-0139 Award: $79,824
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point



Initial Award Abstract (2002)
Normal mammary gland function depends on proper interactions of breast epithelial cells with the surrounding tissue. Via these interactions, breast cells get guidance from their environment that regulates their responses to hormones, such as prolactin and estrogen, and enables normal growth and function. Disruption of these delicately balanced interactions will result in loss of tissue structure and function, resulting in malignant transformation of the cells and breast cancer. My objective is to examine the intracellular biochemical events that regulate normal breast cell function in a three-dimensional environment, and determine how inadvertent activation of these events may contribute to malignant transformation and treatment-resistant breast cancer.

I have found that two intracellular signaling molecules termed Rac and STAT5 are likely to function in concert and regulate normal breast function. Based on my preliminary findings, I hypothesize that the Rac/STAT5 complex functions as a molecular switch and controls the appropriate activation of mammary gland genes in response to prolactin and estrogen stimulation, allowing proper mammary gland function. My findings and recent published data further supports the hypothesis that a chronic activation of Rac/STAT5 signaling may in turn result in estrogen-independent growth and antiestrogen resistance, thereby aiding breast cancer development and drug-resistance. These hypotheses will be experimentally tested here.

Three general areas of study will be investigated. First, activation of STAT5 by Rac1 will be studied in normal breast cells in a three-dimensional culture model that mimics normal physiology of mammary gland. Antibodies that bind to activated STAT5, as well as the ability of STAT5 to bind specific DNA sequences, will be utilized to see Rac1 can activate STAT5 in normal breast cells. Second, the ability of activated STAT5 to regulate estrogen receptor action in normal breast cells will be studied. The ability of STAT5 to prevent the estrogen receptor from binding DNA and turning genes on will be studied, thereby elucidating the importance of STAT5-estrogen receptor interaction in normal breast cells. Third, the role of STAT5 in estrogen-independent tumor formation will be studied. A mutant STAT5 that is constantly active will be introduced into normal cells. Tumor formation and ability of the breast cancer cells to grow without estrogen, and in the presence of antiestrogens, such as tamoxifen, will be studied.

Normal breast cells are difficult to study using conventional techniques because their interactions with each other and with proteins outside of the cell are critical for normal behavior. In order to overcome this, I will simulate the environment of breast cells by growing cells in a manner that mimics the 3-dimensional shape and extracellular environment of the cells. This is a novel approach that will allow experimental manipulation within a normal cell context. Hence, the behavior of normal cells can be studied and compared with cells that have become tumorigenic at a molecular level. This approach is anticipated to provide important information of normal breast biology and cancer progression.


Final Report (2004)
Mammary gland epithelial cells integrate signals from the extracellular matrix (ECM) as well as soluble hormones for correct cellular function, and their dysregulation can result in malignant transformation of the breast. However, little is known about the signaling events downstream of integrin signaling, and their interplay with cell responses to hormones such as prolactin (PRL) and estrogen.

My initial goal was to examine the interaction between signaling molecules downstream of integrins, namely the small GTPase Rac1 and the pituitary hormone-responsive transcription factor STAT5 (signal transducer and activator of transcription). A reconstituted COS1 cell system was used to investigate the potential interaction of STAT5 isoforms with Rac1. However, a constitutively-activated form of Rac1 did not significantly alter the tyrosine phosphorylation status, nor DNA binding, of STAT5 as compared to wildtype or dominant-negative Rac1. Nor was a direct interaction between STAT5 and Rac1 seen as judged from pull-down assays. Overexpression of the dominant-negative Rac1 did not modulate activation of STAT5 by PRL, or growth hormone (GH). Because Rac1 and other signaling molecules downstream of integrins are recruited to a cholesterol-rich areas of the cell membrane (lipid raft microdomain), a revised strategy of this research project was to study integrin-dependent modulation of STAT5 signaling mediated by lipid rafts.

Hormone activated STAT5 was markedly reduced when cells were preincubated with -methylcyclodextrin (-MC), a lipid raft disrupting agent, and activation was rescued with excess cholesterol. Sucrose gradient cell fractionation studies show that STAT5 was recruited to putative lipid raft domains only upon stimulation by PRL or GH. The PRL receptor was found constitutively associated with the low density raft domains but was resistant to disruption by -MC. Additionally, hormone-dependent JAK2 tyrosine kinase activation was unaffected by pretreatment with -MC. These studies suggest that STAT5 recruitment to lipid rafts upon hormone stimulation is unlikely to occur via a mechanism involving PRL receptor or JAK2, and may require an additional set of conditions, perhaps the engagement of integrins to the ECM and the sequestration of their intracellular effectors. Hence, the modulation of STAT5 activity by lipid rafts may be a key insight into the dysfunction of concerted hormone and integrin responses that lead to disease.