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    Awarded Grants
    The Role of Growth Factors/Stress Signaling Pathways in Developing Endocrine Resistance of Breast Cancer

    Scientific Abstract:
    The role of growth factors/stress signaling pathways in developing endocrine resistance of breast cancer. Background De novo and acquired resistance to endocrine therapy are major clinical problems in the treatment of breast cancer. My mentor's laboratory has previously reported that stress kinase pathways may be induced coincident with the development of acquired resistance to tamoxifen (Tam) or to prolonged estrogen (E2) deprivation. Activated JNK (stress-activated protein kinase), phospho c-jun, and AP-1 are all increased in these endocrine-resistant tumors, perhaps as a result of cellular stress. Our preliminary data further suggests that inhibiting AP-1, using a dominant negative AP-1 mutant, delays the onset of Tam resistance. JNK can be activated by growth factors as well as by cellular stress. Recent data further indicate that JNK indirectly activates estrogen receptor (ER) transcriptional activity, possibly through phosphorylation of coactivators such as AIB1. ERá levels and p38 MAP kinase (p38) activity are also up-regulated when breast tumors became resistant to E2 withdrawal or Tam. The p38 can phosphorylate ER on at least two different sites in vitro, and our preliminary data suggests that it can also phosphorylate the coactivator AIB1 which we believe plays an important role in Tam resistance in ER+ tumors amplified for HER-2. ER is subject to phosphorylation at several sites, and this modification can cause ligand-independent activation, and/or a synergistic increase in transcriptional activation of ER in the presence of E2 or antiestrogen, suggesting a role in treatment resistance. Hypothesis My hypothesis is that kinases in the stress/cytokine signaling pathway contribute to the failure of endocrine therapy by phosphorylating and activating ER and/or key ER coregulatory proteins like AIB1. The modifications of ER result in a receptor that can function at very low levels of E2 (hypersensitive receptor) or one which has significant E2-like agonist rather then antagonist activity when bound by Tam. Furthermore, I suggest that these processes can be delayed or reversed by inhibitors of these pathways, resulting in more effective combined “endocrine therapy”. Specific Aims: 1) Demonstrate in cultured breast cancer cells that modulating JNK and p38 activity changes the transcriptional effects of ER on estrogen target genes in presence of E2 or Tam. 2) Confirm that AIB1 is a substrate for JNK and p38, map the phosphorylation sites on AIB1, and determine which of these sites affect transcriptional activity of ER and especially the agonist activity of Tam. 3) Determine whether in vivo modulation of JNK and p38 activity both by genetic approaches and by recently developed small molecular inhibitors of JNK and p38, can prevent, delay, or overcome Tam resistance. Study Design: 1) An ERE-luciferase reporter gene and western blots of endogenous estrogen-responsive gene products will be used to examine the effects of JNK and p38 activity on ER transcriptional activity. Chromatin immunoprecipitation (ChIP) assays will be used to detect transcriptional complex components (coactivator/corepressor) on the promoter of target genes as a function of JNK or p38 activities in the presence or absence of different E2 concentrations or Tam. JNK and p38 activity in these cells will be modulated both by genetic approaches (expression of active and dominant-negative forms, gene transfection) and by specific small molecule inhibitors. 2) Native or Flag-tagged recombinant immunoprecipitated AIB1 will be used as substrates in in vitro kinase assays to assess phosphorylation by JNK and p38. Phosphorylation of AIB1 by JNK and p38 in vivo will be further confirmed in tissue culture by (32P)orthophosphate labeling. Several specific potential phosphorylation sites, identified primarily by sequence analysis, will be an initial focus. Mass spectrometry, if needed and phosphopeptide mapping will be used to determine the phosphorylation sites. Based on data from the mapping, relevant AIB1 domains and mutants (at specific phosphorylation sites) will be constructed and tested for their ability to modulate ER-transcriptional activity (in luciferase and ChIP assays) in HeLa, COS-1, and breast cancer cells (with low levels of AIB1) in conditions of induced or repressed JNK and p38. 3) An MCF-7 Tet–Off cell line will be used to generate inducible stable transfectants carrying dominant-negative p38 to complement a similar transfectant already made expressing dominant-negative AP1. These transfectants will then be studied in our MCF-7 xenograft model of endocrine resistance to determine if inhibition of p38 activity in the tumor cells can circumvent the development of resistance. We will also treat the animals with Tam or E2, without or with specific p38 and/or JNK inhibitors, to determine if these agents can delay or prevent the development of resistance. Relevance These experiments will determine if crosstalk between stress kinase pathways and ER can modify ER to cause acquired hormone therapy resistance in breast cancer. We will determine the molecular mechanism of this effect and investigate a new treatment strategy designed to prevent resistance which could next be tested in clinical trials in patients. Understanding the mechanism of resistance and identifying creative strategies to overcome it would be a major step in further reducing mortality.

    Lay Abstract:
    The role of growth factors/stress signaling pathways in developing endocrine resistance of breast cancer. Endocrine (hormone) therapy, and especially tamoxifen (Tam), is the most important systemic treatment of estrogen receptor (ER)-positive breast cancer at all stages. We know that Tam binds ER in the tumor cell and doesn’t allow estrogen, a potent growth factor for these cells, to bind the receptor. Blocking ER by Tam causes arrest of growth and eventually regression of the tumor. This, of course, translates into clinical benefit for the patients. Unfortunately many patients eventually become resistant to the treatment, and we still don’t know why this happens. My mentor's laboratory has developed a mouse model of endocrine resistance, in which human breast cancer cells form tumors in mice, and stop growing or regress with endocrine treatment (either Tam treatment or estrogen withdrawal). Eventually, as in patients, the tumors become resistant and grow again in spite of continued treatment. In this model, we have recently discovered that two stress-signaling proteins called p38 MAP kinase (p38) and stress-activated protein kinase (JNK) are tremendously increased in resistant tumors. In addition, we have showed that, when one of these proteins, JNK, is inactivated, resistance to endocrine therapy no longer appears. Based on these exciting findings I hypothesize that these two proteins may play a role in the development of the endocrine resistance. Based on our preliminary data, I hypothesize that these proteins change directly the ER function by phosphorylating it, or by activating other proteins, called co-activators of ER, that then cause the same or similar changes on the ER structure and function. When these changes in the ER happen, Tam no longer acts as an antagonist but rather as an agonist, thus mimicking the estrogen activity. Therefore the tumor starts to grow again. To test my hypothesis, I will first study the mechanism we described in cell culture. In the tumor cells, treated either with Tam or estrogen, I will modulate the activity of p38 and JNK in order to see how the ER activity changes. I will measure the ER activity by measuring proteins that are generally regulated by ER. In addition, I will test whether JNK phosphorylates AIB1, the co activator for ER which has been recently shown by our lab to be important in breast cancer and Tam resistance. I will further map the phosphorylation sites of AIB1 and study the role of JNK-phosphorylated AIB1 in acquired Tam resistance. Then I’ll test whether active p38 and JNK could serve as therapeutic targets. I will study whether specific inhibition of p38 and JNK can delay the development of resistance to Tam treatment, or can overcome already established resistance, in our in vivo xenograft model of human breast cancer endocrine resistance. Relevance: Doing this project I hope to clarify the role of these stress kinase pathways in the development of endocrine resistance in breast cancer, and, more importantly, show that specific inhibitors of p38 and JNK may delay or even reverse this resistance. Thus, I hope to develop this study into a novel approach to circumvent the problem of endocrine resistance of breast cancer.