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Part I
“Induction of Differentiation of Human Leukemia Cells by Combinations
of COX Inhibitors and 1,25-dihydroxyvitamin D3 Involves Raf1 but not Erk 1/2 Signaling”

Part II
“Mechanisms of Integration for Androgen Receptor and Phosphatidylinositol 3-kinase Signaling in Estrogen Receptor Negative Class A Breast Cancer”

by
Faith Jamshidi
Interdisciplinary Biomedical Sciences Program
B.A. 2001, University of Missouri Kansas City
M.S. 2003, University of North Carolina


Thesis Advisor: Jacqueline Bromberg, M.D., Ph.D.

Professor

Department of Medicine
Memorial Sloan Kettering Cancer Center

Wednesday, July 28, 2010
Cancer Center, G-1196, 1:30 P.M.


Abstract

Cell-cell communication is a sophisticated process that involves a variety of signaling proteins. The main goal of cell signaling is to facilitate cellular functions such as cell growth, differentiation, migration, proliferation, survival, and development. Modification of cellular signaling events such as gene mutations can lead in tumor development. Understanding of cellular signaling pathways is a crucial step in the generation of cancer therapies.
The traditional way of studying cell signaling pathways is the study of single linear pathway. However, the emergence of new evidence suggests that signaling proteins operating in isolation through linear pathways is a scarce phenomenon. Nevertheless, signaling pathways are in communication though cross-talk in large and complex networks.
The first project described in this thesis, involves differentiation therapy of cancer which is being explored as a potential modality for treatment of myeloid leukemia, and derivatives of vitamin D are gaining prominence as agents for this form of therapy. Cyclooxygenase (COX) inhibitors have been reported to enhance 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation of promyeloblastic HL60 cells, but the mechanisms of this effect are not fully elucidated.
We determined that combination treatment with 1,25D and non-specific COX inhibitors acetyl salicylic acid (ASA) or indomethacin can robustly potentiate differentiation of other types of human leukemia cells, i.e., U937, THP-1, and that ASA +/- 1,25D is effective in primary AML cultures. Increased cell differentiation is paralleled by arrest of the cells in the G1 phase of the cell cycle, and by increased phosphorylation of Raf1 and p90RSK1 proteins. However, there is no evidence that this increase in phosphorylation of Raf1 is transmitted through the ERK module of the MAPK signaling cascade. Transfection of small interfering (si) RNA to Raf1 decreased differentiation of U937 cells induced by a combination of ASA or indomethacin with 1,25D. However, phosphorylation levels of ERK1/2, though not of p90RSK, were increased when P-Raf1 levels were decreased by the siRNA, suggesting that in this system the ERK module does not function in the conventional manner. Identification of the strong antiproliferative activity of ASA/1,25D combinations associated with monocytic differentiation has implications for cancer chemoprevention in individuals who have a predisposition to myeloid leukemia.
The second project involves the cross-talk between androgen receptor (AR) as well as PI3K/Akt pathways in a subset of breast cancers. Investigation of estrogen receptor-negative (ER-), progesterone receptor-negative (PR-) breast cancer so far has only led to systemic chemotherapy and currently targeted therapies remain elusive. Previously, we identified a new subclass of ER- / PR- / AR+ breast cancers as class A breast cancers. We studied PIK3CA gene mutations and their response to AR and PI3K/AKT signaling pathways.
The expression of AR in ER- / PR- breast cancers is positively correlated with PIK3CA mutations, pAkt levels, and HER2 expression. Also there is a strong association between HER2 expression and pAkt levels in ER- / PR- / AR+ breast cancers. The mutations of PIK3CA gene are associated with ER- class A breast cancer compared to ER- class B or ER+. Class A breast cancer cell lines harboring PIK3CA mutations are more responsive to PI3K inhibitor for growth inhibition compared to class B cell lines lacking PIK3CA mutations. Induction of proliferation by synthetic androgen in class A breast cancer cell lines is decreased by androgen antagonist as well as PI3K inhibitors and the combination of both inhibitors had an additive effect. Three elements, serine phosphorylated AR levels, AR transcriptional activity and the expression of AR target genes GGT1 and CYP4F8 were measured separately in a class A breast cancer cell line MDA-MB-453. When cells were incubated with androgen agonists, the levels of all three elements were increased compared to vehicle control. Treatment with a PI3K inhibitor decreased the levels of all three elements compared to vehicle control. When cells were treated with the combination of the androgen agonist and a PI3K inhibitor, the levels of all three elements were higher than the inhibitor but lower than the agonist alone.
These findings suggest that components of PI3K/AKT pathway can modulate AR activity suggesting a putative cross-talk between AR and PI3K pathway. This potential cooperative effect represents a new paradigm in targeted therapeutic strategies directed at the biological signal integration contributing to AR+ / ER- / PR- cancers.


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