> Research & Grants
> Grants Program
> Research Grants
> Research Grants Awarded
Regulation of PEA-15 During Breast Cancer Oncogenesis
Background: PEA-15 is a protein that is highly expressed in certain breast tumors and breast cancer cell lines. The PEA-15 gene is located at locus 1q21.1, which is a region amplified 50% of the time in aneuploid breast cancers. The PEA-15 locus also encodes for the MAT1 mammary tumor oncogene. PEA-15 regulates integrin function, an important determinant in the metastatic ability of breast cancer. Furthermore, PEA-15 promotes activation of the Ras oncogene; with subsequent activation of ERK (MAPK). However, PEA-15 also suppresses ERK and RSK2 transcriptional activity by binding to and blocking translocation of ERK and RSK2 to the nucleus; thereby suppressing cell proliferation. PEA-15 also associates with components of the Death-Induced Signaling Complex (DISC) and prevents apoptosis. These functions suggest a role for PEA-15 as a regulator of cellular functions important for the development and progression of breast cancer.
Overall hypothesis: PEA-15 is subject to phosphorylation by PKC, Akt, and CamKII. These phosphorylation sites are within the C-terminal tail of PEA-15, immediately adjacent to amino acids critical for binding to both ERK and RSK2. Additionally, phosphorylation of PEA-15 also regulates its recruitment to the DISC. I hypothesize that phosphorylation of PEA-15 modulates its interactions with binding partners such as ERK,RSK2, and DISC proteins; thereby regulating its capacity to enhance integrin and ERK activation, as well as inhibit apopotosis and transcriptional output of the ERK and RSK2 pathways. In this manner, the expression level and phosphorylation state of PEA-15 may govern oncogenesis and the ability of mammary tumor cells to metastasize.
Specific Aims and Study Design: To test this hypothesis, I will analyze the mechanisms that modulate PEA-15’s association with its binding partners: ERK, RSK2, and DISC proteins. (1) In order to test the effect of PEA-15 phosphorylation on this association, I will phosphorylate PEA-15 and test its ability to bind ERK, RSK2 and DISC proteins in vitro. I will also create phospho-mimetic mutants of PEA-15 and analyze their ability to bind ERK, RSK2 and DISC proteins in vitro and in vivo. (2) Additionally, to test the effects of PEA-15 phosphorylation in vivo, I will express phospho-mimetic and non-phosphorylatable mutants in normal fibroblasts and breast cancer cells. In cells transfected with wild-type and mutant PEA-15 constructs, I will assay ERK and RSK2 translocation to the nucleus using fluorescence microscopy, cell fractionation and transcription activation assays. I will study the association of phospho-mimetic PEA-15 with DISC proteins using confocal microscopy and immunoprecipitation. In the same manner, I will express wild type and mutant PEA-15 constructsts, and compare changes in Ras, ERK, and integrin activation in vivo using western blotting and FACS analysis. (3) The known activities of PEA-15 suggest that it has an important role as a regulator of the development and progression of breast cancer. For that reason, I will test the ability of wild type or phospho-mimetic PEA-15 to transform NIH 3T3 fibroblasts, and increase the tumorgenicity of human breast cancer cell lines by using soft agar colony formation assays and implantation in nude mice. Finally, to demonstrate the contribution of PEA-15 expression and phosphorylation in clinical breast cancer, I will screen for the expression and phosophorylation of PEA-15 in breast tumor tissue arrays using pan-PEA-15 and phospho-specific PEA-15 antibodies.
Potential Outcomes and Benefits: The work performed in this study will show that phosphorylation of PEA-15 regulates ERK and integrin activation, transcription, and apoptosis in breast cancer cells. Each of these cellular functions are modulated by PEA-15 and have important roles in breast cancer development and progression. Thus, this study will evaluate the role of PEA-15 in the pathogenesis of breast cancer and identify PEA-15 as a possible therapeutic target.
PEA-15 is a protein whose expression is increased in breast tumors and many breast cancer cell lines. The PEA-15 gene is located on chromosome 1, in a region duplicated 50% of the time in breast cancers that have abnormal chromosomes. The gene for PEA-15 was originally identified as the MAT1 gene, which is a gene that causes breast cancer in mice. PEA-15 regulates the function of integrins, which are cell surface proteins which tightly regulate proper growth and development of all cells in the body. Specific types of integrins have been shown to have important roles in the ability of breast cancer to grow and metastasize. PEA-15 keeps integrins in the “active” state and encourages cell behavior that is necessary for tumors to develop and spread to other regions of the body. Furthermore, PEA-15 activates the ERK signaling pathway, which has also been shown to directly cause cancer and promote cellular behavior that encourages metastasis. PEA-15 also blocks apoptosis (programmed cell death), which is a natural mechanism the body uses to eliminate cells that are growing improperly, as in the case of cancer. These multiple functions of PEA-15 suggest that it contributes to the initiation and progression of breast cancer.
However, PEA-15 simultaneously suppresses cell growth and division by preventing critical signaling of ERK and RSK2. The several functions of PEA-15 that encourage tumor-like behavior of the cell may be diminished by this block in cell division. Thus, there is a balance between these two differing roles of PEA-15 that can be shifted from preventing cell growth to promoting cell growth and progression of cancer. One way that PEA-15 may regulate this shift is by being phosphorylated, which is adding a phosphate to specific sites on the protein. Phosphorylation is a major method of turning “on” or “off” proteins in cells during normal cell behavior. But in the case of cancer, this balance of “on” versus “off” in cellular signaling is often shifted in ways that promote cancer. Similarly, PEA-15 can be phosphorylated, changing the way it behaves in the cell. Phosphorylation of PEA-15 occurs in the exact same area of this protein which is critical for binding between itself and ERK or RSK2. This suggests that phosphorylation of PEA-15 may be the "switch" that blocks its ability to bind ERK and RSK2. This change in PEA-15 can thus alter the signaling inside the in manners which promote breast cancer.
I propose to study how phosphorylation of PEA-15 changes the cell’s behavior. I hypothesize that phosphorylation of PEA-15 alters its ability to associate with ERK, RSK2, and apoptosis related proteins. This, in turn, allows the cell to grow, multiply, and resist programmed cell death. Thus, phosphorylation changes the ability of PEA-15 to target important signaling molecules that are relevant to cancer. This includes its ability to activate the ERK pathway, activate integrins, and block apopotosis. In this manner, phosphorylation of PEA-15 is a potent “switch” that either blocks cell growth, or in contrast, helps breast cancer cells form tumors and spread to other regions of the body. In this manner, the amount of PEA-15 in the cell, as well as its state of phosphorylation, can control the development and progression of breast cancer.
To test this hypothesis, I will investigate how phosphorylation of PEA-15 regulates its association with ERK, RSK2, and apoptosis related proteins. This can be accomplished by looking at their interaction inside the cell using microscopy and other cell-based assays. I am also able to mutate PEA-15 in ways that mimic its phosphorylation, and express them in normal and breast cancer cells. This allows me to study the effect of PEA-15 phosphorylation on ERK and integrin activation in the cell; as well as apoptosis and tumor formation in breast cancer cells. I will also create antibodies that can identify phosphorylated PEA-15 in cells and tissue; and I will use this to screen breast cancer cell lines and tumor samples. This study will allow me to better identify what roles PEA-15 may have during tumor development. Most significantly, this study will demonstrate how breast cancer cells may use PEA-15 to become malignant. From this study, new breast cancer therapies that target PEA-15 may be developed.