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Evaluating Akt Tyrosine Phosphorylation as a Novel Marker for Therapeutic Resistance and Breast Cancer Prognosis
Tumor Cell Biology V
Background : The importance of the AKT kinase pathway in human breast cancer is underscored by frequent findings of pathological changes in mammary tumors and inheritable diseases associated with mammary hyperplasia, and by the ability of mutated AKT to promote mammary tumors in mutant mice. Multiple mechanisms of apoptosis suppression by AKT involve suppression of JNK pathway signaling. However, while some JNK pathway components are direct substrates of AKT, we have identified a more generalized mechanism of JNK interference based on the AKT-dependent destabilization of JNK pathway components. This AKT-dependent inhibition of JNK signaling is independent of its kinase activity and only requires the direct interaction of AKT with JNK pathway components. Importantly, we have identified a conserved tyrosine residue in AKT that is required for the formation of AKT-JNK protein complexes. Objective/Hypothesis : The focus of our laboratory research is to identify molecular mechanisms that link the AKT pathway to the suppression of cell death in mammary cancer. The hypothesis for the proposed project is that the inhibition of JNK signaling by AKT in breast cancer cells depends on its ability to act as a tyrosine-dependent anti-apoptotic scaffold for JNK pathway components. Thus, we propose to characterize the functional importance of the required tyrosine residue in vitro and the significance of its regulation by examining its phosphorylation levels in breast cancer specimens. Specific Aims/Study Design : Our first aim is to examine the relevance of the AKT tyrosine residue in JNK regulation and apoptosis suppression. We will express tyrosine-mutant AKT protein in mammary cancer cells and examine the cellular responses of mammary cells after treatment with chemotherapeutic drugs. In addition, we will generate mammary cell lines expressing tyrosine-mutant AKT and examine them for therapeutic resistances in vitro and in nude mice. Our second aim is to investigate the regulation of the tyrosine residue in primary breast tumors. Using archival mammary tumor tissue specimens, we will examine whether a correlation exists between AKT tyrosine phosphorylation and clinical response. These translational studies are designed to confirm whether tyrosine phosphorylation of AKT can be used as a novel biomarker for breast cancer disease. Relevance : While we plan to utilize established molecular and cellular biology techniques, the observation of a tyrosine-dependent scaffold function for AKT and its role in JNK suppression is both significant and highly innovative, and has not been studied. Although there are more than 8000 published articles on AKT, only a handful have addressed its regulation by tyrosine phosphorylation and none describe an effect. Accordingly, the science underlying our proposal is unique. If tyrosine phosphorylation of AKT in tumor specimens negatively correlates with breast cancer survival, the results from the second part of our study will have defined a new biomarker for breast cancer prognosis and therapeutic outcome. The validation of AKT as an anti-apoptotic scaffold also provides a novel target for therapeutic intervention. Thus, we envision that targeting the protein interactions between AKT and pro-apoptotic members of the JNK pathway will open novel and exciting opportunities to restore therapeutic responses in mammary cancer.
Regulated cell death (also called apoptosis) is a required process for the normal development of multicellular organisms. In breast cancer, the pathways regulating apoptosis are impaired and cancer cells acquire resistance to cancer therapy including irradiation and chemotherapy. A critical anti-apoptotic pathway mediating cell survival is regulated by the AKT protein kinase, which constitutes an important intracellular switch to coordinate the growth and survival of mammary cells with their cellular environment. Not surprisingly, breast cancer cells with increased AKT are resistant to chemotherapeutic drugs, a finding that has defined AKT as a prime target for therapeutic intervention. Observations of pathological dysregulation of AKT in mammary tumors provide additional support for its relevance in breast cancer. Here, we will explore a novel mechanism that links AKT to the inhibition of cell death and involves the suppression of the pro-apoptotic JNK pathway by AKT. The JNK pathway mediates apoptosis after chemotherapy and irradiation but is inhibited in mammary cancer cells, in which AKT decreases the protein levels of its components. Notably, we have identified a single tyrosine residue in AKT that is required for the anti-apoptotic destabilization of JNK components and can be regulated by phosphorylation. To further characterize the functional relevance of our discovery, we will employ in vitro models of tumorigenesis to investigate the role of this tyrosine residue during AKT-dependent cell survival and JNK inhibition in mammary cells. In addition, we will examine tyrosine phosphorylation of this residue in human breast cancer specimens to determine whether it can be used as a novel biomarker for breast cancer diagnosis and prognosis. The most innovative aspect of our proposal is based on the identification of a single residue in AKT that can mediate JNK inhibition. We will characterize this novel mechanism and its relevance for breast cancer with an emphasis on the response of mammary cancer cells to chemotherapeutic drugs. Because of the frequent finding of therapeutic resistance to clinical treatment in advanced stages of mammary cancer, our studies will greatly benefit those patients, whose diseases are no longer responding to currently available therapies. Our preliminary findings also raise a cautionary note, since we find that the AKT-dependent destabilization and inhibition of JNK signaling does not require AKT kinase activity. This may suggest that other therapies under development, which are directed at inhibiting AKT enzymatic activity, are intrinsically flawed, an important concern that requires additional clarification. Thus, by defining the molecular mechanisms that are involved in JNK suppression by AKT, we envision to identify novel therapeutic strategies that will sensitize cancer cells to apoptosis induction by disrupting AKT-dependent protein interactions and restoring JNK signaling.