The development and clinical application of drugs that target intracellular signal-transduction pathways has profoundly changed the treatment of human cancers. Tools enabling complex pathway analysis, particularly those that can identify critical regulatory post-translational modifications, continue to facilitate discovery of novel targets for cancer therapeutics and provide insights into drug-resistance mechanisms.
At the Salk Institute’s “Protein Phosphorylation and Cell Signaling” meeting held recently in La Jolla, CA, academic and industry scientists described specific applications of these technologies, including the use of advanced databases to study disease-associated signaling networks and identification of specific mediators for analysis of signal transduction in animal models of human cancers.
Jon M. Kornhauser, Ph.D., scientific editor for Cell Signaling Technology’s PhosphoSitePlus™ described the application of PhosphoSitePlus (PSP) to the analysis of aberrant signaling through protein kinase networks in human disease states.
Pointing out that abnormal signaling has been implicated in the etiology of many diseases, particularly cancer, Dr. Kornhauser said that PhosphoSitePlus offers an online systems biology resource providing comprehensive information and tools for the study of protein post-translational modifications (PTMs). “PSP identifies over 70,000 sites from published literature, over 500 of which have been implicated (by correlation or causation) in specific diseases.”
PhosphoSitePlus aggregates information about the role of specific PTM sites in regulating biological processes. This aggregated data provides a resource for assembling protein-phosphorylation networks containing site-specific information, and for analysis of these networks to identify specific PTMs that may correlate with disease causation or progression.
Several types of protein interactions are curated in PSP including the protein kinases that catalyze phosphorylation of each site, and associations of specific phosphorylation sites and disease. Data in PSP can be accessed by a variety of flexible queries; for example, “treatment” searches in the user interface allow users to identify those modification sites that respond to specific drugs or ligands.
Dr. Kornhauser said that “PTM-specific interactions from PSP, combined with proteins implicated in disease pathways in other public database sources, can be used to assemble disease-centric signaling networks.”
According to Dr. Kornhauser, “PSP, unlike other pathway databases that are protein-centric, is PTM-specific; that is, it aggregates information about the role of specific modified residues in biological regulation. It is hoped that the data in PSP will allow researchers to identify specific modified residues that may serve as biomarkers of specific disease states, as prognostic markers for drug efficacy predictions, and potentially as therapeutic targets.”
William J. Muller, Ph.D., professor in the department of biochemistry at McGill University, discussed oncogene-mediated signal transduction in transgenic mouse models of human breast cancer. To study the function of the signaling adaptor scaffold protein A (ShcA), Dr. Muller and his colleagues developed mouse models of human breast cancer in which Src gene homology, collagen A expression, or oncogene-coupled ShcA signaling was ablated.
Study results showed that loss of ShcA, a protein required for signal transduction by a group of receptor tyrosine kinases, resulted in local immune responses within mammary tumors. These responses included extensive CD4+ T-cell infiltration, activation, and induction of a humoral immune response. Conversely, the investigators showed that ShcA signaling early during mammary tumor progression is required to establish and maintain an immunosuppressive state that favors tumor growth.
Consistent with studies in transgenic mice, high ShcA levels correlate with poor outcome and reduced CTL infiltration in primary human breast cancers, Dr. Muller said, while elevated expression of a ShcA-regulated immune signature, generated from ShcA-null mammary tumors, predicted a good prognosis in Her2-positive and basal breast cancer patients.
These observations, Dr. Muller said, define a novel role for ShcA in polarizing the immune response to facilitate tumorigenesis. He further observed that in addition to the potential of stratifying patients that might benefit from aggressive treatment, these observations also suggest that inhibition of ErbB2-coupled signaling may synergize with immunotherapy approaches directed against breast cancer.
Dr. Muller also described studies in a second transgenic mouse model of ErbB2-induced mammary tumorigenesis. He and his team had previously established that ErbB2 amplification frequently accompanies loss of the 14-3-3σ gene, a gene that plays a role in the G2-M-phase checkpoint during cell replication and that may act as a tumor-suppressor gene.
Dr. Muller found that ectopic expression of this gene results in restoration of epithelial polarity in ErbB2-transformed mammary tumor cells. Further, targeted deletion of 14-3-3σ within primary mammary epithelial cells increases their proliferative capacity and adversely affects their ability to form polarized structures.
In addition, the scientists showed that 14-3-3σ can specifically form complexes with Par3, a protein essential for the maintenance of a polarized epithelial state. These observations suggest that an important function of the 14-3-3σ tumor suppressor is to ensure retention of normal epithelial integrity, Dr. Muller said.