Send to printer »

Feature Articles : Nov 15, 2010 (Vol. 30, No. 20)

Capitalizing on Cell-Signaling Advances

Revelation of Potential Intervention Points for Therapeutic Development on Upswing
  • Patricia Fitzpatrick Dimond, Ph.D.

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.”

Transgenic Mice

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.

Fluorogenic Cell-Based Assays

Wen-Chieh Liao, research scientist at R&D Systems described a fluorogenic cell-based ELISA that does not require lysate preparation or the multiple steps required to achieve Western blot protocols for analysis of cellular proteins and phosphorylation. This method is amenable to high-throughput applications and may, Dr. Liao said, prove a valuable addition to kinase inhibitor screening strategies.

The relatively rapid and straightforward method, according to Dr. Liao, provides a way to analyze multiple samples and enables analysis of two target cellular proteins or events simultaneously in the same microplate wells to minimize assay variability. Dr. Liao said these cell-based ELISAs have been used to evaluate the effects of stimulators and inhibitors on cultured cells with 10,000 cells, or fewer, per well.

He described phosphorylation of JNK (T183/Y185), Akt (S473), EGFR (Y1068), and TAT3 (Y705) in response to various stimuli or kinase inhibitors. In contrast to other techniques for intracellular protein analysis, including Western blots and sandwich ELISA, Dr. Liao said, total hands-on time for the cell-based ELISA amounts to about three hours, while conventional assays require about two days because of the need to prepare lysates and other laborious steps.

In describing what specific issues with conventional assays this technology addresses, Dr. Liao explained that “cell-based assays are associated with poor accuracy and reproducibility due to well-to-well variations such as cell numbers. However, our assays simultaneously measure two proteins in one well, thus allowing for normalization of well-to-well variations.”

Analyzing phosphoproteins by Western blot requires preparation of cell lysates and does not allow the study of many samples that are essential for high-throughput drug screenings, he said.

“In contrast, our dual-fluorescence cell-based ELISA allows researchers to grow their cells in 96-well plates, treat them with appropriate conditions, and then quickly fix and permeabilize in the wells. This is followed by incubation of cells with two primary antibodies against proteins of interest. Then, two secondary antibodies labeled with HRP and AP, respectively, and spectrally distinct fluorogenic substrates for each enzyme are used for detection.

“In this assay format, two target cellular proteins or events can be analyzed simultaneously in two fluorescence channels, thus allowing users to improve accuracy by normalizing for well-to-well variations.”  Since the assay detection allows for measurement with a standard fluorescence plate reader, there is no need for specialized and expensive equipment.

Although in vitro biochemical kinase assays are routinely used for drug screening, he said, they cannot replicate the intracellular environment. “Our assay allows drug screening of intact cells, and may reflect physiological conditions more closely.” Dr. Liao and his group have used this assay to evaluate phosphorylation of multiple signaling proteins in response to kinase inhibitors, and to determine the IC50 of such inhibitors such as EGFR, ERK1/2, and PI3-kinase inhibitors.

How cells sort out and balance the complex array of signaling events that occur via phosphorylation and dephosphorylation is a prime focus in the laboratory of Alexandra Newton, Ph.D., professor of pharmacology at the University of California, San Diego.

In her presentation, Dr. Newton discussed the role of phosphatase enzymes in controlling protein kinase activation and deactivation. Dr. Newton’s laboratory specifically focuses on the molecular mechanisms of signal propagation and termination in the PI3 kinase and diacylglycerol signaling pathways, and particularly on how protein kinase C (PKC) and Akt (protein kinase B) are activated by phosphorylation events, by second messengers, and inactivated by dephosphorylation via phosphatases.

For example, Akt is a serine/threonine kinase that is the downstream target of PI3K signaling. This kinase mediates most of the PI3K-mediated metabolic actions of insulin through the phosphorylation of several substrates including other kinases, signaling proteins, and transcription factors.

In addition, its activation plays a key role in cell survival. Misregulation of the balance between the phosphorylation and dephosphorylation events leads to pathogenic states, Dr. Newton said. Notably, tipping the balance toward unregulated activation of Akt is a major contributor to cancer.

In her presentation, Dr. Newton focused on the importance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase) in acting as a  “brake” to terminate signaling by PKC and Akt.

These Ser/Thr specific phosphatases, discovered in Dr. Newton’s laboratory, have emerged as important regulators of intracellular signaling pathways by specifically dephosphorylating and inactivating the two pro-survival kinases Akt and PKC. By examining phosphorylation of these two kinases following manipulation of PHLPP levels through genetic depletion by siRNA,  Dr. Newton and her colleagues were able to show that PHLPP controls PKC levels and its activity in the cell.

Because PHLPP is lost in a large number of cancers, thereby conferring a survival advantage to these cells, PHLPP activators may prove useful as cancer therapeutics. Conversely, PHLLP inhibitors would be very useful for diseases where increased Akt activity would be beneficial, such as heart disease and cancer. Dr. Newton further explained that the catalytic domain of this phosphatase belongs to the PP2C family of serine/threonine phosphatases, for which no specific inhibitors exist.

Following medium-throughput chemical screening combined with a virtual approach, her laboratory identified several small molecule PHLPP inhibitors. Treatment of cells with these compounds, she said, caused an increase in both basal and agonist-evoked phosphorylation and activity. These newly discovered inhibitors of PHLPP may be therapeutically relevant in the treatment of diabetes or cardiovascular diseases, in which Akt and PKC pathways are repressed.

Presentations at this meeting confirmed the tremendous progress that has been made in analyzing cell signaling, and pointed out potential intervention points for the development of novel therapeutics.