Phosphoproteomics

Experimental and Molecular Pathology 70, 327–331 (2001) doi:10.1006/exmp.2001.2370, available online at http://www.idealibrary.com on

Phosphoproteomics

David F. Stern Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510

Received January 2, 2001

Pathology is continually being reinvented with the inception of new techniques and strategies for discriminating among disease subtypes and for making prognostic decisions. Analysis of tumors relies upon two overlapping approaches. The first is to develop a taxonomy of tumors that defines discrete disease entities with common tissue of origin and with common biology. For each type, characteristic clinical courses can then be determined. As such data are accrued, they can be used in turn to refine the classification systems, with the goal being to identify classes that are relatively homogeneous with regard to prognosis and responses to therapy. A second strategy has been to identify individual markers or groups of markers that have strong prognostic or predictive significance. In some cases these may be markers that help define categories used in the taxonomic approach. However, in other cases these markers will identify groups of tumors that cross taxonomic boundaries but that have common clinical phenotypes. Such markers may be biological bystanders that are passively activated in parallel with processes that actually drive tumor biology. However, advances in understanding functionality of oncogenes and tumor suppressor genes have revealed a number of markers that are integrally involved in enforcing the carcinogenic phenotypes. These parameters include expression or mutation of genes or secondary changes in regulation of specific processes as a consequence of gene mutations. The more

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intimately a marker is connected with the important carcinogenic processes in a particular tumor, the more likely it is to be tightly associated with a clinical course. As our understanding of biological systems advances, it may eventually become possible to make predictions about behaviors of cells and tissues based upon the status of important intracellular pathways, rather than upon similarity of their phenotypes to antecedant tumors with known outcomes. Hence, it may be anticipated that the taxonomic approach will eventually be superceded by a biologically based marker analysis. In order for this to be feasible, the most important tumor regulatory pathways would need to be identified, algorithms for describing their regulation and their interactions would need to be produced, and the connections of activities of these pathways to tumor behavior would need to be determined. This would permit development of strong predictive models based upon information about activities of the relevant pathways. Acquiring information from tumor specimens. Historically, pathological evaluations were based upon gross and microscopic examination of affected tissues. Over the years, molecular markers have become an important adjunct to this approach. DNA markers are used to identify gene rearrangements in hematopoietic tumors, and markers identified by immunohistochemistry for specific proteins are often useful for typing tumors. The genomic revolution has now made it possible to analyze the expression of hundreds of proteins and thousands of RNAs in parallel. Both of these

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328 techniques hold great promise in analysis of clinical specimens. The hope is that clinically relevant subsets of tumors will be identified by characteristic fingerprints of RNA or protein expression. At minimum, such profiles may include information about markers that can be used to subdivide biologically distinct tumors. For example, expression profiling of diffuse B cell lymphomas revealed a subset, otherwise cryptic to pathologists, that is associated with prognosis (Alizadeh et al., 2000). Ideally, such correlative studies will identify mRNAs for genes that mediate the phenotypes of tumors. For example, mRNA profiling of metastatic variants of a cell line revealed that a subset of genes is associated with metastatic ability (Clark et al., 2000). This subset included a series of genes responsible for cell migration that would logically contribute to a metastatic phenotype. In this instance, mRNA profiling yielded biological information about the mechanism of tumor behavior. Because of the tremendous amount of information made available through RNA profiling, it may turn out to be a an assay of choice for evaluation of pathological specimens. At present, however, its most important utility is as a discovery tool for identification of a subset of interesting genes. Once the most information-rich patterns of expression have been identified, they could then be used as the basis for design of focussed diagnostic kits based on a subset of RNAs or proteins. The phosphoproteome. An alternative to RNA-based genomics work for large-scale profiling of macromolecules is measurement of protein profiles to analyze the proteome. The traditional approach is to use two-dimensional gel separations. However, there are still many technical issues associated with achieving adequate resolution and sensitivity with this method. Continuing advances in mass spectrometry, combined with completion of genomic information required to link mass spectrometry peaks to specific genes, suggest that this will become the method of choice for proteomic analysis in the near future. The major advantages of genomic and proteomic approaches to analysis of clinical specimens are the use of a single robust technology to develop a large number of parallel measurements and the possibility that it will become possible to extract useful clinical information from these enormous data pools. With the intensive efforts underway to exploit RNA profiling in particular, it should only be a few years before the utility of this strategy has been thoroughly evaluated.

DAVID F. STERN

Protein and RNA profiling produce a wealth of information that contains taxonomic as well as functional information. Most RNAs serve only as templates for protein production and are therefore only indirectly linked to phenotype. This means that, in principle, proteomic information might be more valuable. However, in practice, it is much simpler to acquire quantitative information about RNA profiles than it is to acquire protein profiles. At the functional level, results from RNA and protein profiling are limited, since they only read out abundance of individual molecules. This may turn out to be an excellent method for reading out the subset of biological pathways with transcriptional outputs (e.g., the Ras pathway). A biologically based approach toward developing new diagnostic strategies can be based upon information provided by the measurement of activity of important regulatory pathways. Recent work on the molecular genetics of cancer has revealed a modest number of processes and pathways that are dysregulated in cancer. As discussed in a recent review (Hanahan and Weinberg, 2000), the minimal set would include development of independence from growth factor requirements, loss of sensitivity to inhibitory factors, evasion of apoptosis, expanded replicative potential, induction of angiogenesis, and development of invasive and metastatic capabilities. In principle, then, it should be possible to judge tumor progression and invasiveness by measurement of known constituents of these pathways. We hypothesize that measurement of the flux through major growth regulatory pathways will provide a composite view with strong predictive power for analysis of cancer. The majority of markers used for clinical predictions are endpoint markers such as proliferation markers or differentiation markers. However, the greatest potential information would be obtained if an overview of regulatory circuits could be assembled, since they will be associated with long-term cell regulation, and not only the specific status of the cell as it was captured at fixation for histological analysis. Regulation of cellular growth pathways is achieved through a variety of mechanisms. When the Ras pathway is activated by growth factor receptors, changes occur in phosphorylation of receptors, adaptor proteins, and MAP kinases; subcellular localization of adaptor proteins, SOS, and MAP kinases; guanine nucleotide loading of Ras; and activation of transcription factors. Although changes in levels of the constituent components (except for those of the transcriptional targets) are unimportant, changes in phosphorylation occur at several levels of the pathway. Because phosphorylation is such an important aspect of protein regulation, we hypothesize that the phosphorylation status of proteins contains an extraordinary amount of information

PHOSPHOPROTEOMICS

about the cellular state. The “phosphoproteome” is the entire assemblage of protein phosphorylations in a cell. Because of the paramount importance of reversible protein phosphorylation in regulation of cellular pathways, the information contained within the phosphoproteome that is relevant to carcinogenesis may match or even exceed that contained within the proteome, if protein modifications are excluded. Measurement of protein phosphorylation. Analyzing the phosphoproteome can be done using reagents that recognize specific phosphorylated peptides. The most common reagents are antibodies that recognize individual phosphopeptides (Epstein et al., 1992; Bangalore et al., 1992). Polyclonal antibodies can be raised against phosphopeptide immunogens. Since such sera will include anti-peptide, anti-phosphoamino acid, and the desired anti-phosphopeptide reactivities, it is often necessary to subject crude sera to affinity purification. Monoclonal antibodies that recognize phosphopeptides can also be produced (DiGiovanna and Stern, 1995). Since mass spectroscopy can also be used to resolve phosphopeptides, this tool may ultimately make it possible to analyze numerous phosphorylations in parallel and thereby to interrogate representative domains of the phosphoproteome. Analysis of protein phosphorylation in tissue may face some technical hurdles. The first is that action of cellular protein phosphatases might reduce the signal or introduce inter sample variability in comparing multiple tumors. Moreover, the phospho–peptide linkage may be subject to spontaneous chemical hydrolysis over time. In order to assess the utility of measurements of protein phosphorylation for evaluating protein phosphorylation, we have evaluated the connection between phosphorylation of the receptor tyrosine kinase (RTK) HER2/NEU/ERBB2 and patient outcome in breast cancer (DiGiovanna et al., 1996). HER2/NEU, a prototype for phosphoproteomic analysis. The HER2/NEU/ERB2 gene encodes a RTK in the epidermal growth factor (EGF) receptor family. This gene is amplified in 20–25% of breast and ovarian carcinomas and is overexpressed in other carcinomas. Amplification or overexpression is associated with poor prognosis and, possibly, resistance to chemotherapy. Her2 is now a validated therapeutic target. A humanized monoclonal antibody, Trastuzamab (Herceptin), has been approved by the FDA for use in patients with advanced breast cancer whose tumors overexpress Her2. The frequent amplification of this receptor in breast cancer strongly implies a major role of this receptor in genesis or progression of breast cancer. This is supported by a large body of experimental work showing that HER2 is a potent

329 oncogene in animal models and that HER2 is normally expressed in and active in the mammary gland. This would suggest that HER2 is an oncogene in breast cancer and therefore should have strong prognostic value for this disease. But, although Her2 overexpression is associated with poorer prognosis, the incremental risk has not been great enough to make Her2 a useful determinant for clinicians in most cases (Hynes and Stern, 1994). It is likely that the limited prognostic value of conventionally determined Her2 status is that it quantifies the quantity of Her2 molecules rather than the activity of Her2. Her2 is weakly activated by overexpression, but is strongly activated by EGF family growth factors and possibly by splice alterations and proteolytic processing. Her2 signals in heterodimers with other EGF family receptors, so that the absence or presence of these receptors will also have a strong influence on Her2 signaling. In a group of tumors expressing Her2 at a specific level, heterogeneous activation of the receptor will result in heterogeneity of biological output. For this reason, direct quantification of receptor activity rather than abundance should greatly enhance the linkage with biological outputs, including clinical outcome. RTKs are phosphorylated on specific tyrosine residues when they are activated. The phosphopeptides produced thusly serve as docking sites for downstream signaling proteins, so that Tyr phosphorylation not only marks active receptors, but is also the means by which they transmit their signals. Our laboratory pioneered the development of Tyr phosphopeptide antibodies and has now used a monoclonal anti-phosphoHer2 antibody, PN2A, to establish proof-ofprinciple for the utility of phospho-specific antibodies for analysis of tissue. Monoclonal antibody PN2A recognizes a major terminal Tyr phosphorylation site and is monospecific for phosphorylated Her2 (DiGiovanna and Stern, 1995). Early studies with this antibody confirmed that it detected a heterogeneous signal in breast carcinomas, suggesting considerable signaling heterogeneity of Her2 (DiGiovanna et al., 1996). In a recent collaborative study, 816 invasive breast cancers with a median 16.3 years clinical follow-up were evaluated for P-HER2. Overexpression of Her2 per se only weakly predicted poor prognosis. However, the small subset of patients with high Her2 phosphorylation had a significantly shorter disease-free and disease-significant survival (Thor et al., 2000). These results established that phosphospecific antibodies can have strong predictive power. HER2-regulated pathways. This work with the Her2 phosphopeptide antibody PN2A shows how an oncogene product-directed phospho-antibody can supply important

330 prognostic information. Such information might be augmented by evaluating other Her2 phosphorylation sites (Epstein et al., 1992) and by adding information regarding other HER family receptors that heterodimerize with Her2 (Hynes and Stern, 1994). However, a complementary pathway-directed approach might provide even more information or augment the receptor-specific approach. First, a major function of Her2 in cancer is to stimulate the Ras pathway. However, querying downstream components of the Ras pathway, such as MAP kinase, may prove to be even more useful (Gioeli et al., 1999). One limit on the Her2 work is that biologically significant numbers of activated receptors may fall below the threshold for detection of a single phosphoantibody. The rapid turnover of activated receptors also reduces their steady-state level and may further detract from detection. In principle, measuring MAP kinase functionality may be superior. MAP kinases are near the end of the MAP kinase cascade, so that as a result of amplification during the signaling cascade, a greater number of phosphopeptides may be generated in MAP kinases. Because of pathway cross-talk and the distal location of MAP kinases in the Ras signaling cascade, the MAPK signal will better represent Ras signaling output than receptor activation after other negative regulatory influences (e.g., GAPs) have been processed by the pathway. Hence, even in tumors in which Her2 activation of Ras signaling is paramount, for both technical and biological reasons, MAPK signaling may better predict signaling output and hence cellular response. These arguments do not prove that MAPK is a superior parameter because of the underlying assumptions. Her2 does not signal exclusively through the Ras pathway, so MAP kinase activity may not turn out to be an adequate surrogate for Her2 status. Because of this uncertainty, these issues will have to be worked out empirically. A common property of cancer cells is relief from normal growth factor requirements for cell division. This is commonly achieved through activation of the Ras pathway via Ras mutations, constitutive growth factor receptor activation by autocrine factors, receptor overexpression, and receptor mutation. Although these changes could be measured individually by monitoring receptor changes, Ras, mutations, growth factor production, etc., it is clearly more expedient to use a single measure that reads out total signaling by the pathway. Measurement of activity of MAP kinases, or some other distal signaling constituent in the pathway, will provide a composite measure for pathway activation that will factor in the several modes of stimulation and hence be most general. Thus, while Her2 signaling may be important for a subset of breast cancers, perhaps only those that overexpress

DAVID F. STERN

Her2, we hypothesize that Ras pathway activation will have strong predictive value for all cancers. The Ras pathway is only one of several signaling pathways that will be important for predicting tumor behavior. Ras pathway activation of cell division is antagonized through TGF-␤ response pathways. Hence, in developing a strong prediction for tumor behavior, knowledge of the flux through both pathways will have much greater value than knowledge of each in isolation. This argument applies in turn to other important pathways, such as pro- and anti-apoptotic pathways. In summary, we propose that the phosphorylation status of intermediaries in important signaling pathways relevant to disease states is rich in information. Work by our laboratory and others has demonstrated (i) that it is possible to produce phosphopeptide-specific antibodies; (ii) that phosphorylation-specific information is preserved over decades in archival fixed and paraffin-embedded specimens processed routinely, and (iii) that evaluation of Her2 phosphorylation using this strategy provides significant prognostic information that is inaccessible through other means. Further improvements in application of this strategy will require evaluation of phospho-specific reagents that mark important signaling pathways for clinical utility and determination of the degree to which multipathway analysis using such reagents facilitates prognosis and therapeutic decision-making. Phosphospecific antibodies that identify many of the most important signaling proteins are now available commercially, so work in this area should proceed rapidly.

REFERENCES

Alizadeh, A., et al. (2000). Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, 503–511. Bangalore, L., Tanner, A. J., Laudano, A. P., and Stern, D. F. (1992). Antiserum raised against a synthetic phosphotyrosine-containing peptide selectively recognizes p185neu/erbB-2 and the epidermal growth factor receptor. Proc. Natl. Acad. Sci. USA 89, 11637–11641. Clark, E., Golub, T., Lander, E., and Hynes, R. (2000). Genomic analysis of metastasis reveals an essential role for RhoC. Nature 406, 532–535. DiGiovanna, M. P., Carter, D., Flynn, S. D., and Stern, D. F. (1996). Functional assay for HER-2/neu demonstrates active signaling in a minority of HER-2/neu overexpressing invasive human breast tumors. Br. J. Cancer 74, 802–806. DiGiovanna, M. P., and Stern, D. F. (1995). Activation state specific monoclonal antibody detects tyrosine phosphorylated p185neu/erbB-2

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in a subset of human breast tumors overexpressing this receptor. Cancer Res. 55, 1946–1955. Epstein, R. J., Druker, B. J., Roberts, T. M., and Stiles, C. D. (1992). Synthetic phosphopeptide immunogens yield activation-specific antibodies to the c-erbB-2 receptor. Proc. Natl. Acad. Sci. USA 89, 10435–10439. Gioeli, D., Mandell, J., Petroni, G., Frierson, H. J., and Weber, M. (1999). Activation of mitogen-activated protein kinase associated with prostate cancer progression. Cancer Res. 59, 279–284.

331 Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57–70. Hynes, N. E., and Stern, D. F. (1994). The biology of erbB-2/neu/ HER-2 and its role in cancer. Biochem. Biophys. Acta Rev. Cancer 1198, 165–184. Thor, A., Liu, S., Edgerton, S., Moore, D., Stern, D., and DiGiovanna, M. (2000). Activation (tyrosine phosphorylation) of ErbB-2 (HER2/ neu) as a prognostic factor in node positive breast cancer: A study of incidence and correlation with outcome in breast cancer. J. Clin. Oncol. 18, 3230–3239.