Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

SCOPmap: Automated assignment of protein structures to evolutionary superfamilies

Background  

Inference of remote homology between proteins is very challenging and remains a prerogative of an expert. Thus a significant drawback to the use of evolutionary-based protein structure classifications is the difficulty in assigning new proteins to unique positions in the classification scheme with automatic methods. To address this issue, we have developed an algorithm to map protein domains to an existing structural classification scheme and have applied it to the SCOP database.     

Expanding the nitrogen regulatory protein superfamily: Homology detection at below random sequence identity

Nitrogen regulatory (PII) proteins are signal transduction molecules involved in controlling nitrogen metabolism in prokaryots. PII proteins integrate the signals of intracellular nitrogen and carbon status into the control of enzymes involved in nitrogen assimilation. Using elaborate sequence similarity detection schemes, we show that five clusters of orthologs (COGs) and several small divergent protein groups belong to the PII superfamily and predict their structure to be a (betaalphabeta)(2) ferredoxin-like fold. Proteins from the newly emerged PII superfamily are present in all major phylogenetic lineages. The PII homologs are quite diverse, with below random (as low as 1%) pairwise sequence identities between some members of distant groups. Despite this sequence diversity, evidence suggests that the different subfamilies retain the PII trimeric structure important for ligand-binding site formation and maintain a conservation of conservations at residue positions important for PII function. Because most of the orthologous groups within the PII superfamily are composed entirely of hypothetical proteins, our remote homology-based structure prediction provides the only information about them. Analogous to structural genomics efforts, such prediction gives clues to the biological roles of these proteins and allows us to hypothesize about locations of functional sites on model structures or rationalize about available experimental information. For instance, conserved residues in one of the families map in close proximity to each other on PII structure, allowing for a possible metal-binding site in the proteins coded by the locus known to affect sensitivity to divalent metal ions. Presented analysis pushes the limits of sequence similarity searches and exemplifies one of the extreme cases of reliable sequence-based structure prediction. In conjunction with structural genomics efforts to shed light on protein function, our strategies make it possible to detect homology between highly diverse sequences and are aimed at understanding the most remote evolutionary connections in the protein world.     

Regulation of the EphA2 kinase by the low molecular weight tyrosine phosphatase induces transformation

Intracellular signaling by protein tyrosine phosphorylation is generally understood to govern many aspects of cellular behavior. The biological consequences of this signaling pathway are important because the levels of protein tyrosine phosphorylation are frequently elevated in cancer cells. In the classic paradigm, tyrosine kinases promote tumor cell growth, survival, and invasiveness, whereas tyrosine phosphatases negatively regulate these same behaviors. Here, we identify one particular tyrosine phosphatase, low molecular weight tyrosine phosphatase (LMW-PTP), which is frequently overexpressed in transformed cells. We also show that overexpression of LMW-PTP is sufficient to confer transformation upon non-transformed epithelial cells. Notably, we show that the EphA2 receptor tyrosine kinase is a prominent substrate for LMW-PTP and that the oncogenic activities of LMW-PTP result from altered EphA2 expression and function. These results suggest a role for LMW-PTP in transformation progression and link its oncogenic potential to EphA2.     

Deciphering a novel thioredoxin-like fold family

Sequence--and structure-based searching strategies have proven useful in the identification of remote homologs and have facilitated both structural and functional predictions of many uncharacterized protein families. We implement these strategies to predict the structure of and to classify a previously uncharacterized cluster of orthologs (COG3019) in the thioredoxin-like fold superfamily. The results of each searching method indicate that thioltransferases are the closest structural family to COG3019. We substantiate this conclusion using the ab initio structure prediction method rosetta, which generates a thioredoxin-like fold similar to that of the glutaredoxin-like thioltransferase (NrdH) for a COG3019 target sequence. This structural model contains the thiol-redox functional motif CYS-X-X-CYS in close proximity to other absolutely conserved COG3019 residues, defining a novel thioredoxin-like active site that potentially binds metal ions. Finally, the rosetta-derived model structure assists us in assembling a global multiple-sequence alignment of COG3019 with two other thioredoxin-like fold families, the thioltransferases and the bacterial arsenate reductases (ArsC).     

Ligand binding up-regulates EphA2 messenger RNA through the mitogen-activated protein/extracellular signal-regulated kinase pathway

The EphA2 receptor tyrosine kinase is overexpressed in aggressive cancer cells, where it critically influences many aspects of malignant character. Although high levels of EphA2 have been documented in many different cancers, relatively little is known of the mechanisms that govern EphA2 gene expression in normal or malignant cells. Our present studies demonstrate that EphA2 influences the regulation of its own gene expression. Specifically, ligand-mediated phosphorylation of EphA2 transmits signals to the nucleus via extracellular signal-regulated kinase kinases to up-regulate de novo EphA2 gene expression and synthesis. This mechanism governs EphA2 expression in normal and malignant cells. In normal cells, EphA2 protein expression is balanced by ligand-mediated induction of EphA2 gene expression countered by EphA2 protein turnover. These findings suggest that EphA2 expression and ligand binding are intimately linked in epithelial cells. Increased understanding of this mechanism could have important implications for understanding the causes of EphA2 overexpression and for developing new strategies for therapeutic intervention in the many cancers that overexpress EphA2.     

Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation

Interactions between receptor tyrosine kinases of the Eph family and their ligands, ephrins, are implicated in establishment of organ boundaries and repulsive guidance of cell migration during development, but the mechanisms by which this is achieved are unclear. Here we show that activation of endogenous EphA2 kinase induces an inactive conformation of integrins and inhibits cell spreading, migration and integrin-mediated adhesion. Moreover, EphA2 is constitutively associated with focal-adhesion kinase (FAK) in resting cells. Within one minute after stimulation of EphA2 with its ligand, ephrin-A1, the protein tyrosine phosphatase SHP2 is recruited to EphA2; this is followed by dephosphorylation of FAK and paxillin, and dissociation of the FAK-EphA2 complex. We conclude that Eph kinases mediate some of their functions by negatively regulating integrins and FAK.     

Single-turnover kinetic analysis of Trypanosoma cruzi S-adenosylmethionine decarboxylase

Trypanosoma cruzi S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the pyruvoyl-dependent decarboxylation of S-adenosylmethionine (AdoMet), which is an important step in the biosynthesis of polyamines. The time course of the AdoMetDC reaction under single-turnover conditions was measured to determine the rate of the slowest catalytic step up to and including decarboxylation. Analysis of this single-turnover data yields an apparent second-order rate constant for this reaction of 3300 M(-1) s(-1) in the presence of putrescine, which corresponds to a catalytic rate of >6 s(-1). This rate is minimally 100-fold faster than the steady-state rate suggesting that product release, which includes Schiff base hydrolysis, limits the overall reaction. AdoMetDC exhibits an inverse solvent isotope effect on the single-turnover kinetics, and the pH profile predicts a pK(a) of 8.9 for the basic limb. These results are consistent with a Cys residue functioning as a general acid in the rate-determining step of the single-turnover reaction. Mutation of Cys-82 to Ala reduces the rate of the single turnover reaction to 11 M(-1) s(-1) in the presence of putrescine. Further, a solvent isotope effect is not observed for the mutant enzyme. Reduction of the wild-type enzyme with cyanoborohydride traps the Schiff base between the enzyme and decarboxylated substrate, while little Schiff base species of either substrate or product was trapped with the C82A mutant. These data suggest that Cys-82 functions as a general acid/base to catalyze Schiff base formation and hydrolysis. The solvent isotope and pH effects are mirrored in single-turnover analysis of reactions without the putrescine activator, yielding an apparent second-order rate constant of 150 M(-1) s(-1). The presence of putrescine increases the single-turnover rate by 20-fold, while it has relatively little effect on the affinity of the enzyme for product. Therefore, putrescine likely activates the T. cruzi AdoMetDC enzyme by accelerating the rate of Schiff base exchange.     

Dynamic Interaction of PTPμ with Multiple Cadherins In Vivo

There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPμ associates with the cadherin–catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977– 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPμ interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPμ and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPμ and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPμ from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPμ in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513–1517) have asserted that the association we observed between PTPμ and the cadherin–catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPμ, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPμ obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPμ antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPμ and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.