Impact of Protein Stability, Cellular Localization, and Abundance on Proteomic Detection of Tumor-Derived Proteins in Plasma

Tumor-derived, circulating proteins are potentially useful as biomarkers for detection of cancer, for monitoring of disease progression, regression and recurrence, and for assessment of therapeutic response. Here we interrogated how a protein's stability, cellular localization, and abundance affect its observability in blood by mass-spectrometry-based proteomics techniques. We performed proteomic profiling on tumors and plasma from two different xenograft mouse models. A statistical analysis of this data revealed protein properties indicative of the detection level in plasma. Though 20% of the proteins identified in plasma were tumor-derived, only 5% of the proteins observed in the tumor tissue were found in plasma. Both intracellular and extracellular tumor proteins were observed in plasma; however, after normalizing for tumor abundance, extracellular proteins were seven times more likely to be detected. Although proteins that were more abundant in the tumor were also more likely to be observed in plasma, the relationship was nonlinear: Doubling the spectral count increased detection rate by only 50%. Many secreted proteins, even those with relatively low spectral count, were observed in plasma, but few low abundance intracellular proteins were observed. Proteins predicted to be stable by dipeptide composition were significantly more likely to be identified in plasma than less stable proteins. The number of tryptic peptides in a protein was not significantly related to the chance of a protein being observed in plasma. Quantitative comparison of large versus small tumors revealed that the abundance of proteins in plasma as measured by spectral count was associated with the tumor size, but the relationship was not one-to-one; a 3-fold decrease in tumor size resulted in a 16-fold decrease in protein abundance in plasma. This study provides quantitative support for a tumor-derived marker prioritization strategy that favors secreted and stable proteins over all but the most abundant intracellular proteins

The extracellular domains of ErbB3 retain high ligand binding affinity at endosome pH and in the locked conformation

The extracellular, ligand binding regions of ErbB receptors consist of four domains that can assume at least two alternative conformations, extended and locked. The locked conformation, observed in several crystal structures, is held together by a noncovalent intramolecular tether and is incompatible with current models for receptor dimerization and ligand activation. Based on structures of ligand-receptor complexes in the extended conformation, the high affinity ligand binding pocket between domains I and III is disrupted in the locked conformation. Therefore the biological role of the locked conformation is not clear. To address the impact of the locked conformation on ligand binding, we compared extracellular domains of wild-type ErbB3, mutant domains in a constitutively locked or extended conformation and partial extracellular domain constructs. We found that the constitutively locked receptor domains and truncated constructs carrying only domains I-II or III-IV strongly bind ligand, albeit with reduced affinity compared to wild-type receptor. This suggests that the locked conformation cannot be discounted for ligand binding. The significant binding by both partial interfaces in domains I and III also suggests that "partial bivalency" may be the reason for the low nanomolar and high picomolar binding observed for ErbB3 in the respective "low" and high affinity states. In contrast to EGFR (ErbB1), ErbB3 retains high ligand binding affinity at an endosome-comparable pH in both the extended and locked conformations. Ligand affinity for the locked conformation even improves at low pH. For ErbB3, the contribution of domain I to ligand binding is strong and increases at low pH while its contribution is thought to be minimal for EGFR, regardless of pH. This shift in domain contribution and pH dependency provides a mechanistic explanation for some of the divergent properties of EGFR and ErbB3

The N-terminal domains of neuregulin 1 confer signal attenuation

Degradation of activated ERBB receptors is an important mechanism for signal attenuation. However, compared with epidermal growth factor (EGF) receptor, the ERBB2/ERBB3 signaling pair is considered to be attenuation-deficient. The ERBB2/ERBB3 ligands of the neuregulin family rely on an EGF-like domain for signaling and are generated from larger membrane-bound precursors. In contrast to EGF, which is processed to yield a 6-kDa peptide ligand, mature neuregulins retain a variety of segments N-terminal to the EGF-like domain. Here we evaluate the role of the N-terminal domain of neuregulin 1 in signaling and turnover of ERBB2/ERBB3. Our data suggest that whereas the EGF-like domain of neuregulin 1 is required and sufficient for the formation of active receptor heterodimers, the presence of the N-terminal Ig-like domain is required for efficient signal attenuation. This manifests itself for both ERBB2 and ERBB3 but is more pronounced and coupled directly to degradation for ERBB3. When stimulated with only the EGF-like domain, ERBB3 shows degradation rates comparable with constitutive turnover, but stimulation with full-length neuregulin 1 resulted in receptor degradation at rates that are comparable with activated EGF receptor. Most of the enhancement in down-regulation was maintained after replacing the Ig-like domain with a thioredoxin protein of comparable size but different amino acid composition, suggesting that the physical presence but not specific properties of the Ig-like domain are needed. This sequence-independent effect of the N-terminal domain correlates with an enhanced ability of full-size neuregulin 1 to disrupt higher order oligomers of the ERBB3 extracellular domains in vitro

Abstract C58: Heterotetramers of ERBB2 and ERBB3 generate qualitatively distinct signals from heterodimers and provide most of the neuregulin-induced phosphorylation of ERBB2

Abstract The efficient tyrosine phosphorylation of the orphan ERBB2 (HER2) receptor in heterodimer with the kinase-impaired ERBB3 presents a highly potent source of mitogenic signal. However, it is also a conceptual challenge to our established model of dimer-based signal generation through transphosphorylation. Proposed and mutually not exclusive models for ERBB2 phosphorylation in complex with ERBB3 include autophosphorylation in cis after complex formation and allosteric activation of its kinase domain by ERBB3 in trans, phosphorylation of ERBB2 in trans by the low but detectable kinase activity of ERBB3, or phosphorylation of ERBB2 in proxy. Proxy activation refers to phosphorylation between ERBB2 receptors of activated heterodimers in higher order complexes. We previously reported that the ligand-induced activation and growth stimulation through ERBB2/ERBB3 can be efficiently inhibited by an aptamer directed against the extracellular portion of ERBB3. Studies of the exact mode of inhibition now reveal that the aptamer neither acts by interfering with ligand binding nor by suppressing heterodimer formation. Combined biochemical studies and homology models identified the binding site of the aptamer and suggest that it interferes with the transition from activated heterodimers to heterotetramers. This inhibits the proxy activation of ERBB2 and associated MAPK activation while heterodimerization, the phosphorylation of ERBB3, and downstream activation of AKT are largely insensitive to inhibition. The fact that ERBB2 appears to receive most of its tyrosine phosphorylation through a proxy mechanism is consistent with reports of a prevalence of tetramers in the context of EGFR activation. Furthermore, our study demonstrates for the first time that mechanistically definable and dissectible higher order ERBB complexes contribute to signaling in a manner that is qualitatively distinct from the signal that emanates from isolated dimers. This forces us to fundamentally reinterpret approaches to therapeutic intervention that are at present conceptually centered around modulating the formation of dimers but often generate experimental and clinical outcomes that do not readily reconcile with dimer-based models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C58.</jats:p

Quantification of cancer cell migration with an integrated experimental-computational pipeline

We describe an integrated experimental-computational pipeline for quantifying cell migration in vitro. This pipeline is robust to image noise, open source, and user friendly. The experimental component uses the Oris cell migration assay (Platypus Technologies) to create migration regions. The computational component of the pipeline creates masks in Matlab (MathWorks) to cell-covered regions, uses a genetic algorithm to automatically select the migration region, and outputs a metric to quantify cell migration. In this work we demonstrate the utility of our pipeline by quantifying the effects of a drug (Taxol) and of the extracellular Anterior Gradient 2 (eAGR2) protein on the migration of MDA-MB-231 cells (a breast cancer cell line). In particular, we show that inhibiting eAGR2 reduces migration of MDA-MB-231 cells

Oligomers of ERBB3 have two distinct interfaces that differ in their sensitivity to disruption by heregulin

ErbB receptors associate in a ligand-dependent or -independent manner, and overexpression of epidermal growth factor receptor (ErbB1) or ErbB2 results in ligand-independent activation. Ligand-independent activation is poorly understood, and dimerization alone is not sufficient for activation. ErbB receptors also form higher order oligomers, but the mechanism of oligomer formation and their contribution to signaling are not known. The kinase-deficient ErbB3 as well as its extracellular domains are particularly prone to ligand-independent oligomerization, and oligomers are destabilized by binding of the ligand heregulin. In contrast, ligand binding facilitates heterodimerization with ErbB2 and is expected to stabilize an extended conformation of the ErbB3 extracellular domain (ECD) in which the dimerization interface is exposed. In the absence of ligand, ErbB3 can adopt a closed conformation that is held together by an intramolecular tether. We used a constitutively extended form of the ErbB3-ECD to analyze the conformation of the ECD in oligomers and the mechanism of oligomer disruption by heregulin. The extended conformation of the ECD forms oligomers more readily, suggesting the crystallographically defined dimer interface is one of the interfaces involved in oligomerization. Heregulin destabilizes oligomeric complexes but not dimers, which are neither stabilized nor disrupted by ligand binding, indicating a distinct second interface in oligomers of ErbB3. Cross-linking and activation studies on membrane-embedded ErbB3/ErbB2 chimeras confirm this dual effect of heregulin. Most of the ErbB3-ECD on the cell surface is apparently kept in an open conformation through oligomerization, and the resulting oligomers adopt a conformation representing a state of reduced activity

Identification, characterization and application of a new peptide against anterior gradient homolog 2 (AGR2)

The cancer-associated protein Anterior Gradient 2 (AGR2) has been described, predominantly in adenocarcinomas. Increased levels of extracellular AGR2 (eAGR2) have been correlated with poor prognosis in cancer patients, making it a potential biomarker. Additionally, neutralizing AGR2 antibodies showed preclinical effectiveness in murine cancer models suggesting eAGR2 may be a therapeutic target. We set out to identify a peptide by mRNA display that would serve as a theranostic tool targeting AGR2. This method enables the selection of peptides from a complex (>10 11 ) library and incorporates a protease incubation step that filters the selection for serum stable peptides. We performed six successive rounds of enrichment using a 10-amino acid mRNA display library and identified several AGR2 binding peptides. One of these peptides (H10), demonstrated high affinity binding to AGR2 with a binding constant (K D ) of 6.4 nM. We developed an AGR2 ELISA with the H10 peptide as the capture reagent. Our H10-based ELISA detected eAGR2 from cancer cell spent media with a detection limit of (20-50 ng/ml). Furthermore, we investigated the therapeutic utility of H10 and discovered that it inhibited cell viability at IC 50 (9-12 μmoles/L) in cancer cell lines. We also determined that 10 μg/ml of H10 was sufficient to inhibit cancer cell migration in breast and prostate cancer cell lines. A control peptide did not show any appreciable activity in these cells. The H10 peptide showed promise as both a novel diagnostic and a potential therapeutic peptide

Protein Mimetic and Anticancer Properties of Monocyte-Targeting Peptide Amphiphile Micelles

Monocyte chemoattractant protein-1 (MCP-1) stimulates the migration of monocytes to inflammatory sites, leading to the progression of many diseases. Recently, we described a monocyte-targeting peptide amphiphile micelle (MCP-1 PAM) incorporated with the chemokine receptor CCR2 binding motif of MCP-1, which has a high affinity for monocytes in atherosclerotic plaques. We further report here the biomimetic components of MCP-1 PAMs and the influence of the nanoparticle upon binding to monocytes. We report that MCP-1 PAMs have enhanced secondary structure compared to the MCP-1 peptide. As a result, MCP-1 PAMs displayed improved binding and chemoattractant properties to monocytes, which upregulated the inflammatory signaling pathways responsible for monocyte migration. Interestingly, when MCP-1 PAMs were incubated in the presence of prostate cancer cells in vitro, the particle displayed anticancer efficacy by reducing CCR2 expression. Given that monocytes play an important role in tumor cell migration and invasion, our results demonstrate that PAMs can improve the native biofunctional properties of the peptide and may be used as an effective inhibitor to prevent chemokine–receptor interactions that promote disease progression