Noncatalytic Receptor Research Articles

CDCP1 is a novel marker of the most aggressive human triple-negative breast cancers.

CDCP1, a transmembrane noncatalytic receptor, the expression of which has been associated with a poor prognosis in certain epithelial cancers, was found to be expressed in highly aggressive triple-negative breast cancer (TNBC) cell models, in which it promoted aggressive activities—ie, migration, invasion, anchorage-independent tumor growth, and the formation of vascular-like structures in vitro. By immunohistochemical (IHC) analysis of 100 human TNBC specimens, CDCP1 was overexpressed in 57% of samples, 38% of which exhibited a gain in CDCP1 copy number by fluorescence in situ hybridization (FISH). CDCP1 positivity was significantly associated between FISH and IHC. CDCP1 expression and gains in CDCP1 copy number synergized with nodal (N) status in determining disease-free and distant disease-free survival. The hazard ratios (HRs) of the synergies between CDCP1 positivity by IHC and FISH and lymph node positivity in predicting relapse did not differ significantly, indicating that CDCP1 overexpression in human primary TNBCs, regardless of being driven by gains in CDCP1, is for a critical factor in the progression of N-positive TNBCs. Thus, CDCP1 is a novel marker of the most aggressive N-positive TNBCs and a potential therapeutic target.

Abstract P5-07-11: CDCP1 as a new marker of aggressiveness in triple-negative breast cancers

Abstract Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with high recurrences and mortality rate, for which no therapies besides chemotherapy are available to date. Lacking specific markers for an effective targeted therapy, TNBCs continue to represent the most important challenge for clinical oncologists. Here, we investigated the expression of CDCP1, a transmembrane non-catalytic receptor reportedly associated with poor prognosis in some solid tumors (e.g., lung and pancreatic cancer), and its association with tumor aggressiveness in a cohort of 115 human TNBC primary specimens obtained from women surgically treated in our Institute from the beginning of 2002 to the end of 2006 and selected based on immunohistochemical (IHC) criteria (<1% cell positivity for estrogen receptor, progesterone receptor and HER2 expression classified as 0 or 1+). CDCP1 was overexpressed in 56.5% of human primary TNBCs. FISH analysis of 75 TNBCs for which material was available delineated four different genetic categories: 1) disomic, with only two copies of CDCP1 and centromere (CDCP1<3, CEP3<3) (50/75, 67%); 2) amplified CDCP1 (CDCP1 ≥3, CEP3<3) (4/75, 5%); 3) polysomic CDCP1 (CDCP1≥3, CEP3≥3) (15/75, 20%); and 4) CDCP1 deleted of its centromere (CDCP1<3 CEP3≥3) (6/75, 8%). FISH positivity (polysomy or amplification) was significantly associated with IHC positivity (p=0.003). Permutation accuracy variable importance estimated by Random Survival Forests identified both CDCP1 protein expression and FISH positivity for CDCP1 as prognostic factors for DFS (HR=2.67, 95%CI 1.25-5.71, and HR= 2.95, 95%CI 1.33-6.53, respectively) and DDFS (HR=2.40, 95%CI 1.01-5.73, and HR= 3.40, 95%CI 1.44-8.04, respectively), together with age, lymph node involvement, tumor size, DCIS and Ki67 expression. Multivariate Cox survival analysis revealed a synergistic interaction between CDCP1 FISH/IHC status and N-status in DFS and DDFS. Indeed, while the 5-year relapse probability in N-negative patients did not differ according to CDCP1 IHC expression in tumor cells (18% and 13% in CDCP1 IHC negative and positive, respectively), the probability of developing distant metastases at 5 years of follow-up was 82% in N-positive/CDCP1 IHC-positive patients versus only 29% in N-positive/CDCP1 IHC-negative patients. Similarly, the probability of developing distant metastases at 5 years in the N-positive subgroup was 88% for CDCP1 FISH-positive versus 35% for CDCP1 FISH-negative patients, but only 16% and 14% in N-negative/CDCP1 FISH negative and positive, respectively. Together, our results strongly suggest that CDCP1 is a marker of aggressiveness able to identify cases with poorer prognosis among N-positive TNBCs and, noticeably, overexpression of CDCP1 in human primary TNBCs can reflect a CDCP1 genetic gain. Supported by AIRC. Citation Format: Tagliabue E, Turdo F, Bianchi F, Sandri M, Forte L, Casalini P, Gasparini P, Agresti R, Triulzi T, Sozzi G, Campiglio M. CDCP1 as a new marker of aggressiveness in triple-negative breast cancers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-11.

Structural basis of death domain signaling in the p75 neurotrophin receptor.

Death domains (DDs) mediate assembly of oligomeric complexes for activation of downstream signaling pathways through incompletely understood mechanisms. Here we report structures of complexes formed by the DD of p75 neurotrophin receptor (p75(NTR)) with RhoGDI, for activation of the RhoA pathway, with caspase recruitment domain (CARD) of RIP2 kinase, for activation of the NF-kB pathway, and with itself, revealing how DD dimerization controls access of intracellular effectors to the receptor. RIP2 CARD and RhoGDI bind to p75(NTR) DD at partially overlapping epitopes with over 100-fold difference in affinity, revealing the mechanism by which RIP2 recruitment displaces RhoGDI upon ligand binding. The p75(NTR) DD forms non-covalent, low-affinity symmetric dimers in solution. The dimer interface overlaps with RIP2 CARD but not RhoGDI binding sites, supporting a model of receptor activation triggered by separation of DDs. These structures reveal how competitive protein-protein interactions orchestrate the hierarchical activation of downstream pathways in non-catalytic receptors.

Detection of p75NTR Trimers: Implications for Receptor Stoichiometry and Activation.

The p75 neurotrophin receptor (p75(NTR)) regulates a wide range of cellular functions, including apoptosis, neuronal processes remodeling, and synaptic plasticity. The goal of our work was to inquire whether oligomers of the receptor are required for function. Here we report that p75(NTR) predominantly assembles as a trimer, similar to other tumor necrosis factor receptors. Interestingly, monomers and trimers coexist at the cell surface, but trimers are not required for p75(NTR) activation in a functional assay. However, monomers are capable of inducing acute morphological effects in neurons. Identification of the oligomerization state of p75(NTR) begins to provide insights to the mechanisms of signal initiation of this noncatalytic receptor, as well as to develop therapeutic interventions to diminish its activity.

Toll-like Receptor Signaling during Myocardial Ischemia

Acute myocardial infarction and myocardial ischemia are leading causes of short- and long-term morbidity and mortality in the perioperative period. In part, this relates to the fact that treatment options for a myocardial infarction in surgical patients are extremely limited. For example, anti-coagulation or treatment with highly effective inhibitors of platelet aggregation may not be an option due to the risk for bleeding from the operative site. Therefore, it is not surprising that the search for new pharmacologic approaches to treat myocardial ischemia or to render the myocardium more resistant to limited oxygen availability is an area of intense investigation for perioperative scientists.1-4 In fact, the introduction of intraoperative beta-blockers for cardio-protection from ischemia was one of the most significant alterations in anesthesia practice during the past fifteen years. In line with the search for novel therapeutic approaches to render the myocardium more resistant to ischemia, a very exciting research study by Wang et al. from the research laboratory of Dr. Wei Chao from the Anesthesia Center for Critical Care Research of the Massachusetts General Hospital in Boston provides new insight into signaling pathways involving toll-like receptor (TLR)-elicited cardio-protection from ischemia.1

A Mechanism for Src Kinase-Dependent Signaling by Noncatalytic Receptors

A fundamental issue in cell biology is how signals are transmitted across membranes. A variety of transmembrane receptors, including multichain immune recognition receptors, lack catalytic activity and require Src family kinases (SFKs) for signal transduction. However, many receptors only bind and activate SFKs after ligand-induced receptor dimerization. This presents a conundrum: How do SFKs sense the dimerization of receptors to which they are not already bound? Most proposals for resolving this enigma invoke additional players, such as lipid rafts or receptor conformational changes. Here we used simple thermodynamics to show that SFK activation is a natural outcome of clustering of receptors with SFK phosphorylation sites, provided that there is phosphorylation-dependent receptor-SFK association and an SFK bound to one receptor can phosphorylate the second receptor or its associated SFK in a dimer. A simple system of receptor, SFK, and an unregulated protein tyrosine phosphatase (PTP) can account for ligand-induced changes in phosphorylation observed in cells. We suggest that a core signaling system comprising a receptor with SFK phosphorylation sites, an SFK, and an unregulated PTP provides a robust mechanism for transmembrane signal transduction. Other events that regulate signaling in specific cases may have evolved for fine-tuning of this basic mechanism.

Sequence variants of natriuretic peptide receptor C are common and their frequency differs between African Americans and European Americans

Introduction: The growing importance of B-type natriuretic peptide (BNP) as a diagnostic and therapeutic modality in heart failure is well known. One of the primary clearance mechanisms is a non-catalytic receptor, natriuretic peptide receptor C (NPR3). NPR3 sequence variants and haplotypes have not been described in African Americans (AA) and European Americans (EA). Hypothesis: Common polymorphisms in the NPR3 gene exist, and their frequencies differ between African Americans (AA) and European Americans (EA). Methods: Genomic DNA was extracted from 95 African American (AA) and 95 European American (EA) healthy volunteers. Genotype was determined using pyrosequencing. Haplotypes were inferred from genotype results using the Polymorphism and Haplotype Analysis Suite (http://ilya.wustl.edu/∼pgrn/programs.html). Results: Four polymorphisms were validated in the NPR3 gene. One in the promoter region 2664 bases upstream of the transcription start site (A-2664G), one non-synonymous coding polymorphism in exon 8 at codon 521 (A1779G N521D), and two polymorphisms within intron 2; 79 and 84 bp upstream of the exon 3 splice junction (Intron 2 -84G>A, and -79C>T). Each polymorphism occurred commonly with minor allele frequencies ranging from 4.4% to 28%. Genotype distribution differed significantly between AA and EA at 2 of 4 loci (A-2664G and N521D, p=0.037 and p=0.045 respectively). The A1779G and Intron 2 -79C>T polymorphisms were significantly linked in EA (|D|=0.84; p<0.001) but not in the AA group. Of 16 theoretical haplotypes, the 5 most common accounted for >90% of subjects within each racial group. The distribution of haplotypes is shown in figure 1. The two most common haplotypes were the same for both AA and EA. One common haplotype among AA (haplotype 5, 9%) was not observed among EA. Conclusions: Genetic variation in NPR3 is common. The frequency of two variants (A-2664G, and A1779G-N521D) differ significantly between AA and EA. The haplotypes present among AA and EA were generally similar but occurred at differing frequencies. Clinical and functional assessment of these polymorphisms is warranted to evaluate their influence on BNP physiology and pharmacology.

The noncatalytic TrkCNC2 receptor is cleaved by metalloproteases upon neurotrophin-3 stimulation.

The trkC locus encodes catalytic and noncatalytic receptors, generated by alternative splicing. These primary high-affinity neurotrophin-3 (NT-3) receptors may act in concert to modulate responsiveness to NT-3. Signal modulation can also be achieved by receptors that are post-translationally processed. We report that the noncatalytic TrkC receptor, TrkCNC2, is cleaved at the membrane-proximal region of its extracellular domain. This generates a soluble ectodomain (gp90(TrkCNC2)) recovered in the cell culture medium and a membrane-bound fragment (p20(TrkCNC2)), which contains the transmembrane and intracellular regions including the juxtamembrane and the NC2-specific cytoplasmic domains. We also show that this processing, which does not occur in the TrkC catalytic counterpart, is upregulated by NT-3 and upon treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Moreover, cleavage inhibition after EDTA or 1.10 phenanthroline treatment suggests involvement of a metalloprotease(s). Finally, this post-translational processing was observed not only in TrkCNC2-overexpressing NIH3T3 cells but also in primary cultures of cortical neurons and brain extracts. This study shows that, in addition to alternative splicing, ectodomain shedding represents a novel means of regulating TrkC receptor signaling, and consequently NT-3 biological effects on target cells.

Trk C Signaling Is Required for Retinal Progenitor Cell Proliferation

Although neurotrophin actions in the survival of specific retinal cell types have been identified, the biological functions for neurotrophin-3 (NT-3) in early retinal development remain unclear. Having localized NT-3 and trk C expression at early developmental stages when retinal neuroepithelial progenitor cells predominate, we sought to modulate NT-3 signaling in these cells by overexpressing a truncated isoform of the NT-3 receptor, trk C. We have demonstrated that this non-catalytic receptor can inhibit NT-3 signaling when coexpressed with the full-length kinase-active trk C receptor. Using a replication-deficient retrovirus to ectopically express the truncated trk C receptor to limited numbers of progenitor cells in ovo, we examined the effects of disrupted trk C signaling on the proliferation or differentiation of retinal cells. Clones expressing truncated trk C exhibited a 70% reduction in clone size, compared with clones infected with a control virus, indicating that inhibition of trk C signaling decreased the clonal expansion of cells derived from a single retinal progenitor cell. Additionally, impaired NT-3 signaling resulted in a reduction of all retinal cell types, suggesting that NT-3 targets retinal precursor cells rather than differentiated cell types. BrdU labeling studies performed at E6 indicate that this reduction in cell number occurs through a decrease in cell proliferation. These studies suggest that NT-3 is an important mitogen early in retinal development and serves to establish the size of the progenitor pool from which all future differentiated cells arise.

Quantitative evaluation of neurotrophin andtrk mRNA expression in visual and limbic areas along the occipito-temporo-hippocampal pathway in adult macaque monkeys

The neurotrophins have been implicated in shaping and remodeling the connectivity of neural circuits. To explore the role of neurotrophins and their receptors, Trks, in cortical neural circuits of adult macaque monkeys, we determined mRNA expression levels of neurotrophins and Trk receptors in various visual and limbic areas along the occipito-temporo-hippocampal pathway by using a quantitative reverse-transcription polymerase chain reaction technique. The expression level of brain-derived neurotrophic factor (BDNF) mRNA was lowest in the primary visual cortex (V1), moderate in the temporal visual association area, and highest in the hippocampus. The expression levels of trkB mRNA isoforms, the full-length form that encodes a receptor tyrosine kinase and the truncated form that encodes a noncatalytic receptor, were also low in V1, moderate in the visual association area, and high in the entorhinal cortex. However, in contrast to their ligand BDNF, the expression levels of both trkB isoforms in the hippocampus were significantly lower than those in the entorhinal cortex. NT-3 mRNA was detectable only in the hippocampus and the entorhinal cortex, whereas both the full-length and the truncated forms of trkC mRNA were widely distributed throughout the neocortex and the limbic cortex. The expression levels of NGF and trkA mRNAs in these cortical areas were too low to determine quantitatively. The present findings suggest that, among neurotrophin/Trk signaling systems, the BDNF/TrkB-mediated signal most likely contributes to stabilization, remodeling, or both, of neural circuits in cortical areas along the occipito-temporo-hippocampal pathway in the adult macaque monkey.

Quantitative evaluation of neurotrophin and trk mRNA expression in visual and limbic areas along the occipito‐temporo‐hippocampal pathway in adult macaque monkeys

The neurotrophins have been implicated in shaping and remodeling the connectivity of neural circuits. To explore the role of neurotrophins and their receptors, Trks, in cortical neural circuits of adult macaque monkeys, we determined mRNA expression levels of neurotrophins and Trk receptors in various visual and limbic areas along the occipito-temporo-hippocampal pathway by using a quantitative reverse-transcription polymerase chain reaction technique. The expression level of brain-derived neurotrophic factor (BDNF) mRNA was lowest in the primary visual cortex (V1), moderate in the temporal visual association area, and highest in the hippocampus. The expression levels of trkB mRNA isoforms, the full-length form that encodes a receptor tyrosine kinase and the truncated form that encodes a noncatalytic receptor, were also low in V1, moderate in the visual association area, and high in the entorhinal cortex. However, in contrast to their ligand BDNF, the expression levels of both trkB isoforms in the hippocampus were significantly lower than those in the entorhinal cortex. NT-3 mRNA was detectable only in the hippocampus and the entorhinal cortex, whereas both the full-length and the truncated forms of trkC mRNA were widely distributed throughout the neocortex and the limbic cortex. The expression levels of NGF and trkA mRNAs in these cortical areas were too low to determine quantitatively. The present findings suggest that, among neurotrophin/Trk signaling systems, the BDNF/TrkB-mediated signal most likely contributes to stabilization, remodeling, or both, of neural circuits in cortical areas along the occipito-temporo-hippocampal pathway in the adult macaque monkey. J. Comp. Neurol. 408:378–398, 1999. © 1999 Wiley-Liss, Inc.

Catalytic and non-catalytic forms of the neurotrophin receptor xTrkB mRNA are expressed in a pseudo-segmental manner within the early Xenopus central nervous system.

The induction of anterior-posterior and medio-lateral patterning within the Xenopus neural plate leads to the rapid establishment of a functional nervous system. Here we describe two Xenopus TrkB neurotrophin receptor genes which are expressed in discrete sets of neuroblasts during this developmental process. The xTrkB mRNAs encode both catalytic and non-catalytic receptors and exhibit membrane-spanning-domain proximal splicing. Expression begins at neural tube closure within the trigeminal ganglion and within the Rohon-Beard neurons of the dorsal spinal cord, providing an excellent dorsal marker of early neural tube patterning. Expression occurs later in the facial ganglia and possibly within the Kolmer-Agduhr neurons. The predominant xTrkB transcripts within the trigeminal and Rohon-Beard neurons and the exclusive early transcripts of the facial ganglia encode C-terminally truncated non-catalytic receptors. Such Trk mRNAs have previously been observed in rodents. However, our observations suggest that they may play a specific role during early development. Anterior-posterior segmentation of the neural tube occurs rostrally within the prospective brain, but previous studies have suggested that segmentation does not extend caudally into the spinal cord. We show that the xTrkB positive Rohon-Beard neurons of the spinal cord do in fact display clear segmental groupings soon after neural tube closure. This is consistent with a role for segmentation in the anterior-posterior patterning of the trunk central nervous system.

Structural and functional properties of the TRK family of neurotrophin receptors.

The Trk family of tyrosine-protein kinases, TrkA, TrkB, and TrkC, are the signaling receptors that mediate the biological properties of the NGF family of neurotrophins. This family of growth factors includes in addition to NGF, BDNF, NT-3, and NT-4. TrkA is the NGF receptor. TrkB serves as a receptor for both BDNF and NT-4, and TrkC is the primary receptor for NT-3. NT-3 is a somewhat promiscuous ligand that can also activate TrkA and TrkB receptors at high concentrations. The trkB and trkC genes also encode noncatalytic receptor isoforms of an, as yet, unknown function. In addition to the Trk receptors, the NGF family of neurotrophins also binds with low affinity to an unrelated molecule, designated p75, a member of the TNF-receptor superfamily. Recently, we have generated strains of mice lacking each of these tyrosine-kinase receptors by gene targeting in embryonic stem cells. Characterization of these mutant mice is providing relevant information regarding the critical role that these receptors play in the ontogeny of the mammalian nervous system.