Sara Lavi

ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network.

ABSTRACT The recently isolated second family of neuregulins, NRG2, shares its primary receptors, ErbB-3 and ErbB-4, and induction of mammary cell differentiation with NRG1 isoforms, suggesting functional redundancy of the two growth factor families. To address this possibility, we analyzed receptor specificity of NRGs by using an engineered cellular system. The activity of isoform-specific but partly overlapping patterns of specificities that collectively activate all eight ligand-stimulatable ErbB dimers was revealed. Specifically, NRG2-β, like NRG1-α, emerges as a narrow-specificity ligand, whereas NRG2-α is a pan-ErbB ligand that binds with different affinities to all receptor combinations, including those containing ErbB-1, but excluding homodimers of ErbB-2. The latter protein, however, displayed cooperativity with the direct NRG receptors. Apparently, signaling by all NRGs is funneled through the mitogen-activated protein kinase (MAPK). However, the duration and potency of MAPK activation depend on the identity of the stimulatory ligand-receptor ternary complex. We conclude that the NRG-ErbB network represents a complex and nonredundant machinery developed for fine-tuning of signal transduction.

Persistent elimination of ErbB-2/HER2-overexpressing tumors using combinations of monoclonal antibodies: Relevance of receptor endocytosis

Monoclonal antibodies (mAbs) to ErbB-2/HER2 or to its sibling, the epidermal growth factor receptor (EGFR), prolong survival of cancer patients, especially when combined with cytotoxic therapies. However, low effectiveness of therapeutic mAbs and the evolution of patient resistance call for improvements. Here we test in animals pairs of anti-ErbB-2 mAbs and report that pairs comprising an antibody reactive with the dimerization site of ErbB-2 and an antibody recognizing another distinct epitope better inhibit ErbB-2-overexpressing tumors than other pairs or the respective individual mAbs. Because the superiority of antibody combinations extends to tumor cell cultures, we assume that nonimmunological mechanisms contribute to mAb synergy. One potential mechanism, namely the ability of mAb combinations to instigate ErbB-2 endocytosis, is demonstrated. Translation of these lessons to clinical applications may enhance patient response and delay acquisition of resistance.

Conjugation to Nedd8 Instigates Ubiquitylation and Down-regulation of Activated Receptor Tyrosine Kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.

Role of Type 2C Protein Phosphatases in Growth Regulation and in Cellular Stress Signaling

ABSTRACT A number of interesting features, phenotypes, and potential clinical applications have recently been ascribed to the type 2C family of protein phosphatases. Thus far, 16 different PP2C genes have been identified in the human genome, encoding (by means of alternative splicing) for at least 22 different isozymes. Virtually ever since their discovery, type 2C phosphatases have been predominantly linked to cell growth and to cellular stress signaling. Here, we provide an overview of the involvement of type 2C phosphatases in these two processes, and we show that four of them (PP2Cα, PP2Cβ, ILKAP, and PHLPP) can be expected to function as tumor suppressor proteins, and one as an oncoprotein (PP2Cδ /Wip1). In addition, we demonstrate that in virtually all cases in which they have been linked to the stress response, PP2Cs act as inhibitors of cellular stress signaling. Based on the vast amount of experimental evidence obtained thus far, it therefore seems justified to conclude that type 2C protein phosphatases are important physiological regulators of cell growth and of cellular stress signaling.

Inhibition of triple-negative breast cancer models by combinations of antibodies to EGFR

Breast tumors lacking expression of human epidermal growth factor receptor 2 (HER2) and the estrogen and the progesterone receptors (triple negative; TNBC) are more aggressive than other disease subtypes, and no molecular targeted agents are currently available for their treatment. Because TNBC commonly displays EGF receptor (EGFR) expression, and combinations of monoclonal antibodies to EGFR effectively inhibit other tumor models, we addressed the relevance of this strategy to treatment of TNBC. Unlike a combination of the clinically approved monoclonal antibodies, cetuximab and panitumumab, which displaced each other and displayed no cooperative effects, several other combinations resulted in enhanced inhibition of TNBC’s cell growth both in vitro and in animals. The ability of certain antibody mixtures to remove EGFR from the cell surface and to promote its intracellular degradation correlated with the inhibitory potential. However, unlike EGF-induced sorting of EGFR to lysosomal degradation, the antibody-induced pathway displayed independence from the intrinsic kinase activity and dimer formation ability of EGFR, and it largely avoided the recycling route. In conclusion, although TNBC clinical trials testing EGFR inhibitors reported lack of benefit, our results offer an alternative strategy that combines noncompetitive antibodies to achieve robust degradation of EGFR and tumor inhibition.

Small polydispersed circular DNA (spcDNA) in human cells: association with genomic instability

Small Polydispersed Circular DNA (spcDNA) was suggested to be associated with genetically unstable cells. However, until now, qualitative and quantitative research has been limited due to the lack of efficient methods for detection and analysis. We developed a two-dimensional (2-D) neutral-neutral gel electrophoresis assay for the identification, characterisation and quantitation of spcDNA. Using this method, we established the relation of spcDNA to genetic and induced genomic instability in human cells, both in vitro and in vivo. Enhanced amounts of spcDNA were found in genetically unstable cells and tissues. spcDNA was detected in a tumor cell-line (HeLa) and in tumor tissue (colon carcinoma) as well as in fibroblasts derived from patients suffering from the genomic instability disease, Fanconis Anemia. We failed to detect spcDNA in the genetically stable normal human fibroblasts. However, following treatment with the initiating carcinogen MNNG, an induction of spcDNA was observed. The level of spcDNA was quantified according to mitochondrial DNA (mtDNA) standards. In light of these findings, we discuss the possible role of spcDNA as a marker and an enhancer of genomic instability.

Telomeric repeats on small polydisperse circular DNA (spcDNA) and genomic instability.

Small polydisperse circular DNA (spcDNA) is a heterogeneous population of extrachromosomal circular molecules present in a large variety of eukaryotic cells. Elevated amounts of total spcDNA are related to endogenous and induced genomic instability in rodent and human cells. We suggested spcDNA as a novel marker for genomic instability, and speculated that spcDNA might serve as a mutator. In this study, we examine the presence of telomeric sequences on spcDNA. We report for the first time the appearance of telomeric repeats in spcDNA molecules (tel-spcDNA) in rodent and human cells. Restriction enzyme analysis indicates that tel-spcDNA molecules harbor mostly, if not exclusively, telomeric repeats. In rodent cells, tel-spcDNA levels are higher in transformed than in normal cells and are enhanced by treatment with carcinogen. Tel-spcDNA is also detected in some human tumors and cell lines, but not in others. We suggest, that its levels in human cells may be primarily related to the amount of the chromosomal telomeric sequences. Tel-spcDNA may serve as a unique mutator, through specific mechanisms related to the telomeric repeats, which distinguish it from the total heterogeneous spcDNA population. It may affect telomere dynamics and genomic instability by clastogenic events, alterations of telomere size and sequestration of telomeric proteins.

Evolution of the metazoan protein phosphatase 2C superfamily.

Members of the protein phosphatase 2C (PP2C) superfamily are Mg2+/Mn2+-dependent serine/threonine phosphatases, which are essential for regulation of cell cycle and stress signaling pathways in cells. In this study, a comprehensive genomic analysis of all available metazoan PP2C sequences was conducted. The phylogeny of PP2C was reconstructed, revealing the existence of 15 vertebrate families which arose following a series of gene duplication events. Relative dating of these duplications showed that they occurred in two active periods: before the divergence of bilaterians and before vertebrate diversification. PP2C families which duplicated during the first period take part in different signaling pathways, whereas PP2C families which diverged in the second period display tissue expression differences yet participate in similar signaling pathways. These differences were found to involve variation of expression in tissues which show higher complexity in vertebrates, such as skeletal muscle and the nervous system. Further analysis was performed with the aim of identifying the functional domains of PP2C. The conservation pattern across the entire PP2C superfamily revealed an extensive domain of more than 50 amino acids which is highly conserved throughout all PP2C members. Several insertion or deletion events were found which may have led to the specialization of each PP2C family.

Modulation of the cellular phenotype by integrated adeno-associated virus.

The adeno-associated virus (AAV) rep gene encodes a series of overlapping, multifunctional, nonstructural proteins (Rep proteins) which regulate the viral life cycle and which are also capable of trans-regulating nonviral gene expressions (reviewed in Berns, 1990, Microbiol. Rev. 54, 316-329). To investigate the expression of the AAV rep gene in a cellular chromosomal context, SV40-transformed Chinese hamster embryo (OD4) cells were infected with an AAV/neo hybrid virus and progeny resistant to the antibiotic G418 were selected and amplified. Chromosomal integration and RNA transcription of the AAV and neo DNA inserts were confirmed by Southern and Northern blotting procedures. One of the G418R cell lines stably expressed a protein which reacted specifically with AAV anti-Rep antiserum in Western immunoblots. The stable integration of AAV rep DNA, which did not interfere with cell proliferation under normal growth conditions, was associated with two changes in cellular phenotype: eight of nine lines were markedly more sensitive to UV light (254 nm) than were the parental OD4 cells; and seven of the nine lines had lost the capacity to promote SV40 origin DNA amplification in vitro, in contrast to the parental OD4 cells. OD4 cells transformed to G418R by AAV/neo DNA constructs with a deleted rep gene, or by a neo DNA construct lacking AAV DNA, did not display these phenotypic changes. It is suggested that stable integration of the AAV rep gene interferes with cellular processes connected with DNA repair and gene amplification.

Role of PP2Cα in cell growth, in radio- and chemosensitivity, and in tumorigenicity

BackgroundPP2Cα is the representative member of the type 2C family of protein phosphatases, and it has recently been implicated in the regulation of p53-, TGFβ-, cyclin-dependent kinase- and apoptosis-signaling. To investigate the role of PP2Cα in cell growth and in radio- and chemosensitivity, wild type and PP2Cα siRNA-expressing MCF7 cells were subjected to several different viability and cell cycle analyses, both under basal conditions and upon treatment with radio- and chemotherapy. By comparing the growth of tumors established from both types of cells, we also evaluated the involvement of PP2Cα in tumorigenesis.ResultsIt was found that knockdown of PP2Cα did not affect the proliferation, the clonogenic survival and the membrane integrity of MCF7 cells. In addition, it did not alter their radio- and chemosensitivity. For PP2Cα siRNA-expressing MCF7 cells, the number of cells in the G0/G1 phase of the cell cycle was reduced, the induction of the G1 block was attenuated, the number of cells in G2/M was increased, and the induction of the G2 block was enhanced. The tumorigenic potential of PP2Cα siRNA-expressing MCF7 cells was found to be higher than that of wild type MCF7 cells, and the in vivo proliferation of these cells was found to be increased.ConclusionBased on these findings, we conclude that PP2Cα is not involved in controlling cell growth and radio- and chemosensitivity in vitro. It does, however, play a role in the regulation of the cell cycle, in the induction of cell cycle checkpoints and in tumorigenesis. The latter notion implies that PP2Cα may possess tumor-suppressing properties, and it thereby sets the stage for more elaborate analyses on its involvement in the development and progression of cancer.